The results of experiments with indirect methods have suggested that various interventions reduce infarct size after coronary artery occlusion. To determine and quantify directly both the short- and long-term effects of several interventions on myocardial salvage without relying on indirect methods, the left coronary artery was occluded in 880 rats; they were then given either no treatment or one of the following interventions: (a) hyaluronidase, an enzyme that hydrolyzes interstitial glycoproteins, 1,500 National Formulary (NF) U/kg i.v. 5 min and 24 h after occlusion; (b) cobra venom factor, a protein that depletes the third component of complement, 20 U/kg i.v. 5 min after occlusion; (c) a glucocorticoid: hydrocortisone, 50 mg/kg i.v. 5 min after occlusion; or the five-fold more potent methylprednisolone (MP): (i) 50 mg/kg i.v. 5 min after occlusion or (ii) 50 mg/kg i.v. 5 min after occlusion followed by 50 mg/kg i.m. 3, 6, and 24 h after occlusion; or (d) reserpine, an agent that depletes the heart of catecholamines, 0.5 mg/kg i.m. once on each of the 3 days before occlusion. The animals were sacrificed either 2 days after occlusion, i.e., at the time of peak necrosis, or after 3 wk, i.e., after the infarct was completely healed. The amount of preserved myocardium was then assessed by two independent techniques: planimetric measurement of serial histologic sections and creatine kinase activity of the whole left ventricle. The amount of normal myocardium preserved at 21 days postocclusion was significantly increased, by 22.3±7.8% (P < 0.025) after the administration of hyaluronidase, by 25.3±5.8% (P < 0.005) after cobra venom factor, by 14.5±6.9% (P < 0.05) after hydrocortisone, by 20.8±8.2% (P < 0.025) after the single dose of MP, by 20.9±3.9% (P < 0.001) after the four doses of MP, and by 10.2±3.7% (P < 0.05) as a result of pretreatment with reserpine. The four doses of MP significantly thinned the infarct—by 25.6±2.9% (P < 0.001)—and although ventricular rupture did not occur, the intervention caused distension of the left ventricle as a result of stretching of the infarcted tissue during scar formation. Thus, myocardium acutely jeopardized by ischemia can be preserved on a long-term basis.
Derek Maclean, Michael C. Fishbein, Eugene Braunwald, Peter R. Maroko
To investigate the role of hepatic glucagon receptors in the hypersensitivity to glucagon observed in insulin-deprived diabetics, liver plasma membranes were prepared from control rats and from streptozotocin-induced diabetic rats some of whom were treated with high-dose and low-dose insulin. The untreated diabetic animals exhibited hyperglycemia, weight loss, hypoinsulinemia, and hyperglucagonemia. High-dose insulin treatment (2 U Protamine-zinc-insulin/100 g per day) resulted in normoglycemia, normal weight gain, mild hyperinsulinemia, and return of glucagon levels toward base line. The low-dose (1 U protamine-zinc-insulin/100 g per day) insulin-treated diabetic group demonstrated chemical changes intermediate between the untreated and the high-dose insulin-treated animals.
Vijay Soman, Philip Felig
The effect of increased capillary permeability on glomerular immune complex localization was studied in rats immunized with proximal tubular antigen (Fx1A) to induce autologous immune complex nephropathy (AICN). AICN rats were made proteinuric by injection or unilateral renal perfusion with aminonucleoside of puromycin (PA) before developing subepithelial complex deposits. Control AICN kidneys developed diffuse granular deposits of IgG and Fx1A on the subepithelial surface of the glomerular basement membrane (GBM) at 3 wk by immunofluorescence and electron microscopy, and deposits increased in subsequent weekly biopsies. In contrast, PA-nephrotic AICN kidneys developed few or no GBM deposits and a significant increase in mesangial localization of IgG and Fx1A during the period of PA-induced proteinuria. These alterations in complex localization were documented both in rats with PA nephrosis and in unilaterally PA-nephrotic kidneys compared with contralateral controls in the same animals, thus excluding any effect of PA on the immunopathogenetic mechanism in AICN as an explanation for these findings. The absence of GBM deposits closely correlated with reduced staining for polyanionic glomerular sialoprotein in proteinuric kidneys, since PA-perfused kidneys studied 2 wk after resolution of proteinuria demonstrated return of normal staining for sialoprotein and development of subepithelial complex deposits similar to those in contralateral control kidneys. These studies demonstrate that properties of the glomerulus itself play an important role in determining the site of complex deposition in experimental AICN and suggest that electrophysical characteristics of the glomerular capillary wall may influence complex localization on the GBM.
