Evidence to support an important role of oxidative modification in mediating the atherogenicity of LDL continues to grow. New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDL can affect many components of the atherogenic process, including vasomotor properties and thrombosis, as well as lesion initiation and progression itself. These ideas suggest new approaches, that in combination with lowering of plasma cholesterol, could lead to the prevention of atherosclerosis and its complications.
J L Witztum, D Steinberg
We analyzed the pre-C and core region of hepatitis B virus (HBV) DNA by a polymerase chain reaction in 22 chronic carriers. In 9 hepatitis B e antigen-positive asymptomatic carriers, a single DNA band was detected at the expected size, whereas additional shorter DNA bands were observed in 7 out of 11 patients with chronic hepatitis. The smaller-sized DNAs from one chronic hepatitis patient had various lengths of deletions spanning from 105 to 183 bp in the middle of the core gene, and all deletions included common nucleotide sequences. All of the smaller-sized DNAs from the other patients proved to be variant core genes. They were deleted in similar regions by Southern analysis using oligonucleotide probes. A follow-up study revealed that four out of seven chronic hepatitis patients with a short core gene seroconverted to antibody to hepatitis B e antigen, but those with only a "wild type" did not. In another set of sequence studies, clones isolated from two chronic carriers displayed heterogeneity of the pre-C and core gene which was more often present in sera with normal alanine aminotransferase levels than with abnormal levels. These results suggest that mutant HBV alters the host immune response, and may modulate the clinical course of HBV infection. An alternative possibility is that chronic hepatitis selects for mutant forms.
T Wakita, S Kakumu, M Shibata, K Yoshioka, Y Ito, T Shinagawa, T Ishikawa, M Takayanagi, T Morishima
Neutrophil migration into the airspaces of the lung is thought to contribute to the alveolar damage and subsequent fibrosis in idiopathic pulmonary fibrosis (IPF). Interleukin 8 (IL-8), a monocyte- and macrophage-derived cytokine, displays potent chemotactic and activating properties towards neutrophils and thus may contribute to the pathogenesis of IPF. The objective of this investigation was to quantify the spontaneous expression of IL-8 transcripts by alveolar macrophages from normal healthy volunteers and individuals with IPF. A quantitative assay employing reverse transcription of mRNA and the polymerase chain reaction was utilized. The level of IL-8 mRNA in alveolar macrophages was found to be significantly elevated in individuals with lone IPF or with lung fibrosis associated with connective tissue disorders compared to normal healthy controls. Moreover, the level of IL-8 mRNA in the 23 individuals with IPF correlated with the number of neutrophils per milliliter in their bronchoalveolar lavage (BAL) and with the degree of disease severity. In addition, the level of IL-8 protein in BAL was found to reflect the pattern of IL-8 mRNA expression by alveolar macrophages. These data suggest that IL-8 derived from alveolar macrophages may significantly contribute to neutrophil involvement in the pathogenesis of IPF.
P C Carré, R L Mortenson, T E King Jr, P W Noble, C L Sable, D W Riches
Human antibodies specific for the Haemophilus influenzae b polysaccharide (Hib PS) frequently express a cross-reactive idiotype (CRI), and commonly utilize a VL region that is the product of the V kappa II gene A2. To examine further anti-Hib PS V region expression and to determine whether CRI expression is correlated with the V kappa IIA2 chain, we isolated a monoclonal antibody (MAb) reactive with an idiotypic determinant of anti-Hib PS antibodies. This MAb inhibited Hib PS binding but did not react with Ig isotypic determinants. The CRI recognized by this MAb, designated HibId-1, was associated with the Hib PS-combining site since the reactivity of the MAb with anti-Hib PS antibodies could be inhibited by Hib PS. HibId-1 was expressed by 17 of 17 clonally purified and sequence-defined anti-Hib PS antibodies having V kappa IIA2 L chains. In contrast, 0 of 10 anti-Hib PS antibodies having either V lambda, V kappa I, or V kappa III chains expressed HibId-1. Western blot analysis showed that the MAb anti-CRI reacted with isolated anti-Hib PS V kappa IIA2 L chains but not with H chains or other L chains, indicating that the HibId-1 determinant is localized to the V kappa IIA2 chain, and does not require pairing with H chain for expression. Anti-Hib PS antibodies bearing HibId-1 were present in at least 85% of subjects immunized with either free Hib PS or Hib PS coupled to diphtheria toxoid (Hib PS-DT), and comprised on the average 60% of the total vaccine-induced serum anti-Hib PS. HibId-1 expression was not related to age at vaccination inasmuch as infants, children, and adults had similar distributions of HibId-1-positive anti-Hib PS after vaccination with Hib PS-DT. HibId-1 was expressed at a lower frequency and comprised a smaller fraction of the total anti-Hib PS antibody in adult preimmunization sera as compared to post-Hib PS immunization sera, suggesting that immunization preferentially stimulates HibId-1-positive B cells. These data demonstrate that antibodies bearing HibId-1/V kappa IIA2 comprise a predominant component of the anti-Hib PS response induced by immunization, and that this pattern of VL expression is established early in ontogeny.
A H Lucas, R J Langley, D M Granoff, M H Nahm, M Y Kitamura, M G Scott
Lysophosphoglyceride accumulation in ischemic myocardium has been implicated as a cause of arrhythmias. We examined the effects of lysophosphatidylcholine (LPC) in isolated guinea pig ventricular myocytes. In paced myocytes loaded with the Ca2+ indicator Indo-1-AM and studied at room temperature, 20 microM LPC caused an initial positive inotropic effect followed by spontaneous automaticity, a decline in active cell shortening, and progressive diastolic shortening (contracture) leading to cell death. These changes were accompanied by a progressive increase in cytosolic [Ca2+]i. In patch-clamped myocytes dialyzed internally with high EGTA concentrations, LPC caused membrane depolarization, shortening of the action potential duration, and abnormal automaticity as seen in multicellular preparations. Voltage clamp experiments revealed the appearance of a nonselective leak conductance without significant changes in the delayed rectifier K+ current, inward rectifier K+ current, L-type Ca2+ current, and T-type Ca2+ current. Pretreatment with 20 mM caffeine and [Ca2+]o-free solution did not prevent the leak current. In patch clamped myocytes loaded with 0.1 mM Fura-2 salt, the [Ca2+]i transient induced by either voltage clamps or brief caffeine exposure remained normal until the nonselective leak current developed. The Na(+)-Ca2+ exchange current elicited during caffeine-induced [Ca2+]i transients also did not appear to be altered by LPC. Qualitatively similar results were obtained in myocytes studied at 35 degrees C. The membrane detergent saponin (0.005% wt/wt) mimicked all of the effects of LPC. We conclude that under these experimental conditions the effects of LPC are most compatible with a detergent action causing membrane leakiness with resultant depolarization, [Ca2+]i overload, and contracture.
