Studies in man have shown that the episodic release of growth hormone (GH) is infrequent and erratic, and unlike that in the rat does not appear to have discernible ultradian periodicities. However, these observations in nonfasted subjects may be invalid since mixed nutrients have unpredictable effects on GH release. Moreover, in the fed state basal GH levels are frequently undetectable, thus rendering the identification of low amplitude pulses unreliable. Accordingly, the 24-h pulsatile pattern of GH secretion obtained from repetitive venous sampling in six normal adult male subjects was examined during a control fed day and during the first and fifth days of a 5-d fast. The GH data were analyzed using two distinct methods: a discrete pulse detection algorithm (Cluster analysis) and Fourier expansion time-series, which allows fixed periodicities of secretory activity to be resolved. The 5-d fast resulted in a significant increase in discrete GH pulse frequency (5.8 +/- 0.7 vs. 9.9 +/- 0.7 pulses/24 h, P = 0.028), 24 h integrated GH concentration (2.82 +/- 0.50 vs. 8.75 +/- 0.82 micrograms.min/ml; P = 0.0002), and maximal pulse amplitude (5.9 +/- 1.1 vs. 12.3 +/- 1.6 ng/ml, P less than 0.005). While multiple low-amplitude sinusoidal periodicities were present on the control fed day, time-series analysis revealed enhancement of circadian and ultradian cycles on the first and fifth days of fasting. Concomitantly, fasting resulted in a decline (day 1 vs. day 5) in serum concentrations of somatomedin C (1.31 +/- 0.22 vs. 0.77 +/- 0.18 U/ml) and glucose (4.9 +/- 0.2 vs. 3.2 +/- 0.2 mmol/liter), and a marked rise in free fatty acid (0.43 +/- 0.12 vs. 1.55 +/- 0.35 mmol/liter) and acetoacetate (35 +/- 6 vs. 507 +/- 80 nmol/liter). We conclude that the acute nutritional status is an important determinant of spontaneous pulsatile GH secretion in man. Fast-induced enhancement of GH release is achieved through combined frequency (discrete pulses) and amplitude (sinusoidal periodicities) modulation. Such alterations in somatotropic hormone release may play an important role in substrate homeostasis during starvation.
K Y Ho, J D Veldhuis, M L Johnson, R Furlanetto, W S Evans, K G Alberti, M O Thorner
Insulin-like growth factor-I (IGF-I) in human hepatoma cells (HEP-G2) has, in addition to its effect on cell growth, short-term metabolic effects acting through its own receptor. We have demonstrated that normal human hepatocytes, compared with HEP-G2 cells, have virtually no IGF-I binding sites. Because the rate of growth is the major difference between the hepatoma and the normal liver, we asked if normal liver might express IGF-I binding sites under physiologic growth conditions. Indeed, whereas adult rat hepatocytes have low IGF-I binding sites similar to those in human liver, hepatocytes from regenerating liver after 3 d subtotal hepatectomy have an approximately sixfold increase (P less than 0.005) and those from fetal rat liver a approximately 12-fold increase (P less than 0.005), to levels comparable to those in the HEP-G2 cells. The specificity of 125I IGF-I binding to its receptor was demonstrated by competition studies with monoclonal antibodies directed toward the IGF-I and the insulin receptors, with unlabeled IGF-I and insulin and by affinity labeling experiments. Thus, if IGF-I has any short-term metabolic functions in the adult human liver, it is not through interaction with its own receptor. Autocrine regulation by IGF-I of liver growth appears possible since IGF-I binding sites are expressed under pathological and physiological conditions of growth. The mechanism that couples these two phenomena remains to be elucidated.
J F Caro, J Poulos, O Ittoop, W J Pories, E G Flickinger, M K Sinha
In vivo PTH administration to rats resulted in increased brain synaptosomal Ca++ transport, while parathyroidectomy (PTX) resulted in decreased transport. To determine the mechanism of action of PTH on Ca++ transport in rat brain synaptosomes, we performed transport studies by the Na-Ca exchanger and also measured cAMP generation in synaptosomes from PTX rats. Ca++ transport was studied after in vivo additions of either bovine (b)PTH, cAMP, or forskolin, and adenylate cyclase activity was assessed after additions of either bPTH, forskolin, sodium fluoride (NaF), or isoproterenol. In the presence of 1-34 bPTH [10(-7) M], Ca++ uptake was significantly increased by 55% (P less than 0.001) above control, while 3-34 bPTH [10(-7) M] had no effect on uptake. Both 8br,cAMP [10(-6) M] and dibut,cAMP [10(-6) M] also significantly increased (P less than 0.001) Ca++ uptake above control by 63 and 44%, respectively. Similarly, forskolin [10(-5) M], the adenylate cyclase activator, increased Ca++ uptake by 41%. We next evaluated Ca++ efflux, and found that 1-34 bPTH [10(-7) M], 1-84 bPTH [10(-7) M], and forskolin [10(-5) M] also increased Ca++ efflux by 50, 73, and 120%, respectively, above control. Since Ca++ transport was increased by either PTH, cAMP, or forskolin, we decided to determine if PTH action on Ca++ transport in synaptosomes was dependent on cAMP. This was investigated by measuring cAMP production during the conversion of 32P-ATP to 32P-cAMP in the presence of an ATP regenerating system (30 micrograms creatine phosphokinase, 10 mM creatine phosphate), and the cyclic nucleotide phosphodiesterase inhibitor (1 mM IBMX). Whereas forskolin [10(-4) M] and NaF [100 mM] significantly increased (P less than 0.001) adenylate cyclase activity in synaptosomes by eight- and fourfold, respectively, neither 1-34 bPTH nor 1-84 bPTH increased synaptosomal cyclase activity. However, in canine renal cortical plasma membranes (CRCPM), we observed significant increases in cAMP production with either forskolin, NaF, or PTH. Finally, to determine if synaptosomes contain an intact adenylate cyclase system, we measured cAMP production in the presence of the beta adrenergic agent, isoproterenol. Isoproterenol significantly increased adenylate cyclase activity in both synaptosomes (90%) and CRCPM (50%). These data suggest that although there is an intact adenylate cyclase system in rat brain synaptosomes, PTH-stimulated calcium transport in synaptosomes appears to be independent of this system.
C L Fraser, P Sarnacki, A Budayr
We have analyzed the configuration of the T cell receptor (TCR) alpha gene using newly developed genomic joining region (J alpha) probes, which cover approximately 80 kb of the J alpha region upstream from the constant region in 19 patients with T cell acute lymphoblastic leukemia (T-ALL) and in three CD3- leukemic T cell lines (HSB2, CEM, and MOLT4). In parallel, transcription of the TCR-alpha, beta, and gamma genes was examined in 11 of these patients and in the T cell lines. All T-ALL and the three T cell lines exhibited both TCR-gamma and beta gene rearrangements. 8 of 10 T-ALL and all T cell lines expressed TCR-gamma transcripts. All samples tested expressed both TCR-beta and CD3-gamma transcripts. TCR alpha transcripts were only observed in CD3+ T-ALL but not in CD3- T-ALL or the CD3- cell lines. Among the CD3+ T-ALL, eight had TCR-alpha gene rearrangements. In addition, TCR-alpha gene rearrangements were detected in one CD3- T-ALL and all three T cell lines. These leukemic cells may represent a transient stage between rearrangement and expression and provide an opportunity for analyzing the mechanism regulating the expression of the TCR-alpha gene.