W G Couser, N B Jermanovich, S Belok, M M Stilmant, J R Hoyer
The effects of phosphate depletion on magnesium (Mg) homeostasis were evaluated in rats fed a diet containing 0.03% phosphorus for periods up to 8 wk. Plasma phosphorus fell significantly (P < 0.01) from 10.1±0.27 (SE) to 5.0±0.54 mg/100 ml within 1 day and continued to fall gradually to a level of 1.2±0.21 mg/100 ml by the end of the 8th wk. A significant (P < 0.01) increment in urinary Mg excretion (UMgV) from 46±2.7 to 126±24 μeq/24 h occurred during the 1st day of phosphate depletion; UMgV reached a peak of 300±24 μeq/24 h by the 3rd day and remained high ranging between 150-300 μeq/24 h, thereafter. The magnitude of the magnesuria was related to the degree of hypophosphatemia and was not affected by lowering the calcium intake and reducing the hypercalciuria. The concentration of plasma Mg fell significantly (P < 0.01) from 1.2±0.02 to 0.79±0.10 meq/liter by the 1st day of the study and remained low throughout.
Wilhelm J. Kreusser, Kiyoshi Kurokawa, Enrique Aznar, Ellen Sachtjen, Shaul G. Massry
Complement appears to be involved in the destruction of platelets in certain clinical disorders, such as quinidine purpura and post-transfusion purpura. In both disorders, the classical complement sequence is activated by antigen-antibody complexes. It has been suggested that the terminal components of the complement sequence insert into the hydrophobic core of cell surface membranes and that this process leads to cell lysis. Fluidity is a fundamental property of lipids within the membrane's hydrophobic core. To examine the interaction of complement with membranes, we investigated the effect of complement activation on the fluidity of human platelet membranes. Complement was fixed to platelets using a post-transfusion purpura antibody, and membrane lipid fluidity was assessed in terms of fluorescence anisotropy using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene and 9-(12-anthroyl) stearic acid. Microviscosity, expressed in poise, was derived from the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.
Sanford J. Shattil, Douglas B. Cines, Alan D. Schreiber
Studies were carried out in anesthetized dogs to characterize the increase in cation excretion which occurs after acute unilateral nephrectomy (AUN). 60 min after AUN, cation excretion had increased from 31.5±2.7 to 66.3±12.0 μeq/min (P < 0.005) and fractional cation excretion had increased from 0.56±0.05 to 1.03±0.14% (P < 0.005), as the glomerular filtration rate was unchanged and renal blood flow fell. The increased cation excretion was accompanied by an increase in fractional phosphate excretion, no change in chloride excretion, and a fall in renin secretion. These alterations in renal function were associated with marked changes in systemic hemodynamics: cardiac output fell from 2.52±0.24 to 1.85±0.16 liters/min (P < 0.001), as diastolic pressure rose without an overall increase in mean arterial pressure, and heart rate fell.
Michael H. Humphreys, J. Carlos Ayus
In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.
P K Peterson, B J Wilkinson, Y Kim, D Schmeling, S D Douglas, P G Quie, J Verhoef
The mechanism whereby the vasoconstrictor response to angiotensin II (AII) is influenced by sodium balance or disease is unclear. To explore this question, the renal vascular responses (RVR) to intrarenal injections of subpressor doses of AII and norepinephrine were studied in dogs with an electromagnetic flowmeter. Acute and chronic sodium depletion increased plasma renin activity (PRA) and blunted the RVR to AII, while acute sodium repletion and chronic sodium excess plus desoxycorticosterone acetate decreased PRA and enhanced the RVR to AII. The magnitude of the RVR to AII was inversely related to PRA. The RVR to norepinephrine was unaffected by sodium balance and was not related to PRA. Inhibition of the conversion of angiotensin I to AII by SQ 20,881 during sodium depletion lowered mean arterial blood pressure (MABP), increased renal blood flow (RBF), and enhanced the RVR to AII but not to norepinephrine. Administration of bradykinin to chronically sodium-depleted dogs also lowered the MABP and increased RBF but had no effect on the RVR to AII. SQ 20,881 had no effect on MABP, RBF, or the RVR to AII in the dogs with chronic sodium excess and desoxycorticosterone acetate. Administration of indomethacin to chronically sodium-depleted dogs lowered RBF but did not influence the RVR to AII. The results indicate that the RVR to AII is selectively influenced by sodium balance and that the magnitude of the response is inversely related to the availability of endogenous AII. The data did not suggest that the variations in the RVR to AII were because of direct effects of sodium on vascular contraction, changes in the number of vascular AII receptors, or the renal prostaglandins. The results are consistent with the hypothesis that the vasoconstrictor effect of AII in the renal vasculature is primarily dependent upon the degree to which the AII vascular receptors are occupied by endogenous hormone.
J A Oliver, P J Cannon
The effect of ethanol feeding on ovarian function and structure in female rats was studied in alcohol-fed animals, isocalorically fed controls, and two ad libitum-fed control groups. Ovarian weight was reduced by 60% in alcohol-fed animals compared with the control groups. Gross disruption of ovarian architecture was noted, characterized by the absence of any corpus hemorrhagicum and corpus albicans. Moreover, plasma levels of estradiol were significantly reduced in the alcohol-fed animals (P < 0.01) compared with the levels found in isocaloric controls. Plasma levels of estrone and corticosterone were increased in alcoholfed and isocaloric control animals relative to those of ad libitum-fed animals suggesting a primarily adrenal, rather than ovarian, origin for these two steroids. Despite the increase in estrone, the secondary sex organs (uterus and fallopian tubes) reflected marked estrogen deprivation presumably as a result of estradiol insufficiency.