E Liu, J I Goldhaber, J N Weiss
Terminal complement protein complexes C5b-9 have been found in human atherosclerotic lesions. Insertion of C5b-9 in the endothelial cell membrane alters permeability, induces membrane vesiculation, and activates secretion. We hypothesized that complement might also alter interactions of the endothelial surface with lipoproteins, particularly high density lipoprotein (HDL), which is reported to inhibit C5b-9-induced hemolysis. We now demonstrate that exposure to C5b-9 increases (by 2- to 50-fold) specific binding of HDL and its apolipoproteins (apo) A-I and A-II to endothelial cells. Binding to cells exposed to antibody, C5b67, and C5b-8 was virtually unchanged. Enhanced binding was also dependent on the number of C5b-9 complexes deposited on the cells. Other agonists that activate endothelial secretion did not augment binding. Calcium was required for full exposure of new binding sites by C5b-9. The C5b-9-induced increase in binding was independent of the increase observed after cholesterol loading. In addition, apo A-I and A-II appear to compete for the same binding sites on untreated and C5b-9-treated cells. In contrast to the data reported for red cells, we were unable to detect significant inhibition of C5b-9-mediated endothelial membrane permeabilization by HDL (up to 1 mg/ml) or by apo A-I (up to 100 micrograms/ml). These data demonstrate that the C5b-9 proteins enhance endothelial binding of HDL and its apoproteins, suggesting that intravascular complement activation may alter cholesterol homeostasis in the vessel wall.
K K Hamilton, P J Sims
Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.
T Hongo, J Kupfer, H Enomoto, B Sharifi, D Giannella-Neto, J S Forrester, F R Singer, D Goltzman, G N Hendy, C Pirola
Malaria parasites, unable to synthesize purine de novo, use host-derived hypoxanthine preferentially as purine source. In a previous study (1990. J. Biol. Chem. 265:6562-6568), we noted that xanthine oxidase rapidly and completely depleted hypoxanthine in human erythrocytes, not by crossing the erythrocyte membrane, but rather by creating a concentration gradient which facilitated hypoxanthine efflux. We therefore investigated the ability of xanthine oxidase to inhibit growth of FCR-3, a chloroquine-resistant strain of Plasmodium falciparum in human erythrocytes in vitro. Parasites were cultured in human group O+ erythrocytes in medium supplemented, as required, with xanthine oxidase or chloroquine. Parasite viability was assessed by uptake of radiolabeled glycine and adenosine triphosphate-derived purine into protein and nucleic acid, respectively, by nucleic acid accumulation, by L-lactate production, and by microscopic appearance. On average, a 90% inhibition of growth was observed after 72 h of incubation in 20 mU/ml xanthine oxidase. Inhibition was notably greater than that exerted by 10(-7) M chloroquine (less than 10%) over a comparable period. The IC50 for xanthine oxidase was estimated at 0.2 mU/ml, compared to 1.5 x 10(-7) M for chloroquine. Inhibition was completely reversed by excess hypoxanthine, but was unaffected by oxygen radical scavengers, including superoxide dismutase and catalase. The data confirms that a supply of host-derived hypoxanthine is critical for nucleic acid synthesis in P. falciparum, and that depletion of erythrocyte hypoxanthine pools of chloroquine-resistant malaria infection in humans. of chloroquine-resistant malaria infection in humans.
P A Berman, L Human, J A Freese
The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations. Images
T Gotoda, N Yamada, M Kawamura, K Kozaki, N Mori, S Ishibashi, H Shimano, F Takaku, Y Yazaki, Y Furuichi, T Murase
The accumulation of dicarboxylic acids, particularly long chain, is a prominent feature of Reye's syndrome and diseases of peroxisomal metabolism. We assessed the omega-oxidation of a spectrum of fatty acids in rats and asked whether pretreatment of rats with aspirin, which is known to predispose children to Reye's syndrome, would affect omega-oxidation of long chain fatty acids. We found that aspirin increased liver free fatty acids and increased the capacity for omega-oxidation three- to sevenfold. Omega-oxidation of long chain substrate was stimulated to a greater degree than medium chain substrate and was apparent within one day of treatment, at serum aspirin concentrations below the therapeutic range in humans. The apparent Km for lauric acid was 0.9 microM and 12 microM for palmitate. We also found a difference in the storage stability of activity toward medium and long chain substrate. Saturating concentrations of palmitate had no effect on the formation of dodecanedioic acid, whereas laurate decreased but never eliminated the omega-oxidation of palmitate. 97% of the total laurate omega-oxidative activity recovered was found in the microsomes, but 32% of palmitate omega-oxidative activity was present in the cytosol. These results demonstrate that aspirin is a potent stimulator of omega-oxidation and suggest that there may be multiple enzymes for omega-oxidation with overlapping substrate specificity.
R K Kundu, J H Tonsgard, G S Getz
The influence of pyrene-fatty acids on the resistance of cells to ultraviolet (UV) radiation was investigated in cultured fibroblasts from patients with five types of peroxisomal disorders. All showed reduced survival compared to control. The effect varied with the biochemical defect involved and the chain length of the pyrene fatty acid. Reduced survival was observed in cells deficient in plasmalogens (rhizomelic chondrodysplasia punctata) and in cells deficient in peroxisomal fatty acid oxidation (bifunctional enzyme deficiency), which accumulated pyrene-fatty acids. X-linked adrenoleukodystrophy fibroblasts accumulated pyrene-fatty acids and showed increased UV sensitivity only when exposed to longer-chain pyrene fatty acids. UV radiation resistance was lowest in cells with combined impairment of plasmalogen synthesis and fatty acid oxidation (Zellweger syndrome, neonatal adrenoleukodystrophy), suggesting that UV sensitivity correlates inversely with the ratio of plasmalogens to radical producing substances. Fibroblasts deficient in plasmalogens gained normal UV resistance when their plasmalogen levels were normalized by hexadecylglycerol. UV resistance increased when Zellweger cells were fused with X-linked adrenoleukodystrophy cells, and also when Zellweger cells belonging to different complementation groups were fused. The results provide leads to the pathogenesis of the multiple malformations associated with peroxisomal disorders and a method for the selection of cells in which the metabolic defect has been corrected.
G Hoefler, E Paschke, S Hoefler, A B Moser, H W Moser
Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas.
A Hamosh, B C Trapnell, P L Zeitlin, C Montrose-Rafizadeh, B J Rosenstein, R G Crystal, G R Cutting
Hydrogen peroxide (H2O2) contributes to renal cellular injury. alpha-Keto acids nonenzymatically reduce H2O2 to water while undergoing decarboxylation at the 1-carbon (1-C) position. We examined, in vitro and in vivo, the protective role of sodium pyruvate in H2O2-induced renal injury. Pyruvate effectively scavenged H2O2 in vitro, and suppressed H2O2-induced renal lipid peroxidation. Injury to LLC-PK1 cells induced by hydrogen peroxide was attenuated by pyruvate to an extent comparable to that seen with catalase. Studies utilizing [1-14C]pyruvate further demonstrated 1-C decarboxylation concurrent with cytoprotection by pyruvate from H2O2-induced injury. Pyruvate was also protective in vivo. Infusion of pyruvate before and during the intrarenal infusion of H2O2 attenuated H2O2-induced proteinuria. Systemic administration of pyruvate was also protective in the glycerol model of acute renal failure, a model also characterized by increased generation of H2O2. These findings indicate that pyruvate, a ubiquitous alpha-keto acid, scavenges H2O2 and protects renal tissue in vitro and in vivo from H2O2-mediated injury. These data suggest a potential therapeutic role for pyruvate in diseases in which increased generation of H2O2 is incriminated in renal damage.