J Hara, S H Benedict, E Champagne, T W Mak, M Minden, E W Gelfand
To examine the renal tubular sites and mechanisms involved in the effects of hypooncotic volume expansion (VE) on renal electrolyte excretion, we performed clearance and isolated tubular perfusion studies using intact and thyroparathyroidectomized (TPTX) rabbits. We also examined the effect of VE on luminal brush border transport. In the microperfusion studies, proximal convoluted (PCT) and straight (PST) tubules were taken from rabbits without prior VE or after 30 min of 6% (body wt) VE. Acute VE increased the percentage excretion of Na, Ca, and P in TPTX animals and the percentage and absolute excretions of these ions in intact rabbits. In PST from VE animals, fluid flux (Jv) was depressed compared with Jv in PST from nonVE rabbits: Jv = 0.18 +/- 0.03, (VE) vs. 0.31 +/- 0.03 nl/mm.min, (nonVE) P less than 0.02. Phosphate transport (Jp) in the PST from VE animals was also depressed: JP = 1.58 +/- 0.10 (VE) vs. 2.62 +/- 0.47 pmol/mm.min, (nonVE) P less than 0.05. Similar results were obtained with TPTX animals. In the PCT from VE animals, Jv was decreased (0.49 +/- 0.10 (VE) vs. 0.97 +/- 0.14 nl/mm.min, (nonVE) P less than 0.02), but JP was not affected significantly. Transport inhibition was stable over approximately 90 min of perfusion. In the brush border vesicle studies, sodium-dependent phosphate transport was inhibited compared with that in control animals, at the 9-, 30-, and 60-s time points. These findings indicate that the inhibition of renal ionic transport by VE occurs in both PCT and PST and is, in part, the result of a direct effect of VE on tubular transport mechanisms.
T O Pitts, J A McGowan, T C Chen, M Silverman, M E Rose, J B Puschett
The existence of chloride/bicarbonate exchange across the basolateral membrane and its physiologic significance were examined in rabbit proximal tubules. S2 segments of the proximal straight tubule were perfused in vitro and changes in intracellular pH (pHi) and chloride activity (aCli) were monitored by double-barreled microelectrodes. Total peritubular chloride replacement with gluconate increased pHi by 0.8, and this change was inhibited by a pretreatment with an anion transport inhibitor, SITS. Peritubular bicarbonate reduction increased aCli, and most of this increase was lost when ambient sodium was totally removed. The reduction rates of pHi induced by a peritubular bicarbonate reduction or sodium removal were attenuated by 20% by withdrawal of ambient chloride. SITS application to the bath in the control condition quickly increased pHi, but did not change aCli. However, the aCli slightly decreased in response to SITS when the basolateral bicarbonate efflux was increased by reducing peritubular bicarbonate concentration. It is concluded that sodium coupled chloride/bicarbonate exchange is present in parallel with sodium-bicarbonate cotransport in the basolateral membrane of the rabbit proximal tubule, and it contributes to the basolateral bicarbonate and chloride transport.
S Sasaki, N Yoshiyama
Platelet-adhesive protein-tumor cell interaction was studied in vitro and in vivo. Monoclonal antibody 10E5, which inhibits binding of fibronectin and von Willebrand factor to the platelet membrane glycoprotein GPIIb-GPIIIa complex, inhibited the binding of mouse CT26 and human HCT8 colon carcinoma cells to platelets by 63-65%, whereas an irrelevant monoclonal antibody, 3B2, had no effect. Monoclonal antibody 6D1, which inhibits binding of von Willebrand factor to GPIb, also had no effect. RGDS, a tetrapeptide that represents the adhesive domain of fibronectin and von Willebrand factor inhibited binding of the tumors to platelets by 64-69%. Monospecific polyclonal antifibronectin antibody inhibited binding by 60-82%; anti-von Willebrand factor antibody inhibited binding by 75-81%. In vivo, polyclonal monospecific anti-mouse von Willebrand factor antibody inhibited pulmonary metastases induced by CT26 tumor cells by 53-64%, B16a amelanotic melanoma cells by 45% and T241 Lewis bladder cells by 46% without induction of thrombocytopenia. Pulmonary metastases with CT26 cells could be inhibited by induction of thrombocytopenia, and reconstituted by infusion of either murine or human platelets. Reconstitution of pulmonary metastases with human platelets could be inhibited 77% by preincubation of human platelets with monoclonal antibody 10E5 before infusion of platelets into mice. Thus, platelets appear to contribute to metastases by their adhesive interaction with tumor cells via the adhesive proteins fibronectin and von Willebrand factor.
S Karpatkin, E Pearlstein, C Ambrogio, B S Coller
To examine the biological quality and physiologically pulsatile mode of endogenous luteinizing hormone release in active, healthy aging men, we used the rat interstitial-cell testosterone in vitro bioassay to probe LH bioactivity in response to (a) endogenous gonadotropin-releasing hormone (GnRH) action (basal pulsatile bioactive LH secretion); (b) exogenous GnRH stimulation (10 micrograms IV pulses); and (c) inhibition of endogenous estrogen negative feedback (treatment with a nonsteroidal antiestrogen, tamoxifen). Basally, some healthy older men exhibited evidence of neuroendocrine dysfunction, reflected by irregular bursts of bioactive LH release followed by transiently low plasma bio:immuno (B:I) LH ratios. However, mean basal plasma bioactive LH concentrations, B:I ratios, and spontaneous LH pulse properties (peak frequency, amplitude, duration, and enhanced B:I ratios within LH peaks) were not altered in older men. On the other hand, augmentation of bioactive LH secretion and enhancement of plasma B:I ratios by pulsed injections of exogenous GnRH were either significantly reduced or absent in older men. In addition, although tamoxifen increased bioactive LH pulse frequency in both age groups and facilitated exogenous GnRH action in some subjects, older men increased their 12-h mean bioactive LH concentrations, B:I ratios, and bioactive LH peak amplitudes to a significantly lesser degree than young men. In summary, young and older healthy men exhibit similar mean basal plasma bioactive LH concentrations and spontaneous LH pulse properties. However, pituitary bioactive LH reserve is markedly attenuated in older men challenged with either exogenous GnRH or antiestrogen. Accordingly, we conclude that healthy aging men manifest an impaired secretory reserve for biologically active LH release.
R J Urban, J D Veldhuis, R M Blizzard, M L Dufau
Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.