David H. Van Thiel, Judith S. Gavaler, Roger Lester
Studies were performed in Munich-Wistar rats to determine whether changes in papillary plasma flow might be responsible for the concentrating defect which occurs after exposure of the extrarenal papilla. Papillary plasma flow was measured by 125I-albumin accumulation. Initial studies in hydropenic animals revealed that papillary plasma flow was 40% higher in the kidney with the exposured papilla, 41 vs. 29 ml/min per 100 g of papilla (P < 0.001). This increase in papillary plasma flow was detectable 15 or 45 min after removing the ureter. Because it was unclear whether the rise in papillary plasma flow was a cause or the result of the fall in urine osmolality, similar studies were performed in animals undergoing a water diuresis. In this setting, papillary plasma flow still increased on the exposed side compared to the control side, 81 vs. 60 ml/min per 100 g, despite similarly low urine osmolalities of 155 and 174 mosmol/kg, respectively. This finding is compatible with the possibility that papillary exposure per se causes an increase in papillary plasma flow and that this hemodynamic alteration may lead to a reduction in urinary osmolality secondary to washout of the medullary interstitium. A final group of hydropenic rats was given either indomethacin or meclofenamate before removing the ureter. In these studies, there was no difference in either the papillary plasma flow or the urine osmolality between control and exposed kidneys. It is therefore suggested that opening the ureter induces an increase in papillary plasma flow by some mechanism which may involve an alteration in prostaglandin synthesis.
Elaine L. Chuang, H. John Reineck, Richard W. Osgood, Robert T. Kunau Jr., Jay H. Stein
Using myoelectric recording techniques, we examined the myoelectric effects of castor oil; ricinoleic acid (cis isomer), the active ingredient of castor oil; and ricinelaidic acid (trans isomer) in the small intestine of New Zealand white rabbits. Ricinoleic acid, 2 microgram/kg per min (6mM), was perfused into a distal 12-cm ileal loop. An abnormal myoelectric pattern developed that was similar to the alteration in the electrical activity that has previously been reported for cholera enterotoxin. Castor oil, 0.85 ml/kg, had a similar effect. Ricinelaidic acid, 2 microgram/kg per min, induced no activity. A second preparation consisted of an intraluminal perfusion of ricinoleic acid, 2 microgram/kg per min, into the first section of the duodenum. The abnormal myoelectric pattern was observed in the jejunum and the ileum but not the duodenum. The mean onset time for the development of this altered myoelectric state for all experiments was 3.5 h. These studies suggest that an active motility component in addition to the secretory state exists throughout the small intestine that is exposed to castor oil or ricinoleic acid.
J R Mathias, J L Martin, T W Burns, G M Carlson, R P Shields
We have compared in vivo pyridoxine responsiveness with in vitro cystathionine β-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5′-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five- and sevenfold stimulation of cystathionine β-synthase activity with added pyridoxal-5′-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two- and fivefold with the added cofactor. Cystathionine β-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5′-phosphate were estimated and thermostability (54°C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine β-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5′ phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activity in vitro or with stimulation of in vitro enzyme activity by cofactor.
Brian Fowler, Jan Kraus, Seymour Packman, Leon E. Rosenberg
A procedure was developed for measurement of androgen receptors in cytoplasmic extracts of prostates from intact dogs. The protocol utilized exchange saturation analysis at 15°C employing the synthetic androgen R1881 (17β-hydroxy-17α-methylestra-4,9,11-trien-3-one) as the ligand probe and quantitatively detected total cytoplasmic androgen receptor (Rc, androgen-free receptor, and RcA, androgen-occupied receptor) present at the initiation of the assay. This protocol was employed in conjunction with a tissue mince saturation analysis procedure (for quantitation of nuclear androgen receptor) to quantitate total androgen receptor content of normal and hyperplastic prostates obtained from young (2.5- or 4.6-yr old) and aged (12.5-yr old) purebred dogs of known birth date.
