F Matschinsky, Y Liang, P Kesavan, L Wang, P Froguel, G Velho, D Cohen, M A Permutt, Y Tanizawa, T L Jetton
Clara cell 10-kD protein (cc10kD), a secretory phospholipase A2 inhibitor, is suggested to be the human counterpart of rabbit uteroglobin (UG). Because cc10kD is expressed constitutively at a very high level in the human respiratory epithelium, the 5' region of its gene may be useful in achieving organ-specific expression of recombinant DNA in gene therapy of diseases such as cystic fibrosis. However, it is important to establish the tissue-specific expression of this gene before designing gene transfer experiments. Since the UG gene in the rabbit is expressed in many other organs besides the lung and the endometrium, we investigated the organ and tissue specificity of human cc10kD gene expression using polymerase chain reaction, nucleotide sequence analysis, immunofluorescence, and Northern blotting. Our results indicate that, in addition to the lung, cc10kD is expressed in several nonrespiratory organs, with a distribution pattern very similar, if not identical, to that of UG in the rabbit. These results underscore the necessity for more detailed analyses of the 5' region of the human cc10kD gene before its usefulness in gene therapy could be fully assessed. These data also suggest that cc10kD and UG may have similar physiological function(s).
A Peri, E Cordella-Miele, L Miele, A B Mukherjee
We have examined the hypothesis that TNF may play a pathogenetically important role in the hemolytic uremic syndrome. Specifically, we considered the possibility that shigatoxin, which eventuates this syndrome, might induce TNF biosynthesis, and/or that TNF and shigatoxin might sensitize animals, each to the toxic effects of the other agent. Shigatoxin was found to sensitize mice to the lethal effect of LPS and to the lethal effect of TNF. On the other hand, pretreatment of animals with either TNF or LPS did not noticeably sensitize mice to the lethal effect of shigatoxin. Intraperitoneal injections of shigatoxin did not induce the production of detectable quantities of TNF in the plasma of mice. When shigatoxin was injected into transgenic mice bearing a chloramphenicol acetyltransferase (CAT) reporter gene that indicates TNF synthesis, CAT activity was induced within the kidney, but not in other tissues. We therefore conclude that shigatoxin acts to induce TNF synthesis within the kidney, and at the same time increases renal sensitivity to the toxic effects of TNF. While this mouse model does not reproduce the hemolytic uremic syndrome as it occurs in humans, it does suggest that local synthesis of TNF within the kidney may contribute to renal injury induced by shigatoxin.
Y Harel, M Silva, B Giroir, A Weinberg, T B Cleary, B Beutler
A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.
A M Luo, K M Garza, D Hunt, K S Tung
To study the mechanism of lipoprotein lipase (LPL) regulation by exercise, we recruited 16 healthy athletes to undergo a 2-wk period of detraining. Fasting fat and muscle biopsies were performed both before and after the detraining period. In muscle, detraining resulted in a decrease in LPL activity in both the heparin-releasable (HR) (-45%, P < 0.05) and cellular (extractable [EXT]) (-75%, P < 0.005) fractions, with no significant changes in LPL immunoreactive mass and mRNA levels. However, several subjects demonstrated parallel decreases in LPL mass and mRNA levels with detraining, suggesting that there is some degree of heterogeneity in response. In adipose tissue, detraining had the opposite effects on LPL activity. In the HR fraction, detraining resulted in an 86% increase (P < 0.005) in LPL activity, which was paralleled by a 100% (P = 0.02) increase in HR mass. However, there was no significant change in EXT LPL activity or EXT LPL mass. There were no changes in adipose LPL synthetic rate or LPL mRNA levels with detraining. The ratio of adipose tissue/muscle LPL, which may be an important indicator of the tendency for storage of circulating lipids in adipose tissue, increased significantly after detraining. The adipose/muscle LPL ratio was 0.51 +/- 0.17 in the exercising runners, and 4.45 +/- 2.46 in the same runners after detraining (P < 0.05). Thus, detraining of athletes resulted in a decrease in muscle LPL that occurred through post-translational mechanisms, whereas adipose tissue LPL increased, also due to posttranslational changes. This decrease in muscle LPL, coupled with an increase in adipose LPL, yielded a condition favoring adipose tissue storage.
R B Simsolo, J M Ong, P A Kern
We have evaluated the contribution of depression of individual procoagulant vitamin K-dependent clotting factors to the ability of warfarin to protect rabbits against tissue factor-induced coagulation. Mean activities of individual procoagulant factors were determined, in assays with rabbit substrates, for a group of rabbits achieving a protective degree of anticoagulation with warfarin. Values were: factor VII, 12%; factor IX, 7%; factor X, 14%, and prothrombin, 13%. The effect upon tissue factor-induced coagulation of selective immunodepletion of each factor to a comparable level was then evaluated. Immunodepletion of plasma factor X or prothrombin, but not of factor VII or factor IX, protected otherwise normal rabbits against tissue factor-induced coagulation. Next, we determined the effect upon the protection in warfarin-treated rabbits of selectively restoring factor X or prothrombin before infusing tissue factor. When either factor was selectively restored, warfarin's protective effect was abolished. Moreover, selective restoration of prothrombin sensitized warfarin-treated rabbits to coagulation more severe than observed in nontreated control rabbits. One may extrapolate from these data that depression of both factor X and prothrombin are required for warfarin's clinical antithrombotic efficacy and that depression of plasma prothrombin is particularly important.
A Zivelin, L V Rao, S I Rapaport
We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.
B N Ling, E E Seal, D C Eaton
Neoplastic diseases are frequently associated with metabolic changes collectively known as cancer cachexia. The presence of cachexia complicates therapeutic intervention and is an important cause of death in cancer patients. At present there is no effective treatment for cachexia. Recently, the involvement of interleukin-6 (IL-6) in the wasting of colon-26 adenocarcinoma-bearing mice was demonstrated. The research presented here establishes an anticachectic role for the experimental drug suramin, since it partially blocks (up to 60%) the catabolic effects associated with the growth of this tumor in vivo. Suramin prevents the binding of IL-6 to its cell surface receptor subunits, as demonstrated by radioreceptor binding assay and affinity crosslinking experiments. Furthermore, the uptake of radioactive IL-6 by the liver is significantly reduced in suramin-treated mice. On the other hand, the drug is approximately 10-fold less potent in inhibiting the binding of tumor necrosis factor-alpha to indicator cell line in vitro and fails to block liver uptake of this cytokine in vivo. Collectively, these results suggest that suramin inhibits cancer-associated wasting, in part by interfering with the binding of IL-6 to its receptor. Whether suramin inhibits the action of other factors/cytokines that may also participate in colon-26-mediated cachexia is not yet known.
G Strassmann, M Fong, C E Freter, S Windsor, F D'Alessandro, R P Nordan
We have identified a soluble form of the human urokinase plasminogen activator (uPA) receptor (uPAR) in the ascitic fluids from patients with ovarian cancer. After purification of uPAR from the ascitic fluids by ligand-affinity chromatography (pro-uPA Sepharose), the uPAR was initially identified by cross-linking to a radiolabeled amino-terminal fragment of human uPA. The uPAR purified from the ascitic fluid has no bound ligand (uPA), as similar amounts can be purified by ligand-affinity chromatography as by immuno-affinity chromatography. uPAR from ascitic fluids partitions in the water phase after a temperature-dependent phase separation of a detergent extract. It therefore lacks at least the lipid moiety of the glycophospholipid anchor present in cellular-bound uPARs. It is highly glycosylated and the deglycosylated form has the same electrophoretic mobility as previously characterized cellular uPAR from other sources. The immunoreactivity of the purified uPAR from the ascitic fluid is indistinguishable from that of characterized uPAR, demonstrated by Western blotting with three different anti-uPAR monoclonal antibodies. The uPAR was found in 11 of 11 ascitic fluids from patients with ovarian cancer and in elevated amounts in the plasma from 2 of 3 patients. The concentration of soluble uPAR in the ascitic fluid was estimated to range between 1 and 10 ng/ml. Human soluble uPAR, derived from the tumor cells, was also found in the ascitic fluid and serum from nude mice xenografted intraperitoneally with three different human ovarian carcinomas.