A Rambaldi, N Wakamiya, E Vellenga, J Horiguchi, M K Warren, D Kufe, J D Griffin
Atriopeptin (AP), a natriuretic-diuretic and vasodilatory peptide, is synthesized and secreted from mammalian atria. The definitive role of this peptide on cardiovascular physiology and pathophysiology has yet to be determined. We developed a population of autoimmune rats sensitized against their own AP to evaluate the consequences of prolonged AP deficiency on physiological and pathophysiological processes. Natriuresis in response to acute intravenous volume expansion was inhibited in the autoimmune rat, however, natriuresis produced by chronic oral salt loading was not suppressed in these animals. Plasma AP increased threefold in the spontaneously hypertensive rat when evaluated as a function of blood pressure. Immunization of these rats had no effect on the rate of development, magnitude of their developing hypertension, or their daily sodium excretion when compared with nonimmunized controls. Mineralocorticoid escape occurred during desoxycorticosterone acetate administration to rats. The ability of rats to escape from the sodium-retaining effects of this steroid was not affected by prior immunization against AP. These results suggest that AP is an important natriuretic substance in response to acute intravascular volume loading. However, atriopeptin does not appear to be involved in the natriuretic response to chronic intravascular volume loading, blood pressure regulation, or mineralocorticoid escape.
J E Greenwald, M Sakata, M L Michener, S D Sides, P Needleman
We have used high-pressure freezing techniques to study exocytosis in rat anterior pituitary cells. The cells were either unstimulated or exposed to 1 nM growth hormone releasing factor (GRF) for 10 min before ultrarapid freezing. The magnitude of growth hormone (GH) release was then correlated with the number of exocytotic events observed with freeze-fracture electron microscopy. High-pressure freezing of unfixed and uncryoprotected specimens permits cryofixation of samples up to 1 mm diam (0.5 mm thick) without ice crystal damage, and arrests exocytotic events within 10 ms. Our studies comparing conventionally fixed specimens with those prepared by high-pressure freezing confirm that areas of intramembrane particle clearing at potential exocytotic sites are an artifact of conventional fixation and/or cryoprotection techniques. The cells exposed to 1 nM GRF released approximately fivefold more GH than did unstimulated cells. Morphologically, we have observed a 3.3-fold increase in the number of exocytotic events in GRF-stimulated cells, 33.7 events/100 micron2 compared with 10.4 events/100 micron2 for unstimulated cells. In additional experiments, we studied the effects of two inhibitors of GRF-induced exocytosis, somatostatin and sodium isethionate. Both compounds elicit the same response, a parallel decrease in exocytotic events and in secreted product. We conclude that high-pressure freezing, combined with freeze-fracture and freeze-substitution processing techniques, is an excellent tool for studying the morphological aspects of exocytosis. In the present investigation, it has allowed us to quantitatively relate the biochemistry and morphology of exocytosis in anterior pituitary cells.
B Draznin, R Dahl, N Sherman, K E Sussman, L A Staehelin
Most patients with obstructive sleep apnea have increased pharyngeal collapsibility (defined in the present study as an increased lung volume dependence of pharyngeal area), which predisposes them to upper airway occlusion during sleep. However, there are patients with severe obstructive sleep apnea who have low-normal pharyngeal collapsibility. The factors leading to nocturnal upper airway obstruction in such patients have not been ascertained. We studied 10 overweight male patients with severe obstructive sleep apnea and low-normal pharyngeal collapsibility to determine the site of upper airway pathology in these patients. We found that all 10 patients exhibited paradoxical inspiratory narrowing of the glottis during quiet tidal breathing. This phenomenon was not observed in a matched group of 10 snoring, nonapneic male controls. We conclude that paradoxical glottic narrowing may be a contributing factor in the pathogenesis of upper airway obstruction in patients with severe obstructive sleep apnea who have low-normal pharyngeal collapsibility.
I Rubinstein, A S Slutsky, N Zamel, V Hoffstein
To define the primary effects of aluminum on bone in the mammalian species, we examined the dose/time-dependent actions of aluminum in normal beagles. Administration of low dose aluminum (0.75 mg/kg) significantly elevated the serum aluminum (151.7 +/- 19.9 micrograms/liter) compared with that in controls (4.2 +/- 1.35 micrograms/liter) but did not alter the calcium, creatinine, or parathyroid hormone. After 8 wk of therapy, bone biopsies displayed reduced bone resorption (2.6 +/- 0.63 vs. 4.5 +/- 0.39%) and osteoblast covered bone surfaces (2.02 +/- 0.51 vs. 7.64 +/- 1.86%), which was indicative of low turnover. In contrast, prolonged treatment resulted in increased bone volume and trabecular number (38.9 +/- 1.35 vs. 25.2 +/- 2.56% and 3.56 +/- 0.23 vs. 2.88 +/- 0.11/mm) which was consistent with uncoupled bone formation. Administration of higher doses of aluminum (1.20 mg/kg) increased the serum aluminum further (1242.3 +/- 259.8 micrograms/liter) but did not affect calcium, creatinine, or parathyroid hormone. However, after 8 wk of treatment, bone biopsies displayed changes similar to those after long-term, low-dose therapy. In this regard, an increased trabecular number (3.41 +/- 0.18/mm) and bone volume (36.5 +/- 2.38%) again provided evidence of uncoupled bone formation. In contrast, in this instance poorly mineralized woven bone contributed to the enhanced bone volume. High-dose treatment for 16 wk further enhanced bone volume (50.4 +/- 4.61%) and trabecular number (3.90 +/- 0.5/mm). These observations illustrate that aluminum may stimulate uncoupled bone formation and induce a positive bone balance. This enhancement of bone histogenesis contrasts with the effects of pharmacologic agents that alter the function of existing bone remodeling units.
L D Quarles, H J Gitelman, M K Drezner
We have identified the presence of a putative corneal wound healing substance in mouse tears, which has a molecular size and immunological properties similar to those of epidermal growth factor (EGF). The substance was capable of binding to EGF receptors in mouse parenchymal cells and this binding was inhibited by anti-EGF serum. The concentration of the EGF-like substance in the tears of male and female mice was estimated to be 79.3 +/- 7.0 (SD) ng/ml and 76.5 +/- 8.1 (SD) ng/ml, respectively, by EGF radioimmunoassay. Removal of the submandibular glands, which produce large amounts of EGF, reduced plasma EGF to an undetectable level and also decreased the concentration of the EGF-like substance in tears to 27.3 +/- 3.9 (SD) ng/ml in male mice and 25.8 +/- 3.7 (SD) ng/ml in female mice. Approximately 50% of sialoadenectomized (submandibular glands removed) male mice with deep corneal wounds developed severe ocular lesions or loss of sight whereas none of normal male mice with similar wounds did. Topical application of EGF to deeply wounded eyes of sialoadenectomized mice eliminated the various complications and restored the healing rate and incidence of recovery to virtually normal levels.
O Tsutsumi, A Tsutsumi, T Oka
Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.