A K Salahudeen, E C Clark, K A Nath
The effect of cholesterol enrichment on vascular smooth muscle cell (VSMC) calcium homeostasis was studied by evaluating calcium uptake, efflux, and intracellular content in cultured VSMC derived from the rat pulmonary artery. Incubation of VSMC with liposomes consisting of free cholesterol (FC) and phospholipid (2:1 molar ratio, 1 mg FC/ml medium) for 24 h resulted in a 69 +/- 19% increase (P less than 0.01; n = 10) in FC which was associated with a 73 +/- 11% increase (P less than 0.005; n = 10) in intracellular calcium content as assessed by isotopic equilibrium with 45Ca2+ and a 65 +/- 11% increase (P less than 0.024; n = 3) as assessed by atomic absorption spectroscopy. Cholesterol enrichment caused a marked increase in the unidirectional calcium uptake rate from 0.026 +/- 0.03 to 0.158 +/- 0.022 nmol calcium/s per mg protein (P less than 0.01; n = 3), but had no effect on calcium efflux. Nifedipine (1 microM) reduced (P less than 0.05; n = 6) the effect of cholesterol enrichment on unidirectional calcium uptake by 78 +/- 16%; and verapamil (10 microM), diltiazem (1 microM), and nifedipine (1 microM) each significantly inhibited the effect of cholesterol enrichment on intracellular calcium accumulation. Exposure of cholesterol-enriched VSMC to cholesterol-poor liposomes for 24 h returned both FC and calcium contents to control levels. Serum- and serotonin-stimulated calcium uptakes were potentiated 3.7- and 1.7-fold, respectively, in cholesterol-enriched VSMC, whereas endothelin, vasopressin, and thrombin-stimulated calcium uptakes were not affected. We conclude that VSMC FC content plays a role in regulating cellular calcium homeostasis, both under basal conditions and in response to selected agonists.
R A Bialecki, T N Tulenko, W S Colucci
A case of congenital goiter with defective thyroglobulin synthesis has been studied in molecular terms. The patient is the fifth of a kindred of six, three of which have a goiter. The parents are first cousins. Segregation of thyroglobulin alleles in the family was studied by Southern blotting with a probe revealing a diallelic restriction fragment length polymorphism (RFLP). The results demonstrated that the three affected siblings were homozygous for the RFLP. Northern blotting analysis of the goiter RNA with a thyroglobulin probe suggested that thyroglobulin mRNA size was slightly reduced. Polymerase chain reaction amplification of the 8.5-kb thyroglobulin mRNA as overlapping cDNA fragments demonstrated that a 200-bp segment was missing from the 5' region of the goiter mRNA. Subcloning and sequencing of the cDNA fragments, and of the patient genomic DNA amplified from this region, revealed that exon 4 is missing from the major thyroglobulin transcript in the goiter, and that this aberrant splicing is due to a C to G transversion at position minus 3 in the acceptor splice site of intron 3. The presence in exon 4 of a putative donor tyrosine residue (Tyrosine nr 130) involved in thyroid hormone formation provides a coherent explanation to the hypothyroid status of the patient.
T Ieiri, P Cochaux, H M Targovnik, M Suzuki, S Shimoda, J Perret, G Vassart
Elevated levels of plasma homocysteine are associated with both venous and arterial thrombosis. Homocysteine inhibits the function of thrombomodulin, an anticoagulant glycoprotein on the endothelial surface that serves as a cofactor for the activation of protein C by thrombin. The effects of homocysteine on thrombomodulin expression and protein C activation were investigated in cultured human umbilical vein endothelial cells and CV-1(18A) cells that express recombinant human thrombomodulin. Addition of 5 mM homocysteine to endothelial cells produced slight increases in thrombomodulin mRNA and thrombomodulin synthesis without affecting cell viability. In both cell types, thrombomodulin synthesized in the presence of homocysteine remained sensitive to digestion with endoglycosidase H and failed to appear on the cell surface, suggesting impaired transit along the secretory pathway. In a cell-free protein C activation assay, homocysteine irreversibly inactivated both thrombomodulin and protein C in a process that required free thiol groups and was inhibited by the oxidizing agents diamide or N-ethylmaleimide. By inhibiting both thrombomodulin surface expression and protein C activation, homocysteine may contribute to the development of thrombosis in patients with cystathionine beta-synthase deficiency.
S R Lentz, J E Sadler
The effects of increasing intracellular cAMP levels on IL-1 alpha and IL-1 beta mRNA expression and IL-1 production in human monocytes and nonlymphoid hematopoietic cell lines were examined. Peripheral monocytes and myelomonocytic cell lines could be stimulated by LPS or phorbol myristate acetate (PMA) to express IL-1 mRNA. Dibutyryl cAMP, 8-bromo-cAMP, forskolin, cholera toxin, PGE1, and PGE2 synergized with PMA or LPS to increase the accumulation in cell lines of IL-1 alpha mRNA by up to 50-fold and that of IL-1 beta mRNA by 10- to 20-fold compared to LPS or PMA alone. This increase in IL-1 alpha and IL-1 beta mRNA accumulation was more modest in monocytes. The synergistic stimulation was due to enhanced IL-1 gene transcription rate rather than increased IL-1 mRNA stability. Despite this marked increase in IL-1 mRNA accumulation, IL-1 protein synthesis in these cells was increased by only twofold. Thus, IL-1 synthesis in monocytes and myelomonocytic cell lines is under stringent translational control.
S S Sung, J A Walters
Glioblastoma multiforme, the most malignant astroglial-derived tumor, grows as an adherent mass and locally invades normal brain. An examination of adult cerebral glioblastoma biopsy material for the expression of adhesive proteins that might potentiate adhesion and invasion demonstrated tumor cell-associated vitronectin (5/5). In contrast, vitronectin was not detected associated with glial cells in low grade astroglial tumors (0/4), reactive astrogliosis (0/4), or in normal adult cortex and cerebral white matter (0/5). Also, a wide variety of other adhesive ligands were absent from the glioblastoma tumor parenchyma. The alpha v beta 3 integrin was the only vitronectin receptor identified in glioblastoma tumors in situ, and was also not expressed on low grade astroglial-derived tumors, reactive astrogliosis, or on glia or neurons in normal adult cortex and cerebral white matter. In a cell attachment assay, cultured glioblastoma cells attached to the parenchyma of glioblastoma tumor cryostat sections at the sites of vitronectin expression, but failed to attach to normal brain. This adhesion was inhibited by antibodies directed against vitronectin, the alpha v beta 3 integrin, and with an Arg-Gly-Asp-containing peptide. These data provide evidence for a cell adhesion mechanism in glioblastoma tumors that might potentiate glioblastoma cell invasion of normal brain.
C L Gladson, D A Cheresh
The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.