N Pedersen, M Schmitt, E Rønne, M I Nicoletti, G Høyer-Hansen, M Conese, R Giavazzi, K Dano, W Kuhn, F Jänicke
Bradykinin stimulates diverse functions in endothelial cells including the release of endothelium-derived relaxing factor (EDRF). Little is known, however, regarding the identity of the G protein(s) involved. Here we demonstrate that G proteins of the G alpha i and G alpha q family are coupled to the bradykinin receptor (BKR) in bovine aortic endothelial cells by using specific antisera directed against the COOH-terminal region of G alpha i2 (P4), G alpha i3 (EC), and G alpha q (QL). These antisera are specific since their effects are blocked by the decapeptides from which they were derived. The degree of receptor-G protein coupling was assessed by the formation of high affinity agonist binding sites (HABS) and GTP hydrolysis. In a concentration-dependent manner, the QL antisera reduced HABS and GTPase activity by 65 and 60%, respectively, and effectively abolished them in membranes from pertussis toxin-treated cells. The combination of P4 and EC antisera produced a loss of HABS (41%) and GTPase activity (40%) comparable to the effects of pertussis toxin. These findings indicate that G alpha i and G alpha q proteins mediate the cellular responses to bradykinin in bovine aortic endothelial cells and support the observation that bradykinin-stimulated EDRF release is relatively insensitive to pertussis toxin.
J K Liao, C J Homcy
D C Devor, M C Sekar, R A Frizzell, M E Duffey
Steroid 21-hydroxylase deficiency is a major cause of congenital adrenal hyperplasia and is caused by genetic impairment of this enzyme. Since approximately 80% of cases are caused by point mutations of the CYP21B (CYP21A2) gene, whereas the remaining 20% are due to deletion of this gene, we used the polymerase chain reaction single strand conformation polymorphism technique for rapid and accurate diagnosis of this disease. Of 23 patients examined, 1 had a hemizygous CYP21B gene. 18 patient's genes localized their harmful mutations or deletion on both the alleles, while 4 of them found their causative mutations on one of the two alleles, and 1 failed to find any responsible mutation. All the mutations (four nucleotide substitutions) detected are also found in the CYP21A (CYP21A1) pseudogene. A mutation at the intron 2 site is most prevalent in both salt-wasting and simple virilizing forms of the disease, and accounts for 37% of the patient's genes (17/46). Pedigree analysis of these mutations revealed that the mutations (at least four of them) occurred de novo at a considerable frequency on both the paternally and maternally inherited chromosomes. This result could explain occasional discordance of the diagnosis using HLA typing with the clinical symptoms.
T Tajima, K Fujieda, K Nakayama, Y Fujii-Kuriyama
There are marked variations in the activity of lipoprotein lipase (LPL) among adipose depots, particularly in women. Consistent with data on LPL activity, the level of expression of LPL mRNA was lower in omental (OM) than subcutaneous (SQ) adipose tissue of women. To investigate the cellular basis of these differences, OM and SQ adipose tissues obtained at surgery from obese men and women were placed in organ culture for 7 d with varying concentrations of insulin and dexamethasone. Insulin increased levels of LPL mRNA and LPL activity in abdominal SQ but not OM adipose tissue. Dexamethasone also increased LPL mRNA and LPL activity, and these effects were more marked in the OM adipose tissue, particularly in men. When insulin and dexamethasone were added together, synergistic increases in LPL activity were seen in both depots, and this was in part explained at the level of LPL mRNA. The SQ depot was more sensitive to the effects of submaximal doses of dexamethasone in the presence of insulin. The maximum activity of LPL induced by insulin or insulin plus dexamethasone was higher in the SQ than in the OM depot of women, and this was associated with higher levels of LPL mRNA. Rates of LPL synthesis paralleled LPL mRNA levels. These data show that insulin and glucocorticoids influence human adipose tissue LPL activity at the level of LPL gene expression, as well as posttranslationally, and that responsiveness to these hormonal effects is dependent on adipose depot and gender.
S K Fried, C D Russell, N L Grauso, R E Brolin
An epitope present in the 71-90 sequence of basic protein (BP) has been identified as the dominant epitope recognized by most Lewis rat encephalitogenic T cells isolated during experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the BP epitopes recognized by Lewis rat T cells in naive rats, in rats suffering from acute EAE, and in recovered rats. T cells isolated from the spinal cord lesions and from the lymph nodes were studied using T cell lines and bulk cultures. Virulence of the T cells was assayed by adoptive transfer. We now report that naive and recovered Lewis rats are populated with T cells reactive to a variety of BP epitopes and only a minority are specific for the 71-90 epitope. In contrast, the induction of EAE was associated with a predominance of T cells reactive to the 71-90 epitope. T cells recovered from naive, diseased, or recovered rats were found to be virulent upon passive transfer. Some of these virulent T cells were specific to BP epitopes other than the 71-90 epitope. There was no major difference in the BP specificities of T cells isolated from the lesions and from the lymph nodes. Thus, natural T cell reactivity to BP is heterogeneous and pathogenicity is not confined to one particular epitope, active disease is characterized by a dominant response to the 71-90 epitope, and recovery is marked by a return to heterogeneity.
F Mor, I R Cohen
Human T lymphocytes possess both the type I and II isozymes of protein kinase A (PKA). The type I (PKA-I) isozyme is predominantly associated with the plasma membrane, whereas the type II (PKA-II) isozyme is primarily localized to the cytosol. Because the functions of both PKA-I and PKA-II isozymes in the biochemical events of T lymphocyte activation have not been clearly elucidated, we tested the hypothesis that very early events of normal human T lymphocyte activation are mediated by the PKA-I and/or PKA-II isozyme(s). Fresh normal human T cells and a normal human CD4+ T cell line (GK606) activated with anti-CD3-epsilon and recombinant interleukin 1 alpha (rIL-1 alpha) exhibited a peak six- to sevenfold increase of PKA phosphotransferase activity at 5 min that returned to baseline by 60 min. Similarly, both fresh T cells and the T cell line activated by phorbol myristate acetate and ionomycin demonstrated a peak eightfold increase of PKA activity by 15 min that returned toward baseline by 60 min. Chromatographic separation of the PKA isozymes and quantification of phosphotransferase activities after T cell activation by either agonist pair showed preferential activation of the PKA-I isozyme, resulting in a significant reduction in the ratio of PKA-I to PKA-II isozyme activity from 3.1:1-6.2:1 to 1.1:1-3.2:1. PKA-I isozyme activation resulted in the release of free catalytic (C) subunit, an increase in C subunit phosphotransferase activity, and the phosphorylation of T cell plasma membrane-associated proteins, p14, p17, p20, p21, p38, and p48. However, activation of the PKA-I isozyme did not appear to be required for the transcription of IL-2 mRNA, an event necessary for mitosis. These data indicate that ligand-induced T cell activation is associated with rapid activation of the PKA-I, but not PKA-II, isozyme that results in phosphorylation of plasma membrane-associated proteins. The involvement of the PKA-I isozyme during the very early events of T cell activation suggests that this isozyme may be an antigen- or mitogen-stimulated protein kinase.
D Laxminarayana, A Berrada, G M Kammer
This study was designed to determine whether beta-lactam antibiotics (cephalosporins and penicillins) are all substrates for the renal oligopeptide/H+ symporter and, if so, whether the transport system discriminates among the numerous beta-lactam antibiotics. We used [3H]glycylglutamine, [3H]cephalexin, and [3H]-ampicillin as probes for the transport of oligopeptides, cephalosporins, and penicillins in kidney brush border membrane vesicles, respectively. Among the beta-lactam antibiotics, only those with an alpha-amino group in the phenylacetamido moiety were found to interact with the oligopeptide/H+ symporter. Aminocephalosporins displayed high affinities (KiS generally < 250 microM), whereas aminopenicillins displayed low affinities (Ki 0.78-3.03 mM). These differences in affinities appeared to be a consequence of conformational features of the substrates, especially the sterical location of the carboxy group. The affinities of aminolactams for the oligopeptide/H+ symporter were, furthermore, related to the hydrophobicity of the phenylglycyl chains and the substituents attached to the thiazolidine and dihydrothiazine ring. In sharp contrast to the uptake of [3H]glycylglutamine and [3H]cephalexin, the uptake of [3H]ampicillin was not dependent on a pH gradient and was inhibited by various beta-lactam antibiotics, whether or not they contained an alpha-amino group. Our data suggest that: (a) the transport of aminocephalosporins is largely mediated by the oligopeptide/H+ symporter, which is highly influenced by the substrate structure; and (b) penicillins are transported by another system, which is less discriminative with respect to substrate structure.