S Perkins, R A Fleischman
Isolated human gastric glands from surgical specimens were preincubated in an oxygenated medium with placebo or 16,16 dimethyl prostaglandin E2 (dmPGE2) and incubated at 37 degrees C in either medium alone, medium containing 4.43 mM indomethacin or medium containing 8% ethanol. We assessed the viability of gland cells with fast green exclusion, release of lactate dehydrogenase (LDH) into the medium, and ultrastructural damage by scanning and transmission electron microscopy. Both indomethacin and ethanol significantly reduced the viability of placebo-pretreated glands, increased LDH release into the medium, and produced prominent ultrastructural damage. DmPGE2 significantly reduced both indomethacin and ethanol-induced injury, increased the number of viable cells, reduced LDH release, and diminished the extent of ultrastructural damage. These studies indicate that PG protection of gastric mucosal cells has a direct cellular action that is not limited to replacement of depleted endogenous PGs. PG protection in our experiments did not depend on PG's previously described systemic actions, such as protection of the microvessels, preservation of the mucosal blood flow, or stimulation of bicarbonate and mucus secretion.
A Tarnawski, T Brzozowski, I J Sarfeh, W J Krause, T R Ulich, H Gergely, D Hollander
Two intestinal brush border membrane carboxypeptidases were found to participate in the sequential digestion of proline-containing peptides representing a novel mechanism of hydrolysis from the COOH terminus. NH2-blocked prolyl tripeptides were rapidly hydrolyzed by either brush border membrane angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, E.C. 220.127.116.11) or carboxypeptidase P (E.C.3.4.12-) depending on the position of the proline residue. Furthermore, these two enzymes were found to participate in a concerted manner to sequentially degrade larger proline-containing pentapeptides from the COOH terminus. A brush border membrane associated neutral endopeptidase also participated in the hydrolysis of the prolyl pentapeptides. During in vivo intestinal perfusion, the NH2-blocked prolyl peptides were degraded and their constituent amino acids efficiently absorbed by the intestine. Furthermore, hydrolysis and absorption of these peptides could be dramatically suppressed by low concentrations of captopril, a specific inhibitor of ACE. These studies show that prolyl peptides are efficiently and sequentially hydrolyzed from the COOH terminus by the combined action of ACE and carboxypeptidase P, and that these enzymes may play an important role in the digestion and assimilation of proline-containing peptides.
M Yoshioka, R H Erickson, Y S Kim
Using cDNA probes to human interleukin 2 (IL2) and interleukin 2 receptor (IL2R), the amount of IL2 and IL2R mRNA produced by PHA stimulated peripheral blood mononuclear cells from young (less than 40 yr) and old (greater than 60 yr) donors was quantitated. Stimulated cell cultures from each individual were also examined for proliferative ability, expression of membrane IL2R, membrane IL2R density, and for the amount of IL2R shed into the culture supernatant. Induction of IL2 and IL2R mRNAs were decreased in cells from elderly individuals, as were the levels of IL2 secretion, the percentage of IL2R+ T cells and the density of membrane IL2R per cell. The results suggest that decreased expression of both IL2 and IL2R mRNA contributes to the low synthesis of IL2 and membrane IL2R, respectively, and is partially responsible for the diminished proliferative activity observed in lymphocytes from the elderly.
J E Nagel, R K Chopra, F J Chrest, M T McCoy, E L Schneider, N J Holbrook, W H Adler
We infused dobutamine into the left main coronary artery of 24 patients with severe congestive heart failure (CHF) and 8 normal subjects without hemodynamic dysfunction. The maximal +dP/dt response to intracoronary (IC) dobutamine in CHF patients was only 37% of that in normals. This decrease in maximal response was not associated with a rightshift in the EC50 for dobutamine's effect on +dP/dt, or a decrease in the affinity of myocardial beta adrenergic receptors for dobutamine determined in vitro. In nine of the CHF patients, IC dobutamine infusion was followed by IC infusion of the phosphodiesterase inhibitor milrinone, and subsequently, by a second IC infusion of dobutamine. After IC milrinone, the increase in +dP/dt caused by IC dobutamine (74 +/- 10%) was significantly greater than that caused by the first infusion of dobutamine (52 +/- 11%; P less than 0.003) or milrinone (42 +/- 6%; P less than 0.001). Resting plasma norepinephrine was markedly elevated in CHF patients (837 +/- 208 ng/liter), but not in normal subjects (142 +/- 32 ng/liter); and the increase in +dP/dt caused by IC dobutamine was inversely related to resting plasma norepinephrine levels (r = -0.653; P less than 0.001). IC dobutamine caused a dose-related decrease in plasma norepinephrine (maximal effect, -160 +/- 31 ng/liter; P less than 0.001). Thus, (a) the maximal inotropic response to dobutamine is markedly depressed in patients with severe CHF, and is significantly greater after pretreatment with the phosphodiesterase inhibitor milrinone; (b) the impairment in inotropic response to dobutamine is inversely related to circulating norepinephrine levels; and (c) myocardial stimulation by dobutamine results in withdrawal of sympathetic tone.
W S Colucci, A R Denniss, G F Leatherman, R J Quigg, P L Ludmer, J D Marsh, D F Gauthier
Substance P and related tachykinins contribute to the airway hyperresponsiveness caused by toluene diisocyanate (TDI) in guinea pigs. Neutral endopeptidase (NEP) is an important modulator of substance P-induced responses. To test the hypothesis that exposure to TDI would increase responsiveness to substance P by inhibiting activity of this enzyme, we determined the dose of substance P required to increase pulmonary resistance by 200% above baseline (PD200) before and after administration of the pharmacologic inhibitor phosphoramidon in guinea pigs studied 1 h after a 1-h exposure to air or 3 ppm TDI. TDI exposure increased responsiveness to substance P significantly. However, phosphoramidon caused a significantly greater leftward shift of the substance P dose-response curve in air-exposed animals than it did in TDI-exposed animals, so that after phosphoramidon, mean values of PD200 in animals exposed to air or TDI did not differ. Tracheal NEP activity was significantly less after exposure to TDI than after exposure to air, whereas activity in the esophagus was the same in both groups. These results suggest that TDI exposure increases the bronchoconstrictor responsiveness of guinea pigs to substance P, in large part through inhibition of airway NEP.
D Sheppard, J E Thompson, L Scypinski, D Dusser, J A Nadel, D B Borson
Restriction fragment length polymorphisms (RFLPs), using the enzymes Bgl II and Xba I in conjunction with human von Willebrand factor (vWF) cDNA probes, have been described previously. In the present study we demonstrate the localization of both genetic markers within the vWF gene. The RFLPs were used to study the segregation of alleles associated with von Willebrand's disease (vWD) type IIA in a comprehensive, affected family. Individuals of this family were tested for their bleeding time and their plasma was analyzed for vWF antigen concentration and vWF ristocetin-cofactor activity. Based on these data, the affected members were diagnosed as vWD type-IIA patients; this conclusion was confirmed by the analysis of the multimeric vWF pattern of some of the patients. It was demonstrated that both RFLPs are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect that causes the vWD type IIA is most likely due to a mutation in the vWF gene and not to a mutation in a gene involved in posttranslational processing of the vWF protein.