G G Kelley, O S Aassar, J N Forrest Jr
To determine the effects of chronic coronary artery constriction on the relationship between cardiac function and regulation of beta-adrenoceptor signal transduction, the left main coronary artery was narrowed in rats and the animals were killed 5 mo later. An average reduction in coronary luminal diameter of 44% was obtained and this change resulted in an increase in left ventricular end-diastolic pressure and a decrease in positive and negative dP/dt. Significant increases in left and right ventricular weights indicative of global cardiac hypertrophy were observed. Radioligand binding studies of beta-adrenoreceptors, agonist-stimulated adenylate cyclase activity, and ADP ribosylation of 45-kD substrate by cholera toxin were all depressed in the failing left ventricle. In contrast, in the hypertrophic non-failing right ventricle, beta-adrenoreceptor density was preserved and receptor antagonist affinity was increased. In spite of these findings at the receptor level, agonist stimulated cyclic AMP generation was reduced in the right ventricular myocardium. The quantity of the 45-kD substrate was also decreased. In conclusion, longterm nonocclusive coronary artery stenosis of moderate degree has profound detrimental effects on the contractile performance of the heart in association with marked attenuation of adrenergic support mechanisms.
L G Meggs, H Huang, P Li, J M Capasso, P Anversa
The concept of using thyroid-stimulating hormone (TSH) receptor antagonists in the management of Graves' disease is intriguing. Therefore, we investigated a TSH receptor antagonist derived from human chorionic gonadotropin (hCG) with respect to TSH receptor binding, adenylate cyclase activity, thyroid hormone release, and HLA class II antigen expression in vitro and in an in vivo model. A variant of hCG, asialoagalacto-hCG, like asialo-hCG and unlike hCG itself, inhibited both 125I-bTSH binding and cAMP response to bTSH in human thyroid membranes. However, like intact or deglycosylated hCG and unlike asialo-hCG, asialoagalacto-hCG displayed a limited affinity for hepatic asialoglycoprotein receptors, a likely marker for its in vivo turnover rate. It proved capable of inhibiting bTSH-stimulated thyroid hormone release in human thyroid slices as well as in the nude mouse bearing human thyroid transplants. It also prevented bTSH induced hypertrophy of transplanted thyrocytes. Further, HLA-DR expression induced by bTSH in the presence of gamma-interferon on human thyrocytes was inhibited. In conclusion, we present evidence that asialogalacto-hCG antagonizes bTSH actions on thyroid function and HLA-DR expression in human thyroid in vitro and, more importantly, in an in vivo model. Hence, the hCG variant described here or similar agents should warrant further exploration in the study and treatment of Graves' disease.
R Hoermann, P M Schumm-Draeger, K Rehbach, K Mann
Congenital lipoid adrenal hyperplasia is the most severe form of congenital adrenal hyperplasia. Affected individuals can synthesize no steroid hormones, and hence are all phenotypic females with a severe salt-losing syndrome that is fatal if not treated in early infancy. All previous studies have suggested that the disorder is in the cholesterol side chain cleavage enzyme (P450scc), which converts cholesterol to pregnenolone. A newborn patient was diagnosed by the lack of significant concentrations of adrenal or gonadal steroids either before or after stimulation with corticotropin (ACTH) or gonadotropin (hCG). The P450scc gene in this patient and in a previously described patient were grossly intact, as evidenced by Southern blotting patterns. Enzymatic (polymerase chain reaction) amplification and sequencing of the coding regions of their P450scc genes showed these were identical to the previously cloned human P450scc cDNA and gene sequences. Undetected compound heterozygosity was ruled out in the new patient by sequencing P450scc cDNA enzymatically amplified from gonadal RNA. Northern blots of gonadal RNA from this patient contained normal sized mRNAs for P450scc and also for adrenodoxin reductase, adrenodoxin, sterol carrier protein 2, endozepine, and GRP-78 (the precursor to steroidogenesis activator peptide). These studies show that lipoid CAH is not caused by lesions in the P450scc gene, and suggest that another unidentified factor is required for the conversion of cholesterol to pregnenolone, and is disordered in congenital lipoid adrenal hyperplasia.
D Lin, S E Gitelman, P Saenger, W L Miller
A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.
A K Gupta, K A Kirchner, R Nicholson, J G Adams 3rd, A N Schechter, C T Noguchi, M H Steinberg
Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen- or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental beta-hexosaminidase and partially purified bovine liver beta-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. coli beta-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle.
D B Lew, M C Rattazzi
Acute Respiratory Distress Syndrome (ARDS) is characterized by lung injury and damage to the alveolar type II cells. This study sought to determine if endogenous surfactant is altered in ARDS. Bronchoalveolar lavage was performed in patients at-risk to develop ARDS (AR, n = 20), with ARDS (A, n = 66) and in normal subjects (N, n = 29). The crude surfactant pellet was analyzed for total phospholipids (PL), individual phospholipids, SP-A, SP-B, and minimum surface tension (STmin). PL was decreased in both AR and A (3.48 +/- 0.61 and 2.47 +/- 0.40 mumol/ml, respectively) compared to N (7.99 +/- 0.60 mumol/ml). Phosphatidylcholine was decreased in A (62.64 +/- 2.20% PL) compared to N (76.27 +/- 2.05% PL). Phosphatidylglycerol was 11.58 +/- 1.21% PL in N and was decreased to 6.48 +/- 1.43% PL in A. SP-A was 123.64 +/- 20.66 micrograms/ml in N and was decreased to 49.28 +/- 21.68 micrograms/ml in AR and to 29.88 +/- 8.49 micrograms/ml in A. SP-B was 1.28 +/- 0.33 micrograms/ml in N and was decreased to 0.57 +/- 0.24 micrograms/ml in A. STmin was increased in AR (15.1 +/- 2.53 dyn/cm) and A (29.04 +/- 2.05 dyn/cm) compared to N (7.44 +/- 1.61 dyn/cm). These data demonstrate that the chemical composition and functional activity of surfactant is altered in ARDS. Several of these alterations also occur in AR, suggesting that these abnormalities occur early in the disease process.
T J Gregory, W J Longmore, M A Moxley, J A Whitsett, C R Reed, A A Fowler 3rd, L D Hudson, R J Maunder, C Crim, T M Hyers
Granulocyte-macrophage colony-stimulating factor (GM-CSF) was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with IL-1 alpha (5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of 3.2 to a mean of 8.2 pM (P less than 0.05). Dexamethasone (100 nM) decreased the constitutive GM-CSF elaboration by 49% (P less than 0.001) but did not diminish production by IL-1 alpha-stimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-1 alpha-stimulated endothelial cell-conditioned medium (P less than 0.05), which suggested viability antagonism by glucocorticoids. After 24 h of culture, eosinophil viability for replicate cells in enriched medium alone or with 1 pM GM-CSF decreased from means of 43 and 75% to means of 21 and 54%, respectively, when dexamethasone was included (P less than 0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonuclease-specific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathobiologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentrations that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of select eosinophilic disorders to glucocorticoids.