H Daniel, S A Adibi
High doses of intravenous protamine cause generalized vascular permeability changes in brain and other organs, and concomitant hypoproteinemia. The present investigations test the hypothesis that protamine has a dual action of both binding serum proteins and of undergoing absorptive-mediated transcytosis through microvascular endothelial barriers. Binding of albumin to protamine was demonstrated using equilibrium dialysis, and protamine was shown to selectively augment the uptake of albumin, but not sucrose, in isolated bovine or human brain capillaries. In contrast, the anionic macromolecule, dextran sulfate, resulted in an increased capillary uptake of both albumin and sucrose in vitro. The selective effects of protamine on albumin transport were also documented in vivo using an external organ technique; the intravenous injection of 1.5 mg/kg protamine resulted in a marked and selective influx of albumin into brain, heart, kidney, lung, and liver, and the increased albumin transport exceeded the increased sucrose uptake in some organs by an order of magnitude. The transcytosis of protamine through the cerebral microvascular barrier was documented with an internal carotid artery perfusion technique. In summary, these studies provide evidence for protamine-mediated vectorial transport of albumin through microvascular barriers in brain and other organs.
W M Pardridge, J L Buciak, Y S Kang, R J Boado
Subcutaneous injection of the potent, nonselective opioid antagonist diprenorphine inhibits the vasopressin response to acute hypovolemia. To determine if this inhibition is due to antagonism of opioid receptors in brain pathways that mediate volume control, we determined the vasopressin response to different stimuli when diprenorphine or other opiates were injected into the cerebral ventricles, the nucleus tractus solitarius (NTS), or the lateral parabrachial nucleus (PBN) of rats. We found that the vasopressin response to hypovolemia was inhibited by injection of diprenorphine into the cerebral ventricles at a dose too low to be effective when given subcutaneously. This response also was inhibited when a 20-fold lower dose of diprenorphine was injected into the PBN but not when it was injected into the NTS. The inhibitory effect of diprenorphine in the PBN was not attributable to a decrease in osmotic or hypovolemic stimulation and did not occur with osmotic or hypotensive stimuli. Injecting the PBN with equimolar doses of the mu antagonist naloxone, the delta antagonist ICI-154,129 or the kappa-1 agonist U-50,488H had no effect on basal or volume-stimulated vasopressin. We conclude that the inhibition of vasopressin by diprenorphine is due partially to action at a novel class of opioid receptors that transmit volume stimuli through the PBN.
Y Iwasaki, M B Gaskill, R Fu, C B Saper, G L Robertson
Although regulation of angiotensin II receptor (AT) binding in vascular and uterine smooth muscle is similar in nonpregnant animals, studies suggest it may differ during pregnancy. We, therefore, examined binding characteristics of myometrial AT receptors in nulliparous (n = 7), pregnant (n = 24, 110-139 d of gestation), and postpartum (n = 21, 5 to > or = 130 d) sheep and compared this to vascular receptor binding. We also determined if changes in myometrial binding reflect alterations in receptor subtype. By using plasma membrane preparations from myometrium and medial layer of abdominal aorta, we determined receptor density and affinity employing radioligand binding; myometrial AT receptor subtypes were assessed by inhibiting [125I]-ANG II binding with subtype-specific antagonists. Compared to nulliparous ewes, myometrial AT receptor density fell approximately 90% during pregnancy (1,486 +/- 167 vs. 130 +/- 16 fmol/mg protein) and returned to nulliparous values > or = 4 wk postpartum; vascular binding was unchanged. Nulliparous myometrium expressed predominantly AT2 receptors (AT1/AT2 congruent to 15%/85%), whereas AT1 receptors predominated during pregnancy (AT1/AT2 congruent to 80%/20%). By 5 d postpartum AT1/AT2 congruent to 40%/60%, and > 4 wk postpartum AT2 receptors again predominated (AT1/AT2 congruent to 15%/85%). In studies of ANG II-induced force generation, myometrium from pregnant ewes (n = 10) demonstrated dose-dependent increases in force (P < 0.001), which were inhibited with an AT1 receptor antagonist. Postpartum myometrial responses were less at doses > or = 10(-9) M (P < 0.05) and unaffected by AT2 receptor antagonists. Vascular and myometrial AT receptor binding are differentially regulated during ovine pregnancy, the latter primarily reflecting decreases in AT2 receptor expression. This is the first description of reversible changes in AT receptor subtype in adult mammals.
B E Cox, M A Ipson, P W Shaul, K E Kamm, C R Rosenfeld
To elucidate the effects of insulin-like growth factor I (IGF-I) on fuel oxidation and insulin sensitivity, eight healthy subjects were treated with saline and recombinant human (IGF-I (10 micrograms/kg.h) during 5 d in a crossover, randomized fashion, while receiving an isocaloric diet (30 kcal/kg.d) throughout the study period. On the third and fourth treatment days, respectively, an L-arginine stimulation test and an intravenous glucose tolerance test were performed. A euglycemic, hyperinsulinemic clamp combined with indirect calorimetry and a glucose tracer infusion were performed on the fifth treatment day. IGF-I treatment led to reduced fasting and stimulated (glucose and/or L-arginine) insulin and growth hormone secretion. Basal and stimulated glucagon secretion remained unchanged. Intravenous glucose tolerance was unaltered despite reduced insulin secretion. Resting energy expenditure and lipid oxidation were both elevated, while protein oxidation was reduced, and glucose turnover rates were unaltered on the fifth treatment day with IGF-I as compared to the control period. Enhanced lipolysis was reflected by elevated circulating free fatty acids. Moreover, insulin-stimulated oxidative and nonoxidative glucose disposal (i.e., insulin sensitivity) were enhanced during IGF-I treatment. Thus, IGF-I treatment leads to marked changes in lipid and protein oxidation, whereas, at the dose used, carbohydrate metabolism remains unaltered in the face of reduced insulin levels and enhanced insulin sensitivity.
M A Hussain, O Schmitz, A Mengel, A Keller, J S Christiansen, J Zapf, E R Froesch
Na/K/2Cl cotransport carrier plays an important role in fluid absorption and secretion in many epithelial tissues. The role of the carrier, however, in mammalian choroidal cerebrospinal fluid (CSF) production has been controversial. We used ventriculo-cisternal perfusion (VCP) labeled with blue dextran with or without bumetanide and measured choroidal CSF production in anesthetized, and paralyzed, mechanically ventilated dogs. During 3 h of VCP, mean intracerebroventricular and arterial pressures, PaCO2, pH, [HCO3-], and serum osmolality remained normal in both groups (n = 9 in each group). Beginning 90 min after the start of VCP, choroidal CSF production was measured every 15 min. In group I (control group), values for CSF production (means +/- SD) were 49 +/- 20, 49 +/- 21, 51 +/- 21, 51 +/- 23, 48 +/- 20, 56 +/- 24, and 48 +/- 20 microliters/min, at 90, 105, 120, 135, 150, 165, and 180 min, respectively. These values did not differ significantly from each other. In group II (bumetanide group), after baseline control CSF production had been determined at 90 and 105 min, bumetanide (10(-4) mol/liter) was added to VCP. Mean values for CSF production were 54 +/- 15 and 52 +/- 17 microliters/min before, and 39 +/- 25, 34 +/- 19, 28 +/- 10, 30 +/- 17, and 30 +/- 18 microliters/min after addition of bumetanide at 90, 105, 120, 135, 150, 165, and 180 min, respectively. Comparing the two groups, baseline values for CSF production measured at 90 and 105 min did not differ significantly. After addition of bumetanide (group II), however, decrements in CSF production varied from 30 +/- 27% at 120 min to 47 +/- 14% at 150 min, which were significantly different from changes in group I. The results of this study indicate that NaCl cotransport carrier is involved in secretion of CSF in dogs, and inhibition of the transporter results in approximately 50% reduction in CSF production.