C L Verweij, R Quadt, E Briët, K Dubbeldam, G B van Ommen, H Pannekoek
The disialoganglioside GD2 is expressed on a wide spectrum of human tumor types, including neuroblastomas and melanomas. Upon binding of 3F8, a murine monoclonal antibody (MAb) specific for GD2, neuroblastomas and some melanomas are sensitive to killing by human complement, whereas some melanomas are not. To investigate the mechanism underlying these differences in complement mediated cytotoxicity, complement-insensitive melanoma cell lines were compared with respect to expression of the decay-accelerating factor (DAF), a membrane regulatory protein that protects blood cells from autologous complement attack. While DAF was undetectable among neuroblastomas, it was present in complement-insensitive melanomas. When the function of DAF was blocked by anti-DAF MAb, C3 uptake and complement-mediated lysis of the insensitive melanoma lines were markedly enhanced. F(ab')2 fragments were as effective in enhancing lysis as intact anti-DAF MAb. The DAF-negative and DAF-positive melanoma cell lines were comparably resistant to passive lysis by cobra venom factor-treated serum. The data suggest that in some tumors, DAF activity accounts for their resistance to complement-mediated killing. The ability to render these cells complement-sensitive by blocking DAF function may have implications for immunotherapy.
N K Cheung, E I Walter, W H Smith-Mensah, W D Ratnoff, M L Tykocinski, M E Medof
The microbiostatic action of macrophages was studied in vitro employing peritoneal cytotoxic macrophages (CM) from mice acting against Cryptococcus neoformans cultured in Dulbecco's medium with 10% dialyzed fetal bovine serum. Fungistasis was measured using electronic particle counting after lysis of macrophages with detergent. Macrophage fungistasis failed in medium lacking only L-arginine. Complete fungistasis was restored by L-arginine; restoration was concentration dependent, maximal at 200 microM. Deletion of all other essential amino acids did not abrogate fungistasis provided that L-arginine was present. Of twenty guanido compounds, including D-arginine, only three (L-arginine, L-homoarginine, and L-arginine methylester) supported fungistasis. Known activators or mediators of macrophage cytotoxicity (endotoxin, interferon gamma, tumor necrosis factor) did not replace L-arginine for CM-mediated fungistasis. The guanido analogue NG-monomethyl-L-arginine was a potent competitive inhibitor of CM-mediated fungistasis giving 50% inhibition at an inhibitor/L-arginine ratio of 1:27. Although CM completely blocked fungal reproduction via an L-arginine-dependent mechanism, the majority of the dormant fungi remained viable. Thus, this mechanism is viewed as a microbiostatic process similar or identical to the tumoristatic effect of macrophages. This suggests the production of a broad spectrum biostatic metabolite(s) upon consumption of L-arginine by cytotoxic macrophages.
D L Granger, J B Hibbs Jr, J R Perfect, D T Durack
To study the mechanism of the diabetogenic action of ethanol, ethanol (0.75 g/kg over 30 min) and then glucose (0.5 g/kg over 5 min) were infused intravenously into six normal males. During the 4-h study, 21.8 +/- 2.1 g of ethanol was metabolized and oxidized to CO2 and H2O. Ethanol decreased total body fat oxidation by 79% and protein oxidation by 39%, and almost completely abolished the 249% rise in carbohydrate (CHO) oxidation seen in controls after glucose infusion. Ethanol decreased the basal rate of glucose appearance (GRa) by 30% and the basal rate of glucose disappearance (GRd) by 38%, potentiated glucose-stimulated insulin release by 54%, and had no effect on glucose tolerance. In hyperinsulinemic-euglycemic clamp studies, ethanol caused a 36% decrease in glucose disposal. We conclude that ethanol was a preferred fuel preventing fat, and to lesser degrees, CHO and protein, from being oxidized. It also caused acute insulin resistance which was compensated for by hypersecretion of insulin.
J J Shelmet, G A Reichard, C L Skutches, R D Hoeldtke, O E Owen, G Boden
The nature of the inhibitory neurotransmitter responsible for internal anal sphincter (IAS) relaxation in response to rectoanal reflex is not known. The objective of the present investigation was to examine the role of VIP in IAS relaxation in response to the rectoanal reflex in intact opossums with the use of VIP antagonists, [4CI-D-Phe6,Leu17] VIP and (N-AC-Tyr1,D-Phe2)-GRF (1-29)-NH2. Intraluminal pressures from the sphincter were monitored using low-compliance, continuously perfused catheters. VIP and the antagonists were administered close-intraarterially. The responses to VIP, rectoanal reflex, sacral nerve stimulation, and local intramural stimulation were examined before and after the VIP antagonists. The present studies in intact animals show: (a) VIP causes a dose-dependent fall in the IAS pressures by a direct action at the IAS smooth muscle; (b) VIP antagonists selectively and significantly antagonized the inhibitory action of VIP; and (c) VIP antagonists caused significant antagonism of the IAS relaxation caused by rectoanal reflex and the other neural stimuli. The antagonism of the IAS relaxation by the VIP antagonists, depending upon the volume of rectal distension used, ranged from 40% to 62% (P less than 0.05). From these results, we conclude that VIP acts as an inhibitory neurotransmitter for IAS relaxation during the rectoanal reflex.
S Nurko, S Rattan
Diacylglycerols (DAG) modulate secretory responses by the activation of protein kinase C. Early changes in DAG formation induced by the muscarinic receptor agonist carbachol were compared to those caused by the nutrient secretagogue glucose in pancreatic islets. Turnover rates of DAG were investigated in radiolabeling experiments, whereas changes in total mass and fatty acid composition of DAG were assessed by gas-liquid chromatography. When islet lipids were labeled to steady state in tissue culture with [3H]glycerol, carbachol induced a rapid (10 s) and sustained increase of [3H]DAG generation. In contrast, glucose stimulation failed to increase [3H]glycerol containing DAG, and this was probably due to the isotopic dilution of the label secondary to enhanced glycolysis. This was substantiated by following the transfer of 14C from glucose into DAG. Within 1 min of acute exposure of islets to D-[U-14C]-glucose at stimulatory concentrations, DAG labeling increased fivefold representing up to 2% of total glucose usage. Similar stimulation of 14C incorporation into other neutral lipids and inositol phospholipids was observed, suggesting the enhanced de novo synthesis of phosphatidic acid, the common precursor for DAG, and inositol phospholipids from glycolytic intermediates. Transfer of 14C from glucose was not stimulated by agents such as carbachol and exogenous phospholipase C that act primarily on inositol phospholipid breakdown. The total mass of islet DAG was increased by 60% after both carbachol and glucose stimulation. However, analysis of the fatty acid composition of carbachol-generated DAG revealed at the early time point (10 s) a prevalent stearoyl-arachidonoyl configuration similar to that reported for inositol phospholipids. This pattern shifted to a DAG enriched in palmitic acid at a later time point. Glucose-stimulated islets displayed a predominance of palmitic acid containing DAG, indicating increased de novo synthesis of the putative second messenger rather than its formation by inositol phospholipid hydrolysis. Indeed, steady-state labeling of these phospholipids with [3H]inositol confirmed this idea since only carbachol caused detectable inositol phospholipid hydrolysis. Thus, although protein kinase C may be activated by both carbachol and glucose, the two secretagogues generate diacylglycerols through different mechanisms.