Sydney A. Shain, Robert W. Boesel
Components of the complement system are known to play an important role in the cytolytic process and in chemotaxis of leukocytes. Cobra venom factor specifically cleaves C3 activity via activation of the alternative (properdin) complement pathway. It does not act directly on C3. If C3 is involved in tissue necrosis after ischemic injury, cobra venom factor might reduce tissue damage after acute coronary occlusion. Accordingly, in 14 control dogs occlusion of the left anterior descending artery was carried out for 24 h. Epicardial electrograms were recorded 15 min after occlusion, and 24 h later transmural specimens for creatine phosphokinase activity (CPK) and for histological analysis were obtained from the same sites. In another 14 experimental dogs, 20 U/kg cobra venom factor was given intravenously 30 min after occlusion. Serum complement levels fell within 2-4 h to <20% of normal. In the control dogs, the relationship between ST-segment elevation and CPK activity 24 h later was: log CPK = −0.06 ST + 1.48 (n = 111 specimens, 14 dogs, r = 0.77). In the experimental dogs, log CPK = −0.024 ST + 1.46 (n = 111 specimens, 14 dogs, r = 0.60), showing significantly different slopes (P < 0.001), i.e., less CPK depression for any level of ST-segment elevation. Histologically, 69 of 71 sites (97%) with ST-segment elevation exceeding 2 mV in the control dogs showed signs of necrosis 24 h later, whereas in the experimental group only 43 of 79 sites (54%) with abnormal ST-segment elevations showed signs of necrosis (P < 0.0005). At the same time, it was shown that the administration of cobra venom factor did not alter cardiac performance, collateral blood flow to the ischemic myocardium or the clotting system, but infiltration of polymorphonuclear leukocytes into the myocardium was decreased. It is concluded that cobra venom factor, by reducing the amount of C3 and C5 substrate available for chemotactic factor generation, or other as yet undefined mechanisms, protects the ischemic myocardium from undergoing necrosis, as judged by histology and local CPK activity. Hence, a new approach to limiting the extent of myocardial infarcts after experimental coronary occlusion, based upon inhibition of complement-dependent inflammatory processes, is demonstrated.
Peter R. Maroko, Charles B. Carpenter, Massimo Chiariello, Michael C. Fishbein, Paulo Radvany, James D. Knostman, Sharon L. Hale
Group A streptococci were grown in the presence of [2-3H]glycerol. Concentrated suspensions of the labeled organisms were incubated with and without penicillin. [3H]Glycerol-labeled material accumulated in the supernates in increasing amounts with increasing concentrations of penicillin, ranging from 0 to 50 U/ml. The excretion of labeled material occurred in the absence of nucleic acid synthesis or bacteriolysis indicating that the phenomenon is independent of cell multiplication or decay. The accumulation of label was paralleled by an accumulation of erythrocyte-sensitizing material measured by passive hemagglutination tests for lipoteichoic acid antigen, indicating that a portion of the labeled material possessed the properties of lipoteichoic acid. Culture supernates were fractionated by column chromatography, and the materials obtained were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide, thin-layer chromatography, and paper chromatography. The ability of the same materials to bind to human erythrocytes and epithelial cells was tested. The culture supernate contained lipoteichoic acid, deacylated lipoteichoic acid, glycerol phosphate, and free glycerol. Penicillin caused an increase in the amounts of each of the excreted materials. Streptococci that were stimulated with penicillin to lose their lipoteichoic acid (previously shown to mediate adherence of group A streptococci) lost their ability to adhere to buccal mucosal cells, suggesting that penicillin may influence bacterial ecology by mechanisms other than killing sensitive organisms.
Michael L. Alkan, Edwin H. Beachey
Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.
D W Bilheimer, Y K Ho, M S Brown, R G Anderson, J L Goldstein
Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hank's balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.
R R MacGregor, E J Macarak, N A Kefalides
The present study was undertaken to determine the effect of in vivo hydrocortisone on the kinetics of subpopulations of normal human peripheral blood (PB) thymus-derived (T) cells. Normal volunteers received a single i.v. dose of hydrocortisone, and blood was taken just before, as well as 4, 24, and 48 h after hydrocortisone administration. T cells were purified from each specimen, and proportions and absolute numbers of T lymphocytes bearing receptors for the Fc portion of IgG (T·G) and for the Fc portion of IgM (T·M) were enumerated by rosetting T cells with bovine erythrocytes which had been coated with either antibovine erythrocyte IgG or IgM. 4 h after i.v. administration of hydrocortisone, T·M cells decreased from 52 (±5%) to 23 (±6%) of PB T cells (P < 0.01) and the absolute number of T·M cells decreased from 1,028 (±171) per mm3 to 103 (±23) per mm3 (P < 0.001). In contrast, relative proportion of T·G cells increased from 22 (±4%) to 66 (±7%), while the absolute numbers of T·G cells were essentially unchanged (P > 0.2). In vitro studies involving preincubation of T cells with hydrocortisone before rosette determination of T·G or T·M cells demonstrated that the decrease in absolute numbers of T·M cells did not represent hydrocortisone interference with T·M rosette formation, nor did it represent a switch of T·M cells to T·G cells. Thus, administration of hydrocortisone to normal subjects produces a selective depletion from the circulation of T lymphocytes which possess receptors for the Fc portion of IgM (T·M cells) and of T cells which possess no detectable FC receptor (T·non−M, non−G cells). T·G cells are relatively resistant to the lymphopenic effect of hydrocortisone. These data clearly demonstrate that in vivo corticosteroids have a differential effect on the kinetics of identifiable and distinct subsets of cells in the human T-cell class.