S Javaheri, K R Wagner
A 7-yr-old girl with high density lipoprotein (HDL) deficiency and xanthomas has been identified in a Turkish kindred with repetitive consanguinity. She has severely reduced HDL-cholesterol and no apolipoprotein (apo) A-I. ApoA-II is reduced, whereas apoA-IV and apoC-III are normal. ApoB and low density lipoprotein (LDL)-cholesterol are increased. This is reflected in hypercholesterolemia. VLDL and IDL particles are low, and serum triglycerides are normal. The genetic defect could be identified as a base insertion into the third exon of the apoA-I gene. This leads to a nonsense peptide sequence beginning at amino acid 5 of the mature plasma protein and early termination of translation. The patient is homozygous for this mutation. Pedigree analysis indicated an autosomal dominant inheritance with no evidence of another genetic defect of lipoprotein metabolism in the kindred. In HDL deficiency, HDL binding to leukocytes was increased compared to normal. In the postprandial state, binding of labeled HDL3 to leukocytes is unchanged. This is in contrast to results with postprandially isolated leukocytes from controls or Tangier patients, which have a reduced binding capacity for HDL3. These results indicate that postprandial HDL precursors may compete the binding of labeled HDL3. The metabolic consequences of HDL deficiency were analyzed. There is only a small number of HDL-like particles containing apoA-II, apoA-IV, apoE, and lecithin/cholesteryl acyl transferase. The C-apolipoproteins were normal in the proband. Due to the lack of HDL they can only associate with apoB-containing particles, where they may interfere with cellular uptake. Thus, pure apoA-I deficiency leads to a complex metabolic derangement.
K J Lackner, H Dieplinger, G Nowicka, G Schmitz
We used dextran sulfate (DS) to evaluate barrier charge selectivity in 11 nonproteinuric subjects and in 11 patients with the nephrotic syndrome due to either membranous nephropathy or minimal change nephropathy. The 3H-DS preparation spanned a molecular radius interval of 10-24 A and exhibited size-dependent protein binding in vitro. Urine and ultrafiltrates of plasma were separated by size into narrow fractions using gel permeation chromatography. The sieving coefficient (theta) for ultrafilterable DS of 15A radius averaged 0.68 +/- 0.03 in nonproteinuric vs. 0.95 +/- 0.05 in nephrotic subjects (P < 0.001). Uncharged dextrans of broad size distribution were used to evaluate barrier size-selectivity in separate groups of nonproteinuric subjects (n = 19) and nephrotic patients with either minimal change (n = 20) or membranous nephropathy (n = 27). The value of theta for an uncharged dextran of similarly small radius (approximately 18 A) was significantly larger than that observed for DS in nonproteinuric subjects, but was similar in nephrotic individuals. Further, impaired barrier size-selectivity, as assessed by the sieving profile for uncharged dextrans (18-60 A radius), failed to account fully for the observed level of albuminuria in almost half of the patients with either minimal change (9/20) or membranous nephropathy (12/27). Together these findings suggest that the human glomerular capillary wall normally provides an electrostatic barrier to filtration of negatively charged macromolecules such as albumin, and that impairment of this electrostatic barrier contributes to the magnitude of albuminuria in the nephrotic syndrome.
A Guasch, W M Deen, B D Myers
Glucocorticoid concentrations vary throughout the day. To determine whether an increase in cortisol similar to that present during sleep is of physiologic significance in humans, we studied the disposition of a mixed meal when the nocturnal rise in cortisol was mimicked or prevented using metyrapone plus either a variable or constant hydrocortisone infusion. When glucose concentrations were matched with a glucose infusion, hepatic glucose release (2.6 +/- 0.2 vs. 1.5 +/- 0.4 nmol/kg per 6 h) was higher (P < 0.05) while glucose disappearance (5.9 +/- 0.3 vs. 7.3 +/- 0.9 mmol/kg per 6 h) and forearm arteriovenous glucose difference (64 +/- 24 vs. 231 +/- 62 mmol/dl per 6 h) were lower (P < 0.05) during the variable than basal infusion. The greater hepatic response during the variable cortisol infusion was mediated (at least in part) by inhibition of insulin and stimulation of glucagon secretion as reflected by lower (P < 0.05) C-peptide (0.29 +/- 0.01 vs. 0.38 +/- 0.04 mmol/liter per 6 h) and higher (P < 0.05) glucagon (42.7 +/- 2.0 vs. 39.3 +/- 1.8 ng/ml per 6 h) concentrations. In contrast, the decreased rates of glucose uptake appeared to result from a state of "physiologic" insulin resistance. The variable cortisol infusion also increased (P < 0.05) postprandial palmitate appearance as well as palmitate, beta-hydroxybutyrate, and alanine concentrations, suggesting stimulation of lipolysis, ketogenesis, and proteolysis. We conclude that the circadian variation in cortisol concentration is of physiologic significance in normal humans.
S Dinneen, A Alzaid, J Miles, R Rizza
We examined the genetic basis for adenosine deaminase (ADA) deficiency in seven patients with late/delayed onset of immunodeficiency, an underdiagnosed and relatively unstudied condition. Deoxyadenosine-mediated metabolic abnormalities were less severe than in the usual, early-onset disorder. Six patients were compound heterozygotes; 7 of 10 mutations found were novel, including one deletion (delta 1019-1020), three missense (Arg156 > His, Arg101 > Leu, Val177 > Met), and three splicing defects (IVS 5, 5'ss T+6 > A; IVS 10, 5'ss G+1 > A; IVS 10, 3'ss G-34 > A). Four of the mutations generated stop signals at codons 131, 321, 334, and 348; transcripts of all but the last, due to delta 1019-1020, were severely reduced. delta 1019-1020 (like delta 955-959, found in one patient and apparently recurrent) is at a short deletional hot spot. Arg156 > His, the product of which had detectable activity, was found in three patients whose second alleles were unlikely to yield active ADA. The oldest patient diagnosed was homozygous for a single base change in intron 10, which activates a cryptic splice acceptor, resulting in a protein with 100 extra amino acids. We speculate that this "macro ADA," as well as the Arg156 > His, Arg101 > Leu, Ser291 > Leu, and delta 1019-1020 products, may contribute to mild phenotype. Tissue-specific variation in splicing efficiency may also ameliorate disease severity in patients with splicing mutations.
I Santisteban, F X Arredondo-Vega, S Kelly, A Mary, A Fischer, D S Hummell, A Lawton, R U Sorensen, E R Stiehm, L Uribe
To define the mechanism(s) responsible for the negative inotropic effects of tumor necrosis factor-alpha (TNF alpha) in the adult heart, we examined the functional effects of TNF alpha in the intact left ventricle and the isolated adult cardiac myocyte. Studies in both the ventricle and the isolated adult cardiac myocyte showed that TNF alpha exerted a concentration- and time-dependent negative inotropic effect that was fully reversible upon removal of this cytokine. Further, treatment with a neutralizing anti-TNF alpha antibody prevented the negative inotropic effects of TNF alpha in isolated myocytes. A cellular basis for the above findings was provided by studies which showed that treatment with TNF alpha resulted in decreased levels of peak intracellular calcium during the systolic contraction sequence; moreover, these findings did not appear to be secondary to alterations in the electrophysiological properties of the cardiac myocyte. Further studies showed that increased levels of nitric oxide, de novo protein synthesis, and metabolites of the arachidonic acid pathway were unlikely to be responsible for the TNF alpha-induced abnormalities in contractile function. Thus, these studies constitute the initial demonstration that the negative inotropic effects of TNF alpha are the direct result of alterations in intracellular calcium homeostasis in the adult cardiac myocyte.