B Peter-Riesch, M Fathi, W Schlegel, C B Wollheim
In addition to activating T and B lymphocytes, interleukin 1 (IL-1) induces several hematologic and metabolic changes typical of host responses to infection and injury. We now report a new biological property, namely, the induction of hypotension. Rabbits given a single intravenous injection of recombinant human IL-1-beta (5 micrograms/kg) rapidly developed decreased systemic arterial pressure, which reached the lowest levels after 50-60 min and slowly returned to pre-IL-1 values after 3 h. Associated with the hypotension, systemic vascular resistance and central venous pressure fell, while cardiac output and heart rate increased. These responses were prevented by ibuprofen given 15 min before the IL-1. A bolus injection of IL-1 followed by a 2-h infusion sustained the hypotension and was associated with leukopenia and thrombocytopenia. Ibuprofen given at the mid-point of the infusion reversed the changes in all hemodynamic parameters, but had no effect on the leukopenia or thrombocytopenia. Tumor necrosis factor (TNF) also induced a shock-like state in rabbits. When the dose of IL-1 or TNF was reduced to 1 microgram/kg, no hemodynamic changes were observed; however, the combination of these low doses of both cytokines resulted in a profound shock-like state including histological evidence of severe pulmonary edema and hemorrhage. Pretreatment with ibuprofen prevented the hemodynamic, leukocyte, and platelet changes induced by the low-dose cytokine combination, and ameliorated the pulmonary tissue damage. These results demonstrate that IL-1, like TNF, possesses the ability to induce hemodynamic and hematological changes typical of septic shock, and that the combination of IL-1 and TNF is more potent than either agent alone. These effects seem to require cyclooxygenase products, and suggest that intravenous cyclooxygenase inhibitors may be of therapeutic value in patients with IL-1/TNF-mediated shock.
S Okusawa, J A Gelfand, T Ikejima, R J Connolly, C A Dinarello
Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.
H Ishida, O Kohmoto, J H Bridge, W H Barry
To determine the effect of contraction of the diaphragm on the lower esophageal sphincter (LES) pressure, we studied eight healthy volunteers during spontaneous breathing, maximal inspiration, and graded inspiratory efforts against a closed airway (Muller's maneuver). Electrical activity of the crural diaphragm (DEMG) was recorded from bipolar esophageal electrodes, transdiaphragmatic pressure (Pdi) was calculated as the difference between gastric and esophageal pressures, and LES pressure was recorded using a sleeve device. During spontaneous breathing, phasic inspiratory DEMG was accompanied by phasic increases in Pdi and LES pressure. With maximal inspiration, DEMG increased 15-20-fold compared with spontaneous inspiration, and LES pressure rose from an end-expiratory pressure of 21 to 90 mmHg. Similar values were obtained during maximal Muller's maneuvers. LES pressure fell promptly when the diaphragm relaxed. Graded Muller's maneuver resulted in proportional increases in the Pdi, LES pressure, and DEMG. The LES pressure was always greater than Pdi and correlated with it in a linear fashion (P less than 0.001). We conclude that the contraction of the diaphragm exerts a sphincteric action at the LES, and that this effect is an important component of the antireflux barrier.
R K Mittal, D F Rochester, R W McCallum
The goal of these experiments was to investigate the relationship of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), monobasic phosphate (H2PO4-), and pH to human muscle fatigue. Phosphates and pH were measured in adductor pollicis using 31P nuclear magnetic resonance at 2.0 Tesla. The force of muscle contraction was simultaneously measured with a force transducer. The effects of aerobic and anaerobic exercise were compared using two exercise protocols: 4 min sustained maximal voluntary contraction (MVC) and 40 min of repeated intermittent contractions (75% MVC). The sustained maximal contraction produced a rapid decline of MVC and PCr, and was accompanied by a rapid rise of Pi, H+, and H2PO4-. Intermittent exercise produced steady state changes of MVC, pH, and phosphates. No significant changes of ATP were found in either protocol. During fatiguing exercise, PCr and Pi had a nonlinear relationship with MVC. H+ showed a more linear correlation, while H2PO4- showed the best correlation with MVC. Furthermore, the correlations between MVC and H2PO4- were similar in sustained (r = 0.70) and intermittent (r = 0.73) exercise. The highly significant linear relationship between increases of H+ and H2PO4- and the decline of MVC strongly suggests that both H+ and H2PO4- are important determinants of human muscle fatigue.
R G Miller, M D Boska, R S Moussavi, P J Carson, M W Weiner
The cause of the abnormal active cation transport in erythrocytes of some uremic patients is unknown. In isolated adipocytes and skeletal muscle from chronically uremic chronic renal failure rats, basal sodium pump activity was decreased by 36 and 30%, and intracellular sodium was increased by 90 and 50%, respectively, compared with pair-fed control rats; insulin-stimulated sodium pump activity was preserved in both tissues. Lower basal NaK-ATPase activity in adipocytes was due to a proportionate decline in [3H]ouabain binding, while in muscle, [3H]ouabain binding was not changed, indicating that the NaK-ATPase turnover rate was decreased. Normal muscle, but not normal adipocytes, acquired defective Na pump activity when incubated in uremic sera. Thus, the mechanism for defective active cation transport in CRF is multifactorial and tissue specific. Sodium-dependent amino acid transport in adipocytes closely paralleled diminished Na pump activity (r = 0.91), indicating the importance of this defect to abnormal cellular metabolism in uremia.
W Druml, R A Kelly, R C May, W E Mitch
An electrogenic H-ATpase sensitive to inhibition by N-ethyl-maleimide has been reported to be present in renal distal tubules. In contrast to another H-ATPase (gastric H-K-ATPase), the renal enzyme is not stimulated by K+ and is not inhibited by vanadate. However, our preliminary observations indicated that a K-stimulated ATPase (K-ATPase) sensitive to inhibition by vanadate is present in renal medullary collecting duct (MCD). To localize and further characterize this renal tubular K-ATPase, we measured K-ATPase activity in eight specific segments of the rabbit nephron. K-ATPase activity was the difference in ATPase activity in the presence and absence of KCl but in the presence of ouabain (to inhibit Na-K-ATPase). ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to the oxidation of NADH. There was a significant K-ATPase activity (expressed as pmol.min-1.mm-1) in the connecting tubule (CNT, 17.0 +/- 3.3), cortical collecting duct (CCD, 6.6 +/- 0.7), and MCD (8.8 +/- 1.7), but not in the proximal segments and the thick ascending limbs. The renal tubular K-ATPase was not only inhibited by vanadate but also by omeprazole and SCH 28080 (relatively specific inhibitors of gastric H-K-ATPase). It is concluded that K-ATPase present in the CNT, CCD, and MCD has some properties in common with gastric H-K-ATPase. However, the physiological role of K-ATPase in the distal nephron segments remains to be elucidated.