Barton F. Haynes, Anthony S. Fauci
Dopamine is present in the carotid body and has been postulated to be an inhibitory neurotransmitter. The purpose of this study was to determine the effects of dopamine on ventilation in man and to examine its mechanism of action. Dopamine (0.5-10 μg/kg per min) was infused in eight normal men at different levels of arterial chemoreceptor activity, produced by varying the inspired Po2. During normoxia dopamine produced a small decrease in minute ventilation (V̇e) and an increase in arterial Pco2. When arterial chemoreceptors were stimulated by hypoxia, infusion of dopamine produced a marked initial depression of V̇e followed by a sustained although less pronounced decrease in V̇e. An increase in Paco2 and a decrease in Pao2 were also observed. When arterial chemoreceptor activity was suppressed by hyperoxia, infusion of dopamine did not affect ventilation. Subjects also breathed a hypercarbic, hyperoxic gas mixture. The hypercarbia produces hyperventilation by stimulating central chemoreceptors, whereas the hyperoxia suppresses peripheral chemoreceptors. Dopamine did not alter ventilation while the subjects were breathing this gas mixture.
Michael J. Welsh, Donald D. Heistad, Francois M. Abboud
We have explored the effects of ventromedial hypothalamic lesions on the mobilization of free fatty acids in rats exposed to several stresses. The rise in free fatty acids and glycerol in response to norepinephrine had the same time-course and dose-response characteristics in the sham-operated and lesioned animals, indicating comparable degrees of peripheral responsiveness to this hormone. Forced swimming significantly lowered insulin and increased glycerol and free fatty acids more in control than in ventromedial hypothalamic-lesioned rats. During fasting, the rise in glycerol and free fatty acids was smaller in the lesioned rats, but the fall in insulin was greater. Exposure to cold raised fatty acids and glycerol more in the control than in the sham-operated animals, but had no significant effect on plasma insulin or glucose concentration. Injection of 2-deoxyglucose was done on lesioned or control rats with intact or removed adrenal medullas. The rise in free fatty acids and glycerol was less in the lesioned rats than in the controls, and was not affected by adrenodemedullation. The rise in glucose, however, was completely blocked in the adrenodemedullated rats. Changes in insulin were small and not statistically significant. The reduced mobilization of fatty acids from adipose tissue depots after ventromedial hypothalamic injury is consistent with the hypothesis that the ventromedial hypothalamic region serves to modulate activation of the sympathetic nervous system.
Yoshiki Nishizawa, George A. Bray
The aortas of 11 pigs (aged 1-3 yr) with homozygous von Willebrand's disease (vWd) were compared with those of 11 normal pigs of the same ages. Six of the controls exhibited multiple arteriosclerotic plaques with intimal thickening of 63-130 μm. In contrast, none of the pigs with vWd had multiple plaques, and only one had a lesion >2 mm in diameter.
Valentin Fuster, E. J. Walter Bowie, Jon C. Lewis, David N. Fass, Charles A. Owen Jr., Arnold L. Brown
Ingestion of capsules which contained killed Streptococcus mutans by four healthy human subjects led to the appearance of specific antibodies in external secretions. Salivary and lacrymal antibodies were detected within 1 wk of ingestion and continued to increase throughout a 14-day immunization period, with a gradual decline during the 2 ensuing months. A second period of immunization resulted in a pronounced increase of specific antibody levels which occurred earlier than in the primary immunization period and reached peak levels by day 10. No change was detected in serum antibody levels throughout either immunization period. The antibody activity in all secretions was associated with the immunoglobulin A class, as determined by immunochemical analyses. These data indicate that ingestion of bacterial antigens selectively stimulates the immune response in secretions.
J Mestecky, J R McGhee, R R Arnold, S M Michalek, S J Prince, J L Babb
The mechanism of neutropenia in Felty's Syndrome (FS) was tested. The suppressor capacity of mononuclear cells from patients with FS on normal bone marrow granulopoiesis was tested by the in vitro colony forming unit in culture assay. Peripheral blood, bone marrow, and spleen cells from FS patients with marked neutropenia (less than 1,000 neutrophils/mm3) suppressed the colony forming unit in culture of normal bone marrow. Cells from rheumatoid arthritis patients without neutropenia, cells from patients with drug-induced neutropenia without rheumatoid arthritis, or plasma from FS patients failed to suppress the colony forming unit in culture. Though suppressor cells were predominantly thymus-derived (T) cells, monocytes were also effective in suppression. The suppressor efficiency of cells from the various compartments were spleen greater than bone marrow greater than peripheral blood. Splenectomy in FS transiently corrected the neutropenia and eliminated suppressor cell activity. Hyperactive suppressor cells may be responsible for the neutropenia in some patients with FS. Correction of neutropenia in these patients should be directed at modulating the suppressor cell subpopulation.