T Yokoyama, L Vaca, R D Rossen, W Durante, P Hazarika, D L Mann
We examined pancreas biopsy specimens from 18 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients to elucidate the mechanism underlying beta cell destruction. Pancreas islets were seen in all patients and insulitis in eight patients. Infiltrating mononuclear cells consisted of CD4+T, CD8+T, B lymphocytes, and macrophages. Among them, CD8+T lymphocytes were predominant and macrophages followed. The expression of MHC class I antigens was increased in islet and endothelial cells in nine patients. MHC class II expression was increased in endothelial cells of the same patients. The expression of intercellular adhesion molecule-1 was increased in endothelial cells in two of the nine patients with MHC hyperexpression; in one of them, lymphocyte function-associated antigen-3 expression was also increased. Out of the eight patients with insulitis, seven showed MHC class I hyper-expression, whereas 2 of the 10 patients without insulitis showed the phenomenon (P < 0.05). The relation between insulitis and the hyperexpression of adhesion molecules was not evident. In conclusion, we revealed the close relation between CD8+T lymphocyte-predominant insulitis and MHC class I hyperexpression in islet cells. This suggests that infiltrating CD8+T lymphocytes recognize islet autoantigens in association with increased MHC class I molecules and act as major effector cells in autoimmune response against islet cells in IDDM pancreases. The role of adhesion molecules in the pathogenesis of IDDM still remains to be elucidated.
N Itoh, T Hanafusa, A Miyazaki, J Miyagawa, K Yamagata, K Yamamoto, M Waguri, A Imagawa, S Tamura, M Inada
The effect of IL-1 on expression of the mineralization-related phenotype by chondrocytes was examined. In cultures of rabbit growth plate chondrocytes, IL-1 beta at 0.1 ng/ml caused 95% decreases in alkaline phosphatase activity, alkaline phosphatase mRNA levels, the incorporation of 45Ca into insoluble material, and the calcium content during the hypertrophic stage. These effects of IL-1 beta were dose-dependent and were observed in 24-48 h. Furthermore, IL-1 beta suppressed increase in cell size and the syntheses of 1,25-dihydroxyvitamin D3 receptor and type X collagen, other markers of hypertrophy, but had little effect on the synthesis of total protein including type II collagen. The inhibition of calcification was observed only when chondrocytes were exposed to IL-1 before the onset of calcification: IL-1 treatment from the mineralization stage had a marginal effect on 45Ca incorporation into insoluble material. These results suggest that IL-1 inhibits chondrocyte hypertrophy and the onset of calcification in ossifying cartilage.
Y Kato, K Nakashima, M Iwamoto, H Murakami, H Hiranuma, T Koike, F Suzuki, H Fuchihata, Y Ikehara, M Noshiro
The presence of antiidiotypic antibodies (ab-2) to sperm was investigated in the sera of fertile, infertile, and virgin women using sperm-specific anti-FA-1 monoclonal antibody Fab'.ab-2 were detected in 71% (17/24) of sera from fertile women and in none (0/12) of the sera from virgin females by the enzyme-linked immunosorbent assay, Western blot procedure, and immunoprecipitation procedure. Sera from infertile women that had antisperm antibodies showed a minimal presence of ab-2, with only three sera (13%, 3/23) demonstrating the presence of low levels of ab-2. The ab-2 present in fertile women were capable of neutralizing the fertilization-inhibitory activity of anti-FA-1 antibody in a concentration-dependent manner in a human sperm penetration assay (SPA) of zona-free hamster oocytes. ab-2 were also capable of inhibiting the binding of antisperm antibodies to the sperm surface as determined by the immunobead binding technique. This is the first report demonstrating the presence of ab-2 in the sera of fertile women that are capable of neutralizing antisperm antibodies present in sera of infertile women. These findings suggest that the inability to detect antisperm antibody activity in the sera of fertile women may be due to higher levels of ab-2 present in these sera than levels found in sera of infertile women, although both groups may be producing antisperm antibody response after sexual exposure to sperm.
R K Naz, K Ahmad, A C Menge
Recent studies have revealed that arginine vasopressin (AVP) has at least two types of receptors in the kidney: V1a receptor and V2 receptor. In this study, microlocalization of mRNA coding for V1a and V2 receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction. Large signals for V1a receptor PCR product were detected in the glomerulus, initial cortical collecting duct, cortical collecting duct, outer medullary collecting duct, inner medullary collecting duct, and arcuate artery. Small but detectable signals were found in proximal convoluted and straight tubules, inner medullary thin limbs, and medullary thick ascending limbs. Large signals for V2 receptor mRNA were detected in the cortical collecting duct, outer medullary collecting duct, and inner medullary collecting duct. Small signals for V2 receptor were found in the inner medullary thick limbs, medullary thick ascending limbs, and initial cortical collecting duct. Next, we investigated V1a and V2 receptor mRNA regulation in the dehydrated state. During a 72-h water restriction state, the plasma AVP level increased and V2 receptor mRNA decreased in collecting ducts. In contrast, V1a receptor mRNA did not change significantly. Thus, the two AVP receptor subtypes are distributed differently along the nephron, and these mRNAs are regulated differently in the dehydrated state.
Y Terada, K Tomita, H Nonoguchi, T Yang, F Marumo
The role of interleukin-6 (IL-6) in the growth of B cell derived hairy cell leukemia (HCL) was characterized. Purified hairy cells (HCs) did not increase DNA synthesis in vitro in response to exogenous IL-6; however, they expressed IL-6 receptor (IL-6R) mRNA and bound directly fluorochrome labeled IL-6. IL-6 mRNA was not detectable in tumor cells by Northern blotting, but was evident using PCR amplification. Although intracytoplasmic IL-6 protein was not demonstrable, HCs did secrete low levels of IL-6. Neutralizing antibody to IL-6 did not inhibit HC DNA synthesis. Since tumor necrosis factor (TNF) is a growth factor for HCL, we determined whether the TNF effect could be IL-6-mediated. TNF markedly augmented in vitro DNA synthesis by HCs. TNF did not alter IL-6R expression or IL-6 binding; however, IL-6 mRNA and IL-6 protein were detectable after 3-d culture of HCs with TNF. In addition, IL-6 secretion by HCs was markedly augmented by TNF. Finally, although neither IL-6 nor anti-IL-6 antibody altered TNF-induced DNA synthesis by HCs, IL-6 antisense oligonucleotide inhibited TNF-induced DNA synthesis and IL-6 secretion by HCs. Therefore, IL-6 does not directly affect the growth of HCL, but rather mediates TNF-induced DNA synthesis via an intracytoplasmic mechanism.
B Barut, D Chauhan, H Uchiyama, K C Anderson
During static exercise, heart failure (HF) subjects activate the sympathetic nervous system differently than normal controls. HF causes metaboreceptor desensitization with either enhanced mechanoreceptor activity or central command. In this report, we examined whether increased muscle interstitial pressure, as seen in HF, augments other neural systems. We measured muscle sympathetic nerve activity (MSNA; peroneal nerve) in 10 normals during static exercise (40% maximal voluntary grip) and posthandgrip circulatory arrest (PHG-CA). This was repeated after venous congestion (VC; cuff inflation to 90 mmHg). VC increased forearm volume (plethysmography) by 4.7%. MSNA responses to exercise were greater after VC (150.5 +/- 41.8 vs. 317.3 +/- 69.9 arbitrary units; P < 0.01). However, MSNA responses during PHG-CA were not affected by VC, and 31P nuclear magnetic resonance (n = 5) demonstrated no effect of VC on pH or H2PO4-. Similar effects of VC on MSNA were noted after ischemic exercise (n = 7), excluding flow alterations as the explantation. VC probably sensitized mechanically sensitive afferents since MSNA during involuntary biceps contractions increased after VC (n = 6), and skin sympathetic nerve responses during handgrip, an index of central command, were not increased by VC (n = 6).