E Her, J Frazer, K F Austen, W F Owen Jr
To determine the role of renal sympathetic nerves in influencing renal function during the transition from fetal to newborn life, studies were carried out in conscious, chronically instrumented fetal sheep with either bilateral renal denervation (n = 11) or intact renal nerves (n = 12), 3-6 d after surgery. Endocrine, renal, and cardiovascular parameters were measured before and after delivery of lambs by cesarean section. Blood pressure and heart rate were similar in intact and denervated fetuses, and increased after delivery in both groups. There was also a transient diuresis and natriuresis, in the immediate postnatal period, the response being significantly greater in denervated than intact lambs (P less than 0.05). By 24 h postnatally, fluid and electrolyte excretions were similar in both groups, and significantly less than fetal levels. In the absence of renal nerves, the normal rise in plasma renin activity at birth was attenuated. These data provide evidence that renal sympathetic nerves play an important role during the transition from fetal to newborn life, and support the premise that birth is associated with sympathetic activation.
F G Smith, B A Smith, E N Guillery, J E Robillard
The effect of estrogen and progesterone replacement therapy on the initiating events in atherogenesis was studied in surgically postmenopausal cynomolgus monkeys. Monkeys were ovariectomized and divided randomly into two groups, one receiving 17 beta-estradiol and cyclic progesterone treatment (n = 9) and ovariectomized controls receiving no hormone replacement therapy (n = 8). The monkeys were fed a moderately atherogenic diet for 18 wk to accelerate the early pathogenic processes but not to be of sufficient duration to produce grossly visible atherosclerotic lesions. Sex hormone replacement therapy decreased the accumulation of LDL and products of LDL degradation in the coronary arteries by greater than 70% while having no significant effect on plasma lipid, lipoprotein, or apoprotein concentrations. Arterial intimal lesions were small with no difference between groups. The reduction in arterial LDL metabolism occurred very early in the pathogenesis of atherosclerosis and was independent of indices of endothelial cell injury, such as enhanced endothelial cell turnover or leukocyte adhesion to the endothelium. Results of this study suggest that one mechanism by which sex hormone treatment inhibits the initiation of atherosclerosis is a direct effect at the level of the arterial wall by suppressing the uptake and/or degradation of LDL.
J D Wagner, T B Clarkson, R W St Clair, D C Schwenke, C A Shively, M R Adams
Antiinflammatory therapy has been shown to reduce the adverse pathophysiological consequences that occur in bacterial meningitis and to improve outcome from disease. In the present study, modulation of two principal steps of the meningeal inflammatory cascade was accomplished by concomitant administration of dexamethasone to diminish overproduction of cytokines in response to a bacterial stimulus and of a monoclonal antibody directed against adhesion-promoting receptors on leukocytes to inhibit recruitment of white blood cells into the subarachnoid space. Dexamethasone and antibody therapy produced a marked attenuation of all indices of meningeal inflammation and reduction of brain water accumulation after H. influenzae-induced meningitis in rabbits compared with results of each agent given alone and of untreated animals. In addition, the enhanced host's meningeal inflammatory reaction that follows antibiotic-induced bacterial lysis was profoundly ameliorated when dual therapy was administered without affecting clearance rates of bacteria from cerebrospinal fluid and vascular compartments. The combination of both therapeutic approaches may offer a promising mode of treatment to improve further the outcome from bacterial meningitis.
X Sáez-Llorens, H S Jafari, C Severien, F Parras, K D Olsen, E J Hansen, I I Singer, G H McCracken Jr
Plasmin generation by equimolar concentrations of tissue plasminogen activator (t-PA), pro-urokinase (pro-UK), and urokinase (UK), and a twofold higher concentration of a plasmin-resistant mutant rpro-UK (Ala-158-pro-UK) was measured on a microtiter plate reader. The promoting effects on this reaction of equimolar concentrations of fibrinogen, soluble fibrin (Desafib), CNBr fragment FCB-2 (an analogue of fragment D), or purified fragment E-2 were compared. Plasmin generation by t-PA was moderately promoted by fibrinogen, substantially promoted by Desafib and FCB-2, but not at all promoted by fragment E-2. By contrast, plasmin generation by pro-UK or by Ala-158-pro-UK was not promoted either by fibrinogen, Desafib, or FCB-2, but was significantly promoted by fragment E-2. Plasmin generation by UK was not significantly promoted by any of the fibrin(ogen) preparations. Treatment of fragment E-2 by carboxypeptidase-B (CPB), eliminated its promotion of pro-UK and Ala-158-pro-UK-induced plasmin generation. Pretreatment of FCB-2 with plasmin slightly potentiated its promotion of t-PA activity. This effect of plasmin pretreatment of FCB-2 was reversed by CPB treatment. Plasmin pretreatment of FCB-2 did not induce any promotion of activity in pro-UK or Ala-158-pro-UK. The findings show that the intrinsic activity of pro-UK and the activity of t-PA are promoted by different regions of the fibrin(ogen) molecule. The latter is stimulated primarily by a determinant in the fragment D region, which is available in intact fibrin. By contrast, plasminogen activation by the intrinsic activity of pro-UK was stimulated exclusively by fragment E-2, which is unavailable in intact fibrin. The findings are believed relevant to fibrinolysis and support the concept that t-PA and pro-UK are complementary, sequential, and synergistic in their actions.
J N Liu, V Gurewich
The regulation of heat shock protein 70 (HSP 70) expression was examined in the isolated, red blood cell-perfused rabbit heart by Northern and Western blot analysis. In the isovolumic (balloon in left ventricle), isolated perfused heart, HSP 70 mRNA was increased threefold after 30 min and sevenfold at 2 and 4 h compared to normal, nonperfused hearts. To further elucidate the etiology of the increase in HSP 70 mRNA, the effects of decreased systolic shortening (isovolumic heart) and of a single ventricular stretch were examined. Perfusion without the application of a stretch or the presence of a balloon resulted in no increase in HSP 70 mRNA; while a single stretch resulted in a threefold increase in HSP 70 mRNA. These changes were accompanied by an increase in HSP 70 protein by Western blot analysis. To elucidate the signalling mechanism mediating the increase in HSP 70, hearts were perfused with H7, a protein kinase C inhibitor. H7 did not prevent the induction of HSP 70. These results indicate that initiation of expression of myocardial HSP 70 can be stimulated by a single myocardial stretch or by prevention of systolic shortening. These mechanisms may contribute to the rapid expression of HSP 70 after coronary occlusion when dyskinesis, reduced systolic shortening, and increased diastolic segment length all occur.
A A Knowlton, F R Eberli, P Brecher, G M Romo, A Owen, C S Apstein
Tumor necrosis factor (TNF), a protein released by activated macrophages, is a central mediator of the host response to infection and inflammation. The TNF-binding protein I (TNF-BP-I) is a soluble fragment of the p60 transmembrane TNF receptor and an antagonist to TNF. The level of serum TNF-BP-I was found to be increased in patients with renal insufficiency as a result of a decrease in the glomerular filtration rate. During hemodialysis of patients with renal failure there was a rapid but transient increase in serum TNF-BP-I. This increase was found to be caused by heparin given before dialysis and a similar dose-dependent response to heparin was observed also in healthy individuals. The finding of a repeated release of TNF-BP-I into the circulation with intermittent injections of heparin indicates that TNF-BP-I is present both in a storage pool and in a circulating pool. The mechanism for the heparin-mediated release of TNF-BP-I was not explained; TNF-BP did not show affinity for heparin. On the other hand, TNF was found to have affinity for heparin and it could also be dissociated from heparin by TNF-BP-I. It is suggested that heparin-like molecules of the extracellular matrix can retain TNF in physical proximity with target cells and restrict the actions of TNF and protect against systemic harmful manifestations.