N I Abdou, C NaPombejara, L Balentine, N L Abdou
The effects of hypotensive hemorrhage (HH) on renal hemodynamics and plasma renin activity (PRA) during prostaglandin (PG) synthesis inhibition were examined in three groups of dogs. In each group of animals arterial blood pressure was lowered by a 30% decrement. In the first group of eight control animals, HH was not associated with a significant change in glomerular filtration rate (GFR, 42-36 ml/min, NS); renal blood flow (RBF) declined significantly, from 234 to 171 ml/min, P < 0.05. In the second group of eight animals, pretreated with RO 20-5720 (RO, 2 mg/kg), a competitive inhibitor of PG synthesis, HH was associated with a significant fall in GFR (43-17 ml/min, P < 0.001) and RBF (195-89 ml/min, P < 0.001). In the third group of eight animals, pretreatment with indomethacin (IN, 10 mg/kg), a chemically dissimilar PG inhibitor, HH was also associated with a significant fall in GFR (38-8 ml/min, P < 0.001) and RBF (150-30 ml/min, P < 0.001). Renal denervation attenuated this renal ischemic effect of HH in the presence of PG inhibition. In the RO group, GFR (34 vs. 17 ml/min, P < 0.005) and RBF (145 vs. 89 ml/min, P < 0.025) were significantly greater in denervated vs. innervated kidneys during HH. Similarly, in animals treated with IN, a significantly higher GFR (28 vs. 8 ml/min, P < 0.005) and RBF (101 vs. 30 ml/min, P < 0.005) occurred in denervated as compared to innervated kidneys during HH. With HH, the increase in PRA in the control group (3.34-11.68 ng/ml per h, P < 0.005) was no different than that observed in the RO group (4.96-18.9 ng/ml per h, P < 0.001) or IN group (4.71-17.8 ng/ml per h, P < 0.001). In summary, the present results indicate that renal PG significantly attenuate the effect of HH to decrease GFR and RBF. Furthermore, renal denervation exerts a protective effect against the enhanced renal ischemic effects which occur in the presence of PG inhibition during HH. Finally, PG inhibition does not alter the effect of HH to cause an increase in PRA.
William L. Henrich, Robert J. Anderson, Arnold S. Berns, Keith M. McDonald, Penny J. Paulsen, Tomas Berl, Robert W. Schrier
Assay conditions have been developed for the determination of urinary beta-glucuronidase, beta-galactosidase, alpha-galactosidase, and beta-hexosaminidase using fluorometric substrates. The assay conditions for beta-glucuronidase overcome interference by both low and high molecular weight inhibitors, a problem that has confused earlier studies of enzyme excretion. The four lysosomal enzymes are excreted corrdinately: although their absolute levels (in units per milligram of creatinine) vary during the day and from one day to the next, the ratio of one enzyme to another remains relatively constant. The lack of correlation betweem plasma and urine enzyme levels, together with the high molecular weights of these enzymes, suggests that the urinary enzymes are not derived by glomerular filtration. The lack of coordinacy with lactate dehydrogenase suggests they are not derived from exfoliated cells. by analogy with experimental animals, they may be derived from lysosomes extruded into the lumen of the proximal tubule by epithelial cells. There is considerable variation among a population of 125 healthy adult subjects for total enzyme excretion. Both total enzyme excretion and coordinacy ratios are log-normally distributed, suggesting that they are the resultants of many factors, each of which has a relative, or proportional, effect on enzyme excretion. About one-half the population variation resides in a process common to the excretion of all four enzymes (possibly the lysosome extrusion pathway), and about one-half resides in factors affecting each enzyme independently.
K Paigen, J Peterson
Plasma from some individuals contains substances which are reactive with glucagon antiserum, are larger than 3,500-dalton glucagon, and have been proposed as possible precursors of glucagon. We have evaluated three generations of a kindred in which 9 of 15 members evaluated had elevated plasma levels of large molecular weight immunoreactive glucagon (L-IRG) with an average concentration of 822 pg/ml. The distribution of individuals with elevated L-IRG levels in this pedigree is consistent with autosomal dominant inheritance. Gel filtration of plasma revealed that all affected family members had excessive amounts of two L-IRG peaks, one with a molecular weight of approximately 9,000 daltons and another in the 10,000 to 20,000-dalton range. Oral glucose tolerance tests were nondiabetic and elicited a fall in L-IRG levels, whereas L-IRG concentrations rose dramatically during the infusion of arginine. These L-IRG species may be precursors of 3,500-DALTON GLUCAGON AND MAY BE ELEVATED in this kindred because of an inherited defect in either their synthesis or degradation.