J McClain, C Hardy, B Enders, M Smith, L Sinoway
Polymers used in implantable devices, although relatively unreactive, may degrade in vivo through unknown mechanisms. For example, polyetherurethane elastomers used as cardiac pacemaker lead insulation have developed surface defects after implantation. This phenomenon, termed "environmental stress cracking," requires intimate contact between polymer and host phagocytic cells, suggesting that phagocyte-generated oxidants might be involved. Indeed, brief exposure of polyetherurethane to activated human neutrophils, hypochlorous acid, or peroxynitrite produces modifications of the polymer similar to those found in vivo. Damage to the polymer appears to arise predominantly from oxidation of the urethane-aliphatic ester and aliphatic ether groups. There are substantial increases in the solid phase surface oxygen content of samples treated with hypochlorous acid, peroxynitrite or activated human neutrophils, resembling those observed in explanted polyetherurethane. Furthermore, both explanted and hypochlorous acid-treated polyetherurethane show marked reductions in polymer molecular weight. Interestingly, hypochlorous acid and peroxynitrite appear to attack polyetherurethane at different sites. Hypochlorous acid or activated neutrophils cause decreases in the urethane-aliphatic ester stretch peak relative to the aliphatic ether stretch peak (as determined by infrared spectroscopy) whereas peroxynitrite causes selective loss of the aliphatic ether. In vivo degradation may involve both hypohalous and nitric oxide-based oxidants because, after long-term implantation, both stretch peaks are diminished. These results suggest that in vivo destruction of implanted polyetherurethane involves attack by phagocyte-derived oxidants.
K Sutherland, J R Mahoney 2nd, A J Coury, J W Eaton
The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.
F X Maquart, G Bellon, B Chaqour, J Wegrowski, L M Patt, R E Trachy, J C Monboisse, F Chastang, P Birembaut, P Gillery
Autoimmune polyendocrine syndrome type I (APS I) and idiopathic Addison's disease are both disorders with adrenal insufficiency but with differences in genetic background, clinical presentation, and extent of extraadrenal manifestations. In this study the major adrenal autoantigen identified with sera from patients with APS I was characterized by analyses using indirect immunofluorescence, Western blots of adrenal subcellular fractions and of recombinant proteins, immunoprecipitations of [35S]methionine-labeled lysates of a human steroid-producing cell line, and studies of enzymatic activity. Sera from patients with APS I, identifying cells in adrenal glands and testes involved in steroid synthesis, reacted in Western blots with a 53-kD antigen, which comigrated with the cytochrome P450 cholesterol side chain cleavage enzyme (SCC). The sera also immunoprecipitated this protein from lysates of radiolabeled adrenal cells. The enzymatic activity of SCC was inhibited by the APS I sera but not by control sera. Sera from patients with idiopathic Addison's disease did not react with the SCC. The results show that the autoimmune responses towards adrenal tissue in patients suffering from APS I and Addison's disease are remarkably selective and suggest that a determination of the antigen involved in a patient with autoimmune adrenal insufficiency will have diagnostic as well as prognostic implications.
O Winqvist, J Gustafsson, F Rorsman, F A Karlsson, O Kämpe
Lipofuscin pigment, a terminal oxidation product, accumulates within cells during the normal aging process and under certain pathological conditions. We have analyzed a genetic cross between two inbred mouse strains, BALB/cJ and a subline of C57BL/6J, which differ in lipofuscin deposition. A comparison of the segregation pattern of cardiac lipofuscin with the albino locus (c) on mouse chromosome 7 revealed complete concordance. Analysis of spontaneous mutants of the tyrosinase gene, encoded by the albino locus, confirmed that the tyrosinase gene itself controls lipofuscin formation. Genetic analysis of other strains indicated that one or more additional genes cab contribute to the inheritance of lipofuscin. We also present evidence for an association between cardiac lipofuscin deposition and aortic fatty streak development in the mouse.
J H Qiao, C L Welch, P Z Xie, M C Fishbein, A J Lusis
A system for targeting foreign DNA to epithelial cells in vitro has been developed by exploiting receptor-mediated endocytosis. The polymeric immunoglobulin receptor transports dimeric immunoglobulin A and immunoglobulin M through epithelial cells, including those of the respiratory tract, by binding the immunoglobulins at the basolateral surface and transporting them across the cell. Fab fragments of antibodies directed against the extracellular portion of the receptor, secretory component, are similarly transported. Anti-human secretory component Fab fragments were covalently linked to a polycation, and complexed to various expression plasmids. When bound to an expression plasmid containing the Escherichia coli lacZ gene ligated to the Rous sarcoma virus promoter, the complexes transfected HT29.74 human colon carcinoma cells induced to express polymeric immunoglobulin receptor, but not those lacking the receptor. Primary cultures of human tracheal epithelial cells grown on collagen gels, which induce the expression of polymeric immunoglobulin receptor, were also transfected with the complexes. From 5 to 66% of the respiratory epithelial cells had beta-galactosidase activity after treatment, comparable to the percentage of cultured human tracheal epithelial cells that express polymeric immunoglobulin receptor (8-35%). The addition of excess human secretory component (Fab ligand) to the culture medium at the time of transfection blocked the delivery of DNA. The expression plasmid, either alone, complexed to the polycation, or complexed to a carrier based on an irrelevant Fab fragment, was not effective in transfecting either cell type. This DNA carrier system introduces DNA specifically into epithelial cells that contain pIgR in vitro.
T Ferkol, C S Kaetzel, P B Davis
Pemphigus vulgaris is an autoimmune blistering disease that is induced by binding of antibodies to a 130/85-kD protein complex on epidermal keratinocytes. An in vivo experimental model of this disease was developed by reconstituting severe combined immunodeficiency (SCID) mice with 1-10 x 10(7) PBL from patients with naturally occurring pemphigus vulgaris. Of 49 reconstituted mice, 34 (69%) produced human IgG levels of > 0.1 mg/ml. Circulating anti-pemphigus antibodies were found in 20 of the 34 successfully reconstituted mice; 44% of these animals had deposits of human IgG in their own skin after it was traumatized by either heat or cold. Spontaneous pemphigus vulgaris-like blisters associated with human IgG deposits were rarely found in mouse skin. By contrast, allogeneic human skin grafted to 10 to 12 mice before reconstitution with patients' PBL developed pemphigus vulgaris-like lesions containing human IgG deposits. These results demonstrate that SCID mice can serve as a model of an antibody-mediated human autoimmune skin disease.
I Juhasz, G S Lazarus, G F Murphy, I M Shih, M Herlyn
Because fibroblast growth factors (FGFs) modulate important functions of endothelial cells (EC) and smooth muscle cells (SMC), we studied FGF expression in human vascular cells and control or atherosclerotic arteries. All cells and arteries contained acidic (a) FGF and basic (b) FGF mRNA. Northern analysis detected aFGF mRNA only in one of five control arteries but in all five atheroma tested, while levels of bFGF mRNA did not differ among control (n = 3) vs. plaque specimens (n = 6). Immunolocalization revealed abundant bFGF protein in control vessels (n = 10), but little in plaques (n = 14). In contrast, atheroma (n = 14), but not control arteries (n = 10), consistently exhibited immunoreactive aFGF, notably in neovascularized and macrophage-rich regions of plaque. Because macrophages colocalized with aFGF, we tested human monocytoid THP-1 cells and demonstrated accumulation of aFGF mRNA during PMA-induced differentiation. We also examined the expression of mRNA encoding FGF receptors (FGFRs). All cells and arteries contained FGFR-1 mRNA. Only SMC and control vessels had FGFR-2 mRNA, while EC and some arteries contained FGFR-4 mRNA. The relative lack of bFGF in plaques vs. normal arteries suggests that this growth factor may not contribute to cell proliferation in advanced atherosclerosis. However, aFGF produced by plaque macrophages may stimulate the growth of microvessels during human atherogenesis.