L C Garg, N Narang
The in vitro effect of S-stage-specific drugs on the fetal hemoglobin (Hb F) potential of erythroid precursors and progenitors was tested by exposing bone marrow cells to 5-aza-2'-deoxycytidine, Ara-C, or hydroxyurea in suspension cultures and reculturing the cells in drug-free clonal cultures. Analysis of Hb F in the erythroblasts present at the end of suspension cultures and in the erythroid colonies formed from treated progenitors showed that 1 X 10(-9)-5 X 10(-8) M 5-aza-2'-deoxycytidine produced a concentration-related increase in the proportion of Hb F-positive erythroblasts, of Hb F-positive erythroid CFU (CFUe) colonies, and at the higher doses used, an increased Hb F expression in erythroid burst-forming unit (BFUe)-derived colonies. Preincubation of bone marrow cells with Ara-C produced significant megaloblastic changes by the end of the 2-d incubation and increased the proportion of Hb F-positive erythroblasts, CFUe colonies, and e-clusters, but BFUe-derived progeny was unaffected. Hydroxyurea failed to produce significant changes in Hb F at the range of concentrations used. The data raise the possibility of more than one mechanism underlying the stimulation of Hb F by S-stage drugs.
R Galanello, G Stamatoyannopoulos, T Papayannopoulou
MAb directed to the thyroid microsomal antigen have been developed. All bound to 101- and 107-kD bands in Western blot analysis using thyroid microsomal fraction as antigen. The MAb also bound to microsomal proteins immunoprecipitated by serum from patients having a high titer of anti-microsomal antibody but no antibodies to thyroglobulin or thyrotropin-stimulating hormone receptor. The pattern of binding was related to the amount of reducing agent. The 101- and 107-kD bands were increased by addition of dithiothreitol whereas, in its absence, numerous bands of higher molecular weight were present, suggesting a multimeric protein structure. Despite the inability to immunoprecipitate thyroid peroxidase (TPO) enzymatic activity, the MAb bound intensively in Western blot to denatured purified hog TPO and to denatured immunopurified human TPO. Trypsin digestion of the 101-107-kD antigen produced a doublet of 84-88 kD that was still immunoreactive with MAb. One of five polyclonal sera tested (with a microsomal antibody titer greater than 1/20,480 measured by the tanned red cell hemagglutination technique) also recognized the 84-88 kD trypsin fragments. Addition of V8 protease led to a disappearance of the 107-kD protein, but not the 101-kD protein, proving that this antigen is formed by two different polypeptides. The MAb bound strongly to thyroid epithelium, whereas binding to papillary carcinoma was absent or low and moderate for follicular and Hurthle cell carcinoma. This study indicates that the thyroid microsomal antigen and TPO are identical and are constituted of two different polypeptides. On SDS-PAGE the antigen appears as two contiguous bands which share common epitopes but are not identical, as proven by their size and difference in susceptibility to proteolytic digestion. The immunoreactivity of the molecule is highly dependent on a trypsin-sensitive site, which appears important in the recognition of the antigen by polyclonal sera and may have biological importance. The expression of microsomal antigenicity is variable among various thyroid carcinomas.
L Portmann, F W Fitch, W Havran, N Hamada, W A Franklin, L J DeGroot
Using intact, beating hearts, we have assessed the interaction of insulin with capillary endothelium and the subsequent appearance of insulin in cardiac muscle. Rat hearts were perfused with 125I-insulin (10(-10) M) alone or in combination with unlabeled insulin (10(-9)-10(-5) M). 125I grains (shown to represent greater than 90% intact insulin) over both capillary endothelium and cardiac muscle decreased in a dose-dependent manner when hearts were co-perfused with labeled insulin and increasing concentrations of unlabeled insulin. Perfusion of 125I-desoctapeptide (DOP) insulin, a low affinity insulin analogue, with unlabeled insulin (10(-9)-10(-5) M) had no effect on the appearance of 125I-DOP insulin over microvessel endothelium and muscle. When capillary receptors were first destroyed by trypsin treatment or blocked by anti-receptor antibodies, the appearance of 125I-insulin in cardiac muscle decreased proportional to the inhibition of insulin binding to the capillary receptors. We conclude that insulin binding to capillary endothelial receptors is a central step in the transport of intravascular insulin to rat cardiac muscle.
R S Bar, M Boes, A Sandra
"Perinatal" hypophosphatasia is the most severe form of this inborn error of metabolism, which is characterized by deficient activity of the tissue-nonspecific (liver/bone/kidney) isoenzyme of alkaline phosphatase (ALP) (TNSALP). We report that autopsy tissue from three affected subjects, which was profoundly low in ALP activity, had essentially unremarkable levels of pyridoxal-5'-phosphate (PLP), pyridoxal, and total vitamin B6 content despite markedly elevated plasma PLP levels (5,800, 14,500, and 98,500 nM; adult norm, 5-109 nM). Our findings help to explain the general absence of symptoms of vitamin B6 excess or deficiency in hypophosphatasia, and provide evidence that TNSALP acts as an ectoenzyme to regulate extracellular rather than intracellular concentrations of PLP (the cofactor form of vitamin B6) and perhaps other phosphate compounds.
M P Whyte, J D Mahuren, K N Fedde, F S Cole, E R McCabe, S P Coburn
The hypothesis that intracellular generation of reactive oxygen species in hepatocytes or reticuloendothelial cells may cause ischemia-reperfusion injury was tested in isolated perfused livers of male Fischer rats. GSSG was measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 30 min, the perfusion was stopped (global ischemia) for various times (30, 120 min) and the liver was reperfused for another 60 min. The bile flow (1.48 +/- 0.17 microliters/min X gram liver weight), the biliary efflux of total glutathione (6.54 +/- 0.94 nmol GSH eq/min X g), and GSSG (1.59 +/- 0.23 nmol GSH eq/min X g) recovered to 69-86% after short-term ischemia and to 36-72% after 2 h of ischemia when compared with values obtained from control livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4 +/- 2.1 nmol GSH eq/min X g) and GSSG (0.13 +/- 0.05 nmol GSH eq/min X g) did not change except for an initial 10-30-s increase during reperfusion washout. No increased GSSG secretion into bile was detectable at any time during reperfusion. The liver content of total glutathione (32.5 +/- 3.5 nmol GSH eq/mg protein) and GSSG (0.27 +/- 0.09 nmol GSH eq/mg protein) did not change significantly during any period of ischemia or reperfusion. We conclude, therefore, that at most only a minor amount of reactive oxygen species were generated during reperfusion. Thus, reactive oxygen species are unlikely to cause ischemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation.
H Jaeschke, C V Smith, J R Mitchell
In the absence of antigenic or mitogenic stimulation, certain peripheral blood lymphocytes exhibit proliferative and lymphokine-activated killer (LAK) cell activities when cultured with recombinant IL-2. Both activities were found to be an exclusive property of lymphocytes expressing type 3 complement receptors (CR3) identified by anti-CD11 monoclonal antibodies. CD11+ lymphocytes were then fractionated into three subsets by two-color flow cytometry. These included CD16+ cells, which display distinctive Fc receptors for IgG (CD16). Using anti-CD5, the CD11+ CD16- lymphocytes were separated into non-T cell and T cell subsets. The two non-T cell subsets (CD11+ CD16+ and CD11+ CD16- CD5-), but not the T cell subset (CD11+ CD16- CD5+), could proliferate in response to IL-2. Both CD11+ non-T cell subsets, but not the CD11+ T cell subset, had the capacity to mediate natural killer cell activity. However, all three CD11+ lymphocyte subsets were capable of generating LAK activity. These findings are consistent with the concept that two signals are required to stimulate T cells to proliferate. However, at least a small subset of blood T cells can be activated by IL-2 to become LAK cells.