J P Palmer, P L Werner, J W Benson, J W Ensinck
A peptide of approximately 300-400 daltons exhibiting in vitro chemotactic activity for human polymorphonuclear (PMN) leukocytes, with a preference for the eosinophil series, was isolated from extracts of anaplastic lung carcinomas of the large squamous cell type obtained from three patients with marked peripheral blood hypereosinophilia and eosinophilic infiltration of the tumors and surrounding normal pulmonary tissues. This chemotactic factor was termed ECF-LSC (eosinophil chemotactic factor of lung squamous cell carcinoma). ECF-LSC appeared in the urine of two of the patients in increasing quantities late in the course of their disease and was also elaborated by long-term cultures of dispersed tumor cells from the same two patients. Three anaplastic large cell bronchogenic carcinomas which were not associated with tumor tissue or peripheral blood eosinophilia, a bronchogenic adenocarcinoma from a patient with only peripheral eosinophilia, and a renal cell carcinoma metastatic to the lungs and associated with transient pleural tissue and fluid eosinophilia were all devoid of ECF-LSC. ECF-LSC from tumor tissue extracts, urine, and tumor cell culture medium was comparable to the mast cell-associated tetrapeptides of the eosinophil chemotactic factor of anaphylaxis (ECF-A) in size, but eluted from Dowex-1 at pH 5.0-3.5 in contrast to the more acidic ECF-A tetrapeptides which eluted at pH 3.2-2.2 ECF-LSC, like the tetrapeptides of ECF-A, had a secondary chemotactic activity for neutrophil PMN leukocytes, but not mononuclear leukocytes, and deactivated both eosinophil and neutrophil PMN leukocytes so that they would not respond to a subsequent in vitro chemotactic stimulus. Eosinophils from the two patients with urinary excretion of ECF-LSC and the highest concentrations in tumor extracts were hyporesponsive in vitro to homologous and heterologous chemotactic stimuli, suggesting that ECF-LSC had deactivated the eosinophils in vivo.
Edward J. Goetzl, Armen H. Tashjian Jr., Robert H. Rubin, K. Frank Austen
The effect of efferent, parasympathetic stimulation upon pancreatic polypeptide (PP) secretion was studied in three ways: (a) Plasma PP concentrations increased in response to insulin-induced hypoglycemia in both normal subjects, from 11 pM (9.5-12.5) to 136 pM (118-147), n = 8 (median and interquartile range) and in duodenal ulcer patients, from 33 pM (21-52) to 213 pM (157-233), n = 7. The PP response to hypoglycemia was diminished by atropine in normal subjects (P < 0.005) and completely abolished by vagotomy in the duodenal ulcer patients. (b) Electrical stimulation, 8 Hz, of the vagal nerves in anesthetized pigs induced an increase in portal PP concentrations within 30 s from 32 pM (28-39) to 285 pM (248-294), n = 12. Minimal stimulatory frequency was 0.5 Hz and maximal stimulatory frequency 8-12 Hz. Atropine inhibited the PP response to electrical stimulation. Median inhibition with 0.5 mg of atropine/kg body wt was 74%, range 31-90%, n = 6. The response was eliminated by hexamethonium. Adrenergic alpha and beta blockade did not influence the release of PP in response to vagal stimulation. (c) Acetylcholine stimulated, in a dose-dependent manner, the secretion of PP from the isolated perfused porcine pancreas, half-maximal effective dose being 0.19 μM; maximal PP output in response to 5 min stimulation was 228 pmol, range 140-342 pmol, n = 5. Atropine completely abolished this response.
T. W. Schwartz, J. J. Holst, J. Fahrenkrug, S. Lindkær Jensen, O. V. Nielsen, J. F. Rehfeld, O. B. Schaffalitzky de Muckadell, F. Stadil
Exposure to supralethal total body irradiation and transplantation of bone marrow from a DLA- and pedigree-identical donor have regularly produced successful engraftment and the establishment of stable long-term chimerism in beagles of the Cooperstown colony. Bone marrow allografts performed in pairs of dogs bearing identical DLA haplotypes derived from different pedigree origins (i.e., different classes of the same haplotype) yielded two different results. Depending upon the particular haplotype pedigree combination used, such transplants either led to long-term chimerism or to failures of engraftment, secondary disease, and death of the recipients (i.e., pedigree-incompatible combinations).
F. T. Rapaport, R. J. Bachvaroff, K. Watanabe, H. Hirasawa, N. Mollen, J. W. Ferrebee
The renal handling of oxalate was examined by free-flow micropuncture, intratubular microinjection, and droplet precession techniques in the rat. After the sustained i.v. infusion of [14C]oxalate, the fractional delivery of oxalate from the early portions of the proximal tubule was 120.1±4.4%, indicating net secretion. Fractional delivery rates from the late proximal tubule (124.6±6.1), distal tubule (120.9±2.9), and final urine (126.2±2.9%) were not different from that of the early proximal tubule. Direct intratubular microinjections of oxalate into the early proximal tubule and late proximal tubule yielded urinary recovery rates of 85±3% and 101±2%, respectively, suggesting that oxalate absorption does occur in the mid-portions of the proximal tubule. Droplet precession studies confirmed a secretory flux for oxalate. In contrast to oxalate, para-aminohippurate (PAH), the more traditional marker for organic acid transport, was secreted in the late portions of the proximal tubule and in large measure at a site between the late proximal and distal tubules, presumably the pars recta. Probenecid inhibited PAH secretion but was without effect on net oxalate transport, oxalate absorption, or oxalate secretion.