E Brogi, J A Winkles, R Underwood, S K Clinton, G F Alberts, P Libby
A patient with liver cirrhosis who progressed to hepatocellular carcinoma was found to develop novel antinuclear antibodies. The serum was used to isolate full-length cDNA clones encoding related proteins of 530 amino acids (representative clone HCC1.4) and 524 amino acids (representative clone HCC1.3). Affinity-purified antibodies eluted from recombinant proteins recognized a 64-kD nuclear protein in Western blotting and decorated the nucleoplasm in a speckled-network fashion in immunofluorescence, colocalizing with antibodies to pre-mRNA splicing factor SC35 and uridine-rich small nuclear RNAs. The deduced amino acid sequence contained an arginine/serine-rich (RS) domain and three-ribonucleoprotein consensus sequence domains, two classes of motifs present in several splicing factors. A repeating octapeptide of Arg-Ser-Arg-Ser-Arg(Lys)-Glu(Asp)-Arg-Lys(Arg) was present in RS region of HCC1. This octapeptide sequence called RS-ERK motif was also found in splicing factors U2AF 35- and 65-kD proteins and 70-kD U1 small nuclear ribonucleoprotein. The molecular features and immunolocalization data suggest that the HCC1 autoantigen may be associated with splicing activities and are consistent with observations that autoantibody responses frequently target molecules involved in important cellular biosynthetic functions.
H Imai, E K Chan, K Kiyosawa, X D Fu, E M Tan
The human GPIa/IIa complex, also known as integrin alpha 2 beta 1, serves as a major receptor for collagen in platelets and other cell types. In addition to its role in platelet adhesion to extracellular matrix, GPIa/IIa is also known to bear the clinically important Br(a) and Brb alloantigenic determinants, which can result in antibody-mediated platelet destruction. Immunochemical studies showed that the Br antigenic epitopes reside solely on the GP Ia subunit and do not depend on sialic acid residues. To define the polymorphism responsible for the Br alloantigen system platelet RNA PCR technique, was used to amplify GPIa mRNA transcripts. Nucleotide sequence analysis of the amplified platelet GPIa cDNA from Br(a/a) and Brb/b individuals revealed a single A<-->G polymorphism at base 1648. MnlI RFLP analysis of cDNA from serologically determined individuals confirmed that this polymorphism segregates with Br phenotype. This single base change results in a substitution of Lys (AAG) in Br(a) to Glu (GAG) in Brb at amino acid residue 505 In spite of the reversal in charge at this position, however, we found no difference in the ability of Bra and Brb homozygous platelets to adhere to collagens types I, III, or V, nor did anti-Bra or anti-Brb alloantibodies interfere with platelet adhesion to any of these fibrillar collagens. The identification of the nucleotide substitution that defines the Bra/Brb alloantigen system will now permit both pre- and postnatal diagnosis for Br phenotype.
S Santoso, R Kalb, M Walka, V Kiefel, C Mueller-Eckhardt, P J Newman
Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.
S W Ebbinghaus, J E Gee, B Rodu, C A Mayfield, G Sanders, D M Miller
Alkylating agents can be administered in high dosage to patients with ovarian cancer using autologous bone marrow support, but drug-resistant tumor cells can still persist. Immunotoxins provide reagents that might eliminate drug resistant cells. In the present study, concurrent treatment with alkylators and immunotoxins proved superior to treatment with each agent alone. Toxin immunoconjugates prepared from different monoclonal antibodies and recombinant ricin A chain (rRTA) inhibited clonogenic growth of ovarian cancer cell lines in limiting dilution assays. When alkylating agents and toxin conjugates were used in combination, the addition of the immunotoxins to cisplatin, or to cisplatin and thiotepa, produced synergistic cytotoxic activity against the OVCA 432 and OVCAR III cell lines. Studies performed to clarify the mechanism of action showed that cisplatin and thiotepa had no influence on internalization and binding of the 317G5-rRTA immunotoxin. Intracellular uptake of [195m]Pt-cisplatin was not affected by the immunoconjugate and thiotepa. The combination of the 317G5-rRTA and thiotepa, as well as 317G5-rRTA alone, increased [195m]Pt cisplatin-DNA adduct levels. The immunotoxin alone and in combination with the alkylators decreased intracellular glutathione levels and reduced glutathione-S-transferase activity. Repair of DNA damage induced by the combination of alkylators and 317G5-rRTA was significantly reduced when compared to repair after damage with alkylators alone. These findings suggest that immunotoxins affect levels and activity of enzymes required for the prevention and repair of alkylator damage.
Y J Lidor, K C O'Briant, F J Xu, T C Hamilton, R F Ozols, R C Bast Jr
Low protein diets reverse the urea concentration gradient in the renal inner medulla. To investigate the mechanism(s) for this change, we studied urea transport and cell ultrastructure in initial and terminal inner medullary collecting ducts (IMCD) from rats fed 18% protein or an isocaloric, 8% protein diet for 4 wk. Serum urea, aldosterone, and albumin were significantly lower in rats fed 8% protein, but total protein and potassium were unchanged. Vasopressin stimulated passive urea permeability (Purea) threefold (P < 0.05) in initial IMCDs from rats fed 8% protein, but not from rats fed 18% protein. Luminal phloretin reversibly inhibited vasopressin-stimulated Purea. However, in terminal IMCDs from rats fed either diet, vasopressin stimulated Purea. Net transepithelial urea flux (measured with identical perfusate and bath solutions) was found only in initial IMCDs from rats fed 8% protein. Reducing the temperature reversibly inhibited it, but phloretin did not. Electron microscopy of initial IMCD principal cells from rats fed 8% protein showed expanded Golgi bodies and prominent autophagic vacuoles, and morphometric analysis demonstrated a marked increase in the surface density and boundary length of the basolateral plasma membrane. These ultrastructural changes were not observed in the terminal IMCD. Thus, 8% dietary protein causes two new urea transport processes to appear in initial but not terminal IMCDs. This is the first demonstration that "active" urea transport can be induced in a mammalian collecting duct segment.
T Isozaki, J W Verlander, J M Sands
C Tsigos, K Arai, W Hung, G P Chrousos
Cells from patients with peroxisome-deficient disorders contain membrane ghosts devoid of most matrix contents instead of normal peroxisomes indicating that the underlying molecular defects impair the import of matrix proteins into these peroxisome ghosts. Genetic heterogeneity for the molecular defects was inferred from the assignment of patients with peroxisome-deficient disorders into nine complementation groups. The aim of our studies was to analyze cell lines from six different complementation groups in a systematic manner for the presence of peroxisome ghosts, the ability to import Ser-Lys-Leu-containing proteins into peroxisome ghosts and for the presence of cytosolic factors required for peroxisomal protein import. We show that each of the cell lines analyzed contains peroxisome ghosts, but is unable to import matrix proteins as judged by a peroxisomal import assay using permeabilized cells. The addition of wild type cytosol did not restore the capacity to import matrix proteins but cytosol prepared from these cell lines was functional in stimulation of peroxisomal protein import in a heterologous system. These results implicate organelle-associated molecular defects in each of the six cell lines analyzed.
M Wendland, S Subramani
Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class.
G J Johnson, L A Leis, P C Dunlop
Subepidermal blistering associated with the human skin diseases bullous pemphigoid and herpes gestationis has been thought to be an IgG autoantibody-mediated process; however, previous attempts to demonstrate the pathogenicity of patient autoantibodies have been unsuccessful. An immunodominant and potentially pathogenic epitope associated with these blistering diseases has recently been mapped to the extracellular domain of a human epidermal antigen, BP180. Patient autoantibodies that react with this well-defined antigenic site failed to crossreact with the murine form of this autoantigen and thus could not be assayed for pathogenicity in a conventional passive transfer mouse model. As an alternative, rabbit polyclonal antibodies were generated against a segment of the murine BP180 protein homologous with the human BP180 autoantibody-reactive site and were passively transferred into neonatal BALB/c mice. The injected animals developed a subepidermal blistering disease that closely mimicked bullous pemphigoid and herpes gestationis at the clinical, histological, and immunological levels. Autoantibodies that recognize the human BP180 ectodomain are therefore likely to play an initiatory role in the pathogenesis of bullous pemphigoid and herpes gestationis.