M Lantz, H Thysell, E Nilsson, I Olsson
Airway reactivity has been shown to vary with age; however, the mechanism(s) underlying this process remain unidentified. To elucidate the role of ontogenetic changes in phosphoinositide-linked signal transduction, we examined whether age-related differences in tracheal smooth muscle (TSM) contractility to carbachol (CCh) are associated with developmental changes in the production and metabolism of the second messenger, inositol 1,4,5-trisphosphate (Ins (1,4,5)P3). In TSM segments isolated from 2-wk-old and adult rabbits, both the maximal isometric contractile force and sensitivity (i.e., -logED50) to CCh (10(-10)-10(-4) M) were significantly greater in the immature vs. adult tissues (P less than 0.001). Similarly, Ins(1,4,5)P3 accumulation elicited by either receptor-coupled stimulation with CCh (10(-10)-10(-4) M) or post-receptor-mediated guanine nucleotide binding protein activation of permeabilized TSM with GTP gamma S (100 microM) was also significantly enhanced in 2-wk-old vs. adult TSM. Measurement of the activities of the degradative enzymes for Ins(1,4,5)P3 demonstrated that: (a) mean +/- SE maximal Ins(1,4,5)P3 3'-kinase activity was significantly reduced in the immature vs. adult TSM (i.e., approximately 71.7 +/- 6.0 vs. 137.8 +/- 10.0 pmol/min per mg protein, respectively; P less than 0.005); (b) by contrast, maximal Ins(1,4,5)P3 5'-phosphatase activity was significantly increased in the immature vs. adult TSM (i.e., 27.9 +/- 1.2 vs. 15.6 +/- 1.5 nmol/min per mg protein, respectively; P less than 0.001); and (c) the Km values for Ins(1,4,5)P3 5'-phosphatase were 14- and 19-fold greater than those for Ins(1,4,5)P3 3'-kinase in the 2-wk-old and adult TSM, respectively. Collectively, the findings suggest that the age-related decrease in agonist-induced rabbit TSM contractility is associated with a diminution in Ins(1,4,5)P3 accumulation which is attributed, at least in part, to ontogenetic changes in the relative activities of the degradative enzymes for Ins(1,4,5)P3.
S M Rosenberg, G T Berry, J R Yandrasitz, M M Grunstein
Incubation of cocultures of human aortic endothelial (HAEC) and smooth muscle cells (HASMC) with LDL in the presence of 5-10% human serum resulted in a 7.2-fold induction of mRNA for monocyte chemotactic protein 1 (MCP-1), a 2.5-fold increase in the levels of MCP-1 protein in the coculture supernatants, and a 7.1-fold increase in the transmigration of monocytes into the subendothelial space of the cocultures. Monocyte migration was inhibited by 91% by antibody to MCP-1. Media collected from the cocultures that had been incubated with LDL induced target endothelial cells (EC) to bind monocyte but not neutrophil-like cells. Media collected from cocultures that had been incubated with LDL-induced monocyte migration into the subendothelial space of other cocultures that had not been exposed to LDL. In contrast, media from separate cultures of EC or smooth muscle cells (SMC) containing equal number of EC or SMC compared to coculture and incubated with the same LDL did not induce monocyte migration when incubated with the target cocultures. High density lipoprotein HDL, when presented to cocultures together with LDL, reduced the increased monocyte transmigration by 91%. Virtually all of the HDL-mediated inhibition was accounted for by the HDL2 subfraction. HDL3 was essentially without effect. Apolipoprotein AI was also ineffective in preventing monocyte transmigration while phosphatidylcholine liposomes were as effective as HDL2 suggesting that lipid components of HDL2 may have been responsible for its action. Preincubating LDL with beta-carotene or with alpha-tocopherol did not reduce monocyte migration. However, pretreatment of LDL with probucol or pretreatment of the cocultures with probucol, beta-carotene, or alpha-tocopherol before the addition of LDL prevented the LDL-induced monocyte transmigration. Addition of HDL or probucol to LDL after the exposure to cocultures did not prevent the modified LDL from inducing monocyte transmigration in fresh cocultures. We conclude that cocultures of human aortic cells can modify LDL even in the presence of serum, resulting in the induction of MCP-1, and that HDL and antioxidants prevent the LDL induced monocyte transmigration.
M Navab, S S Imes, S Y Hama, G P Hough, L A Ross, R W Bork, A J Valente, J A Berliner, D C Drinkwater, H Laks
To investigate the mechanism of reduced exercise tolerance in hyperthyroidism, we characterized cardiovascular function and determinants of skeletal muscle metabolism in 18 healthy subjects aged 26 +/- 1 yr (mean +/- SE) before and after 2 wk of daily ingestion of 100 micrograms of triiodothyronine (T3). Resting oxygen uptake, heart rate, and cardiac output increased and heart rate and cardiac output at the same submaximal exercise intensity were higher in the hyperthyroid state (P less than 0.05). However, maximal oxygen uptake decreased after T3 administration (3.08 +/- 0.17 vs. 2.94 +/- 0.19 l/min; P less than 0.001) despite increased heart rate and cardiac output at maximal exercise (P less than 0.05). Plasma lactic acid concentration at an equivalent submaximal exercise intensity was elevated 25% (P less than 0.01) and the arteriovenous oxygen difference at maximal effort was reduced (P less than 0.05) in the hyperthyroid state. These effects were associated with a 21-37% decline in activities of oxidative (P less than 0.001) and glycolytic (P less than 0.05) enzymes in skeletal muscle and a 15% decrease in type IIA muscle fiber cross-sectional area (P less than 0.05). Lean body mass was reduced (P less than 0.001) and the rates of whole body leucine oxidation and protein breakdown were enhanced (P less than 0.05). Thus, exercise tolerance is impaired in short duration hyperthyroidism because of decreased skeletal muscle mass and oxidative capacity related to accelerated protein catabolism but cardiac pump function is not reduced.
W H Martin 3rd, R J Spina, E Korte, K E Yarasheski, T J Angelopoulos, P M Nemeth, J E Saffitz
The effects of FFA on hepatic insulin clearance were studied in the in situ perfused rat liver. Clearance decreased with increasing body weight (age) of the rats. When FFA were added to the perfusate a 40% reduction of hepatic removal of insulin was found over the normal, physiological range (less than 1,000 mumol/liter), less pronounced in heavier rats. When perfusion was started with high concentrations of FFA, inhibition was rapidly reversible, a phenomenon again blunted in heavier rats. In contrast to FFA, different glucose concentrations in the perfusate did not affect the hepatic insulin uptake in the presence of FFA within physiological concentrations. Thus, hepatic clearance of insulin is proportional to rat weight (age) and portal FFA concentrations. Other studies have recently shown that fatty acids inhibit insulin binding, degradation, and function in isolated rat hepatocytes, and that hepatic clearance is inversely dependent on hepatic triglyceride concentrations, both inhibitions reversible by prevention of fatty acid oxidation. It is suggested that the diminished hepatic clearance of insulin in heavier (older) rats is at least partly due to their relative obesity and increased hepatic triglyceride contents. This effect as well as that of portal FFA is probably mediated via fatty acid oxidation in the liver. This mechanism may have implications for the regulation of hepatic metabolism, and peripheral insulin concentrations.