J D Gray, D A Horwitz
When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.
L J Mandel, T Takano, S P Soltoff, S Murdaugh
Numerous studies have suggested that epinephrine may facilitate neural release of NE. There have been no studies in humans that demonstrate the functional significance of this action. To determine whether epinephrine facilitates neurogenic vasoconstriction in humans, we contrasted forearm vasoconstrictor responses to a reflex stimulus (lower body negative pressure [LBNP]) and to intraarterial NE before, during, and 30 min after infusion of epinephrine (50 ng/min) or isoproterenol (10 or 25 ng/min) into a brachial artery. These doses had no systemic effects. We reasoned that if prejunctional stimulation of beta receptors by epinephrine and isoproterenol had functional significance, the vasoconstrictor response to LBNP would be potentiated in comparison to the response to NE (postjunctional mechanism). Studies were done on 23 normal male volunteers. Forearm blood flow was measured with a strain gauge plethysmograph and intraarterial pressure was recorded. The ratio of vasoconstrictor responses to LBNP/NE was used as an index of neural release of the neurotransmitter NE. This ratio increased during infusions of both epinephrine and isoproterenol. 30 min after epinephrine the vasoconstrictor response to LBNP (n = 15) was augmented from +9.9 +/- 2.2 (SE) resistance units (RU) before epinephrine to +16.4 +/- 3.2 RU (P less than 0.05); whereas the response to NE (n = 8) tended to decrease from +8.8 +/- 3.1 RU before to +4.2 +/- 1.2 RU after epinephrine (P greater than 0.05). In contrast, 30 min after isoproterenol the vasoconstrictor responses to LBNP and NE were the same as before isoproterenol. The augmented ratio of responses to LBNP/NE after epinephrine and not after isoproterenol supports the concept that epinephrine, but not isoproterenol, is taken up by the adrenergic terminal, is released subsequently during reflex stimulation, and augments the release of the neurotransmitter NE. These experiments provide the first hemodynamic evidence in humans that epinephrine and isoproterenol facilitate neurogenic vasoconstriction. The sustained effect of epinephrine in contrast to isoproterenol suggests that the late facilitation by epinephrine is related to its neural uptake and subsequent release.
J S Floras, P E Aylward, R G Victor, A L Mark, F M Abboud
Regional variations in the ion transport properties of the colon may have significant physiological and pathophysiological implications. However, only limited studies have been performed in cecum, which comprises 50% of the macrosurface of the rabbit colon. In vitro under short-circuit conditions, cecum actively absorbed Na and Cl (JnetNa = 5.6 +/- 0.3, JnetCl = 1.5 +/- 0.3 mu eq.cm-2.h-1) with a short-circuit current (Isc) of 6.29 +/- 0.2 mu eq.cm-2.h-1.Cl substitution with sulfate decreased both JnetNa and Isc by 1.3 mu eq/cm2.h-1.HCO3 removal decreased both JnetNa and Isc 3.3 mu eq.cm-2.h-1. This effect was due primarily to removal of serosal HCO3. There was both a linear correlation between JNanet and Isc (r = 0.845) and a concentration-dependent stimulation of Isc by increasing [Na] in the bathing media. However, 10(-4) M amiloride did not significantly alter either Isc or JnetNa. In contrast, 10(-4) M phenamil, an amiloride analogue highly specific for the Na channel, significantly blocked both Isc and JnetNa. The sulfhydryl reagent PCMBS increased Isc; this response was reversed by phenamil. Electrogenic Cl secretion was stimulated by 1 mM theophylline, 10(-4) M 8BrcAMP and 10(-4) M 8BrcGMP. None of the secretagogues inhibited JnetNa. Epinephrine (5.5 microM) increased JnetNa from 5.9 +/- 1.3 to 7.8 +/- 1.1 (P = 0.02) and JnetCl from 0.1 +/- 1.2 to 2.0 +/- 0.8 (P NS) mu eq.cm-2.h-1. Studies of pH stat demonstrated an epinephrine-stimulated increase in Jm-sHCO3 without a change in Js-mHCO3. Thus, cecum exhibits a distinct type of electrogenic Na electrogenic Na absorption which is partially dependent on the presence of Cl and HCO3, not blocked by amiloride but by phenamil. Because of its large surface area and its novel mechanism of electrogenic Na transport, the cecum exerts an important regulatory role in colonic fluid and electrolyte balance.
J H Sellin, H Oyarzabal, E J Cragoe
Localized thrombosis was produced in the left anterior descending (LAD) coronary artery of open chest dogs by constricting a segment so as to produce greater than 90% stenosis (reducing blood flow to 40 +/- 10% of baseline), and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin. Intravenous infusion of recombinant tissue-type plasminogen activator (rt-PA) at a rate of 15-30 micrograms/kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23 +/- 7 min (mean +/- SD), but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7 +/- 5 min. Intravenous injection of 0.8 mg/kg of the F(ab')2 fragment of a monoclonal antibody (7E3) directed against the platelet GPIIb/IIIa receptor, prevented reocclusion in 10/10 dogs during an observation period of 2 h (P less than 0.001 vs. rt-PA alone). The antibody abolished ADP-induced platelet aggregation and markedly prolonged the bleeding time. Intravenous aspirin or dipyridamole prevented reocclusion for 1 h or more in only 2/7 and 1/6 dogs, respectively. We conclude that the monoclonal antibody is very effective in preventing reocclusion after successful thrombolysis of occluded coronary arteries with rt-PA.
T Yasuda, H K Gold, J T Fallon, R C Leinbach, J L Guerrero, L E Scudder, M Kanke, D Shealy, M J Ross, D Collen
Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.
J Y Borg, M C Owen, C Soria, J Soria, J Caen, R W Carrell
Three lines of investigation indicated that hydrogen peroxide (H2O2) from xanthine oxidase (XO) contributes to cardiac dysfunction during reperfusion after ischemia. First, addition of dimethylthiourea (DMTU), a highly permeant O2 metabolite scavenger (but not urea) simultaneously with reperfusion improved recovery of ventricular function as assessed by ventricular developed pressure (DP), contractility (+dP/dt), and relaxation rate (-dP/dt) in isolated Krebs-Henseleit-perfused rat hearts subjected to global normothermic ischemia. Second, hearts from rats fed tungsten or treated with allopurinol had negligible XO activities (less than 0.5 mU/g wet myocardium compared with greater than 6.0 mU/g in control hearts) and increased ventricular function after ischemia and reperfusion. Third, myocardial H2O2-dependent inactivation of catalase occurred after reperfusion following ischemia, but not after ischemia without reperfusion or perfusion without ischemia. In contrast, myocardial catalase did not decrease during reperfusion of ischemic hearts treated with DMTU, tungsten, or allopurinol.
J M Brown, L S Terada, M A Grosso, G J Whitmann, S E Velasco, A Patt, A H Harken, J E Repine