Z Liu, L A Diaz, J L Troy, A F Taylor, D J Emery, J A Fairley, G J Giudice
Elevated [Ca2+]i in deoxygenated sickle cell anemia (SS) red cells (RBCs) could trigger a major dehydration pathway via the Ca(2+)-sensitive K+ channel. But apart from an increase in calcium permeability, the effects of deoxygenation on the Ca2+ metabolism of sickle cells have not been previously documented. With the application of 45Ca(2+)-tracer flux methods and the combined use of the ionophore A23187, Co2+ ions, and intracellular incorporation of the Ca2+ chelator benz-2, in density-fractionated SS RBCs, we show here for the first time that upon deoxygenation, the mean [Ca2+]i level of SS discocytes was significantly increased, two- to threefold, from a normal range of 9.4 to 11.4 nM in the oxygenated cells, to a range of 21.8 to 31.7 nM in the deoxygenated cells, closer to K+ channel activatory levels. Unlike normal RBCs, deoxygenated SS RBCs showed a two- to fourfold increase in pump-leak Ca2+ turnover. Deoxygenation of the SS RBCs reduced their Ca2+ pump Vmax, more so in reticulocyte- and discocyte-rich than in dense cell fractions, and decreased their cytoplasmic Ca2+ buffering. Analysis of these results suggests that both increased Ca2+ influx and reduced Ca2+ pump extrusion contribute to the [Ca2+]i elevation.
Z Etzion, T Tiffert, R M Bookchin, V L Lew
Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our data suggest that in hypertension, genetic overexpression of the ANP A-receptor subtype is related to the exaggerated biological response to ANP in this disease.
J Tremblay, C Huot, R C Willenbrock, F Bayard, F Gossard, N Fujio, C Koch, O Kuchel, W Debinski, P Hamet
In the nonobese diabetic (NOD) mouse, lymphocytic and monocytic infiltration of the pancreatic islets leads to beta cell destruction. To investigate the mechanisms by which lymphocytes enter the NOD pancreas, pancreata were immunostained using monoclonal antibodies to a variety of adhesion molecules known to be involved in lymphocyte binding to vascular endothelium, an initial step in the migration of lymphocytes from blood into organized lymphoid and inflamed tissues. These adhesion molecules include: lymphocyte homing receptors involved in tissue-selective binding of lymphocytes to peripheral lymph node (L-selectin) or mucosal lymphoid tissue (LPAM-1, alpha 4 beta 7-integrin) high-endothelial venules (HEV); and HEV ligands peripheral vascular addressin (PNAd) and mucosal vascular addressin (MAdCAM-1). In NOD pancreata, alpha 4 beta 7 is expressed on most infiltrating cells at all stages of insulitis, whereas L-selectin expression is more pronounced on cells in the islets at later stages. During the development of insulitis, MAdCAM-1 and to a lesser extent PNAd became detectable on vascular endothelium adjacent to and within the inflamed islets. The Stamper-Woodruff in vitro assay was used to examine lymphoid cell binding to such vessels. These functional assays show that both the mucosal (MAdCAM-1/alpha 4 beta 7) and the peripheral (PNAd/L-selectin) recognition systems are involved in this binding. Our findings demonstrate that expression of peripheral and mucosal vascular addressins is induced on endothelium in inflamed islets in NOD pancreas, and that these addressins participate in binding lymphoid cells via their homing receptors. This suggests that these adhesion molecules play a role in the pathogenesis of diabetes in these mice by being involved in the migration of lymphocytes from blood into the inflamed pancreas.
A Hänninen, C Taylor, P R Streeter, L S Stark, J M Sarte, J A Shizuru, O Simell, S A Michie
Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.
J Pfeilschifter, C Schalkwijk, V A Briner, H van den Bosch
IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.
M Konieczkowski, J R Sedor
Pharmaceutically prepared IgG, pooled from sera of over 2,000 normal individuals, contained both monomeric and dimeric IgG. Each type of IgG bound 125I-labeled interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increased binding to IgG was observed if 125I-IL-1 beta was denatured by heating to 39 degrees C. However, the binding of both nondenatured and denatured 125I-IL-1 beta was not inhibited by unlabeled IL-1 beta. In contrast, binding of 125I-IL-1 alpha, 125I-IL-6, and 125I-TNF alpha was inhibited by the corresponding unlabeled cytokine. Papain-digestion of IgG abolished binding of 125I-TNF alpha but failed to influence the displaceable binding of 125I-IL-1 alpha and 125I-IL-6. 125I-TNF alpha was a mixture of trimeric and monomeric forms, the latter being the predominant form at lower concentrations. The apparent saturability of 125I-TNF alpha was explained by a higher nonspecific binding of monomeric than of trimeric 125I-TNF alpha to IgG. The amounts of cytokine antibodies in IgG preparations would contribute approximately 2 micrograms anti-IL-1 alpha IgG and 1 microgram anti-IL-6 IgG per kg body wt during high dose immune globulin therapy. In conclusion, pharmaceutical preparations of human IgG contain specific and neutralizing, high affinity antibodies against IL-1 alpha and IL-6, but not against TNF alpha or IL-1 beta. There are significant methodological pitfalls that hamper detection of IgG autoantibodies against cytokines.
M Svenson, M B Hansen, K Bendtzen
A H Schmaier, L D Dahl, A J Rozemuller, R A Roos, S L Wagner, R Chung, W E Van Nostrand
Parathyroid hormone-related protein (PTHrP) causes hypercalcemia in malignancy. However, the role and regulation of PTHrP in normal physiology is just beginning to be explored. PTHrP is found in the spleen and has several other features common to cytokines. Since endotoxin (LPS) causes many of its effects indirectly by inducing cytokines, studies were undertaken to determine whether LPS might also induce splenic PTHrP expression. LPS (100 ng/mouse) increased splenic PTHrP mRNA levels 3.6-fold in C3H/OuJ mice. This effect was maximal at 2 h and returned to baseline by 4 h. PTHrP peptide levels also increased 3.3-fold in splenic extracts in response to LPS (1 microgram/mouse). Murine TNF-alpha and human IL-1 beta, cytokines that mediate many of the effects of LPS, also increased splenic PTHrP mRNA levels. LPS-resistant C3H/HeJ mice, which produce minimal amounts of TNF and IL-1 in response to LPS, were resistant to LPS induction of splenic PTHrP mRNA, while TNF-alpha and IL-1 beta readily increased PTHrP mRNA levels in C3H/HeJ mice. Anti-TNF antibody blocked LPS induction of splenic PTHrP mRNA in C3H/OuJ mice by 68%, indicating that TNF is a mediator of the LPS induction of PTHrP levels. In contrast, an IL-1 receptor antagonist (IL-1ra) was ineffective. The increase in PTHrP in the spleen during the immune response suggests that PTHrP may play an important role in immune modulation, perhaps by mediating changes in lymphocyte proliferation and/or function.
J L Funk, E J Krul, A H Moser, J K Shigenaga, G J Strewler, C Grunfeld, K R Feingold
An enhancement of sodium-proton exchange activity is a frequently observed ion transport abnormality in essential hypertension. The cellular basis for this has not yet been elucidated. Due to the lack of a specific cell culture system it has been impossible to distinguish between intrinsic cellular abnormalities and influences exerted by the hypertensive neurohumoral milieu. Using Epstein-Barr virus we have immortalized lymphocytes from controls and from patients with essential hypertension that exhibited enhanced sodium-proton exchanger activity. Sodium-proton exchanger activity was determined in cells loaded with the fluorescent cytosolic pH indicator 2'7'-biscarboxyethyl-5,6-carboxyfluorescein acetoxymethylester (BCECF) after pretreatment with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate for 10 min. Cell lines from hypertensive patients displayed higher Vmax values of sodium-proton exchange than those from normotensive controls (129.6 +/- 30.0 vs. 77.1 +/- 13.2 mmol H+/min.; P < 0.001). Hill coefficients for H+ were distinctly lower in hypertension compared to normotension (1.12 +/- 0.12 vs. 1.50 +/- 0.14; P < 0.0001). The enhanced antiporter activity in cell lines from hypertensive patients was not accompanied by a corresponding increase in steady-state NHE-1 mRNA transcript levels, which argues against overexpression of antiporter protein in hypertension. The cells from hypertensive patients with high sodium-proton exchange activity proliferated distinctly faster than those from normotensive controls. These human cell lines represent a novel model to study the mutual interaction between sodium-proton exchange and cell proliferation, and may provide insights into the alterations in ion transport observed in a group of patients with essential hypertension.
D Rosskopf, E Frömter, W Siffert