J Svedberg, G Strömblad, A Wirth, U Smith, P Björntorp
Plasma net cholesteryl ester (CE) transfer and optimum cholesteryl ester transfer protein (CETP) activity were determined in primary hypertriglyceridemic (n = 11) and normolipidemic (n = 15) individuals. The hypertriglyceridemic group demonstrated threefold greater net CE transfer leading to enhanced accumulation of CE in VLDL. This increased net transfer was not accompanied by a change in CETP activity. In normolipidemia, but not in hypertriglyceridemia, net CE transfer correlated with VLDL triglyceride (r = 0.92, P less than 0.001). In contrast, net CE transfer in hypertriglyceridemia, but not in normolipidemia, correlated with CETP activity (r = 0.73, P less than 0.01). Correction of hypertriglyceridemia with bezafibrate reduced net CE transfer towards normal and restored the correlation with VLDL triglyceride (r = 0.90, P less than 0.005) while suppressing the correlation with CETP activity. That net CE transfer depends on VLDL concentration was confirmed by an increase of net CE transfer in normolipidemic plasma supplemented with purified VLDL. Supplementation of purified CETP to normolipidemic plasma did not stimulate net CE transfer. In contrast, net CE transfer was enhanced by addition of CETP to both plasma supplemented with VLDL and hypertriglyceridemic plasma. Thus, in normal subjects, VLDL concentration determines the rate of net CE transfer. CETP becomes rate limiting as VLDL concentration increases, i.e., in hypertriglyceridemia.
C J Mann, F T Yen, A M Grant, B E Bihain
Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells.
A S Cross, D G Wright
In patients with congestive heart failure (CHF), the poor relationship between systemic exercise performance and cardiac function, together with morphologic and metabolic abnormalities in skeletal muscle, raises the possibility that skeletal muscle function may be impaired and limit systemic exercise performance. We assessed strength and endurance of the knee extensors during static and dynamic exercise in 16 patients with Class I-IV CHF and eight age-matched sedentary controls and related these measurements to systemic exercise performance. To assess skeletal muscle function independent of peripheral blood flow, endurance was repeated under ischemic conditions. Strength was not significantly different in the two groups. Dynamic endurance, quantified as the decline in peak torque during 15 successive isokinetic knee extensions, was significantly reduced in the patients compared to controls during aerobic (peak torque 65 vs. 86% of initial for exercise at 90 deg/s and 60 vs. 85% for exercise at 180 deg/s; P less than 0.002 for both), and during ischemic exercise (56 vs. 76% of initial torque; P less than 0.01). Static endurance, defined as the time required for force during a sustained maximal voluntary contraction to decline to 60% of maximal, was reduced in the patients compared to controls (40 +/- 14 vs. 77 +/- 29 s; P less than 0.02). There were highly significant relationships between systemic exercise performance and skeletal muscle endurance at 90 and 180 deg/s in the patients with CHF (r = 0.90 and 0.66, respectively). These findings indicate that skeletal muscle endurance is impaired in patients with CHF, that this abnormality is in part independent of limb blood flow, and that these changes may be important determinants of systemic exercise performance.
J R Minotti, I Christoph, R Oka, M W Weiner, L Wells, B M Massie
In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.
A Hinek, R P Mecham, F Keeley, M Rabinovitch
Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3H]alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 microns away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone. In vitro alendronate bound to bone particles with a Kd of approximately 1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 x 10(-3) fmol/microns 2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.
M Sato, W Grasser, N Endo, R Akins, H Simmons, D D Thompson, E Golub, G A Rodan
Platelet-activating factor (PAF) is a phospholipid with cardiovascular actions at low concentrations (1-100 nM) but with uncertain direct myocardial actions. We investigated the cellular and molecular effects of PAF on heart cells using isolated adult and neonatal rat myocytes. Addition of PAF, in the superfusion solution, decreased twitch amplitude and contractile velocity in both systems. Concentrations of PAF below 1 nM stimulated reproducible responses with maximal effects seen at 100 nM. These functional actions of PAF could be blocked by the known PAF antagonist, BN 50739, in a dose-dependent manner. Parallel biochemical studies showed that nanomolar PAF rapidly stimulated the phosphoinositide pathway in cultured myocytes, evidenced by the accumulation of [3H]inositol phosphates in prelabeled cultured myocytes. The potency and specificity of PAF, as well as the time course, for the response were nearly identical in the biochemical and functional assays. PAF produced no functional changes in protein kinase C-depleted myocytes, but it did stimulate inositol trisphosphate accumulation in such cells. We conclude that: (a) PAF exerts a direct negative inotropic effect on myocardial tissue; (b) the effects of PAF are mediated by a specific, high affinity cardiac receptor; (c) an underlying biochemical mechanism for the action of PAF includes the activation of the phospholipase C/phosphatidylinositol intracellular signaling pathway, which leads to activation of protein kinase C.
C V Massey, T A Kohout, S T Gaa, W J Lederer, T B Rogers
Transforming growth factor-beta (TGF-beta) modulates the growth and differentiation of many cells and often functions in an autocrine or paracrine fashion. The myoepithelial cells of the renal juxtaglomerular apparatus (JGA) synthesize and secrete renin. Under conditions which chronically stimulate renin production, the JGA undergoes hypertrophy and hyperplasia. The molecular factors responsible for these changes in the JGA have not been identified. In the present study, plasma renin activity was stimulated in the mouse by water deprivation. Using immunoperoxidase staining with specific antibodies against TGF-beta 1, beta 2, and beta 3, we found increased TGF-beta 2 accumulation in the JGA and interlobular arteries. Immunostaining with renin antiserum demonstrated colocalization of TGF-beta 2 and renin. TGF-beta 1 and beta 3 expression was not different between control and water-deprived mice. Our results suggest that in the setting of water deprivation, TGF-beta 2 is localized in a manner which would allow it to act either as a growth factor for or as a phenotypic modulator of the JGA and renal arterioles.
S Horikoshi, B K McCune, P E Ray, J B Kopp, M B Sporn, P E Klotman
Genetic analysis in our laboratory of families with generalized thyroid hormone resistance (GTHR) has demonstrated tight linkage with a locus, c-erbA beta, encoding a nuclear T3 receptor. Three point mutations and two deletions in this locus have previously been reported in affected individuals in unrelated families as potential molecular bases for this disorder. In the present study, we have used direct sequencing of polymerase chain reaction-amplified exons of the c-erbA beta gene to rapidly identify novel point mutations from seven previously uncharacterized kindreds with GTHR. Six single base substitutions and one single base insertion were identified and found to be clustered in two regions of exons 9 and 10 in the ligand binding domain of the receptor: in the distal ligand-binding subdomain L2 and across the juncture of the taui and dimerization subdomains. Reduction of T3-binding affinity in each of four mutations tested as well as segregation of all mutations to clinically affected individuals strongly supports the hypothesis that these changes are the cause of GTHR in these kindreds. In view of the diversity of clinical phenotypes manifested, the distinct topographic clustering of the mutations provides an invaluable genetic tool for the molecular dissection of thyroid receptor function.
R Parrilla, A J Mixson, J A McPherson, J H McClaskey, B D Weintraub
Hematopoietic cells from the malignant clone in chronic myelogenous leukemia (CML) maintain and expand a proliferative advantage over normal hematopoietic cells within the bone marrow. This advantage is often ameliorated or reversed in vivo by IFN alpha. Based upon earlier studies suggesting decreased adhesiveness of CML progenitor cells, we asked whether CML progenitor cells are deficient in their expression of the cytoadhesion molecule lymphocyte function antigen-3 (LFA-3, CD58) which is normally expressed on hematopoietic progenitors. Progenitor cells from untreated CML patients showed greatly reduced or absent LFA-3 expression, whereas progenitors from patients treated with IFN alpha in vivo or in vitro expressed surface LFA-3 at more normal levels. LFA-3-deficient CML progenitor cells were unable to stimulate normal regulatory proliferative responses in autologous T cells. We hypothesize that IFN alpha-sensitive LFA-3 deficiency reflects a cell surface cytoadhesion defect which may help explain adhesive abnormalities of CML progenitor cells in vitro and clonal proliferation in vivo.
G Upadhyaya, S C Guba, S A Sih, A P Feinberg, M Talpaz, H M Kantarjian, A B Deisseroth, S G Emerson
Previous studies have suggested that an alteration in the expression of the Na,K-ATPase of muscle may be an important determinant of enhanced insulin sensitivity in chronic renal failure. Therefore, in the present studies we have examined the effect of uremia on the Na,K-ATPase alpha isoforms in skeletal muscle, at the level of mRNA expression and enzymatic activity. The activity of the sodium pump, as measured ouabain-sensitive 86Rb/K uptake in soleus muscle, revealed a reduction in the activity in uremia, related to the increment in plasma creatinine values. The decrement in 86Rb uptake by the rat soleus muscle of experimental animals was associated with changes on Na,K-ATPase gene product. Northern analysis of mRNA revealed isoform-specific regulation of Na,K-ATPase by uremia in skeletal muscle: a decrease of approximately 50% in alpha 1 subunit Na,K-ATPase mRNA, as compared to controls. The decrement in alpha 1 mRNA correlates with the decreased activity of the Na,K-ATPase in uremia, under basal conditions and with the almost complete inhibition of the Na,K-ATPase, of uremic tissue by a concentration of 10(-5) M ouabain. Although the activity of the alpha 2 isoform pump was not modified by uremia, the 3.4-kb message for this enzyme was increased 2.2-fold; this discrepancy is discussed. Altogether these findings demonstrate that the defective extrarenal potassium handling in uremia is at least dependent in the expression of alpha 1 subunit of the Na,K-ATPase.
S Bonilla, I A Goecke, S Bozzo, M Alvo, L Michea, E T Marusic
There are three common C2 protein alleles in caucasians, C2*C, C2*B, and C2*Q0, with allele frequencies of 0.96, 0.03, and 0.01, as well as Sst I RFLP variants of 2.75, 2.7, 2.65, 2.55, and 2.4 kb, with frequencies of 0.017, 0.533, 0.358, 0.017, and 0.075. Thus, C2*C is informatively split by the RFLP. Of 94 nonrandomly ascertained caucasian complotypes, 77 contained C2*C, four contained C2*Q0, and 13 had C2*B. None of the C2*C-containing complotypes carried the 2.75 kb Sst I fragment and all of the complotypes with C2*B or C2*Q0 carried it. All of the C2*Q0 alleles were associated with C4A*4, C4B*2 in the complotype S042 as previously reported. C2*B was usually (9/13) in the complotype SB42, occasionally (1/13 each) in SB45, SB41, SB(4,3)0, and SB31. Thus, the association of the C2 2.75-kb fragment was with C2*B and C2*Q0, not with C4A*4, C4B*2, or even C4A*4 alone. The complotype SC42 was associated with the 2.65-kb Sst I fragment in four of five instances and in a single example with the 2.7-kb fragment. C2*B and C2*Q0 possibly had a common evolutionary ancestor complotype which carried the 2.75-kb Sst I fragment, and BF*S, C4A*4, and C4B*2. C2*B (particularly as the haplotype HLA-Bw62, SB42, DR4) is associated with type 1 diabetes but C2*Q0 is protective.
S Simon, Z Awdeh, R D Campbell, P Ronco 2nd, S J Brink, G S Eisenbarth, E J Yunis, C A Alper
The expression of the basolateral chloride-activated organic anion uptake system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of oocytes with rat liver poly(A)+RNA resulted in the functional expression of chloride-dependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent Km approximately 6.2 microM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 microM; inhibition 53%), 4,4'-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 microM; 80%), taurocholate (100 microM; 80%), and cholate (200 microM; 95%). In contrast to results with total rat liver mRNA, injection of mRNA derived from the Na+/bile acid cotransporter cDNA (Hagenbuch, B., B. Stieger, M. Foguet, H. Lübbert, and P. J. Meier. 1991. Proc. Natl. Acad. Sci. USA. In press.) had no effect on BSP uptake into oocytes. Size fractionation of total rat liver mRNA revealed that a 2.0- to 3.5-kb size-class mRNA was sufficient to express the hepatic chloride-dependent BSP uptake system. These data indicate that "expression cloning" in oocytes represents a promising approach to ultimately clone the cDNA coding for the hepatocyte high affinity, chloride-dependent organic anion uptake system. Furthermore, the results confirm that the Na+/bile acid cotransport system does not mediate BSP uptake.
E Jacquemin, B Hagenbuch, B Stieger, A W Wolkoff, P J Meier
We have recently demonstrated that all-trans retinoic acid (RA), the active metabolite of vitamin A, is an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (AML3). We have further shown that, in these AML3 cells, the gene of the retinoic acid receptor-alpha (RAR alpha) is translocated from chromosome 17 to chromosome 15, and fused to a new gene, PLM. This results in the expression of both normal and chimeric RAR alpha transcripts in AML3 cells. The PLM-RAR alpha protein may account for the impairment of differentiation and thus leukemogenesis, but not for the paradoxical efficacy of RA in these cells. In an attempt to elucidate RA's differentiative effect in AML3 patients, the present work examined the in vitro and in vivo modulation of the normal RAR alpha transcripts by all-trans RA in seven cases of AML3. In all samples, Northern blot analysis revealed a low expression of the two normal RAR alpha transcripts compared with other human myeloid leukemic cells. No modulation was observed after 4-8 d of in vivo therapy with all-trans RA 45 mg/m2 per d. In vitro incubation with all-trans RA, however, increased the level of expression of the normal RAR alpha transcripts in AML3 cells but not in other AML leukemic subtypes. This modulation of the two normal RAR alpha transcripts appeared to be an early and primary event of RA's differentiating effect. We therefore suggest that up-regulation of the normal RAR alpha gene expression by pharmacological concentrations of all-trans RA may restore the normal differentiation pathway in these cells.
C Chomienne, N Balitrand, P Ballerini, S Castaigne, H de Thé, L Degos