E. J. Weinman, S. J. Frankfurt, A. Ince, S. Sansom
Human platelets and platelet particulate fractions were found to emit a burst of chemiluminescence during incubation with arachidonic acid. The magnitude of light emission was directly related to the number of platelets in the reaction mixture and varied little for the same individual from day to day. The chemiluminescence response of platelets was localized to the particulate fraction and was almost totally oxygen dependent. In addition to arachidonate, seven other polyunsaturated fatty acids, including several that are not prostaglandin precursors, also induced platelet chemiluminescence.
Elaine L. Mills, Jonathan M. Gerrard, Dana Filipovich, James D. White, Paul G. Quie
We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.
S E Lux, K M John, T E Ukena
The mixed lymphocyte reaction (MLR) is the proliferative response of one individual's lymphocytes cultured in the presence of another individual's lymphocytes. In man, the MLR is elicited by cell surface antigens coded for by the HLA-D gene locus. This locus is among a cluster of genes which are located on the sixth chromosome and which include genes coding for the major histocompatibility antigens HLA-A, B, and C as well as HLA-D. If the stimulator cell possesses D locus antigens not present in the responder, the lymphocytes of the latter will undergo blast transformation resulting in DNA synthesis which can be measured. A vigorous response in the MLR to allogeneic cells is the rule among healthy individuals.
Edgar G. Engleman, Hugh O. McDevitt
Both the isolated perfused rabbit heart and kidney are capable of synthesizing prostaglandin (PG) I2. The evidence that supports this finding includes: (a) radiochemical identification of the stable end-product of PGI2, 6-keto-PGF1α, in the venous effluent after arachidonic acid administration; (b) biological identification of the labile product in the venous effluents which causes relaxation of the bovine coronary artery assay tissue and inhibition of platelet aggregation; and (c) confirmation that arachidonic acid and its endoperoxide PGH2, but not dihomo-γ-linolenic acid and its endoperoxide PGH1, serve as the precursor for the coronary vasodilator and the inhibitor of platelet aggregation. The rabbit heart and kidney are both capable of converting exogenous arachidonate into PGI2 but the normal perfused rabbit kidney apparently primarily converts endogenous arachidonate (e.g., generated by stimulation with bradykinin, angiotensin, ATP, or ischemia) into PGE2; while the heart converts endogenous arachidonate primarily into PGI2. Indomethacin inhibition of the cyclo-oxygenase unmasks the continuous basal synthesis of PGI2 by the heart, and of PGE2 by the kidney. Cardiac PGI2 administration causes a sharp transient reduction in coronary perfusion pressure, whereas the intracardiac injection of the PGH2 causes an increase in coronary resistance without apparent cardiac conversion to PGI2. The perfused heart rapidly degrades most of the exogenous endoperoxide probably into PGE2, while exogenous PGI2 traverses the heart without being metabolized. The coronary vasoconstriction produced by PGH2 in the normal perfused rabbit heart suggests that the endoperoxide did not reach the PGI2 synthetase, whereas the more lipid soluble precursor arachidonic acid (exogenous or endogenous) penetrated to the cyclooxygenase, which apparently is tightly coupled to the PGI2 synthetase.
Philip Needleman, Sue D. Bronson, Angela Wyche, Mark Sivakoff
The turnover and the catabolic fate of the B apoprotein of very low density lipoprotein (VLDL-B) was studied in 15 normal and hyperlipidemic subjects using reinjected autologous VLDL labeled with radioiodine. The specific radioactivity-time curve of the B apoprotein in total VLDL (Sf20-400) was multiexponential but conformed to a two-pool model during the first 48 h of catabolism. The flux was highest in several hypertriglyceridemic subjects. The mass of pool A exceeded the intravascular content of VLDL-B by 30% on average, indicating extravascular metabolism of VLDL. The two-pool model might reflect the input of several populations of particles or heterogeneity of catabolic processes or pools. The flux of B apoprotein was also measured in several subclasses of VLDL, in smaller intermediate density lipoproteins, and in low density lipoproteins (LDL). In three subjects the flux was similar in Sf 60-400 and in Sf 12-60 lipoproteins, suggesting that VLDL was catabolized at least to a particle in the density range Sf 12-60. Subsequent catabolism appeared to proceed by two pathways: in normotriglyceridemic subjects, B apoprotein flux in the Sf 20-400 and in Sf 12-20 lipoproteins was similar, whereas in hypertriglyceridemic subjects flux through Sf 12-20 accounted for only part of the VLDL-B flux.
Michael F. Reardon, Noel H. Fidge, Paul J. Nestel
Platelets from two patients with Bernard-Soulier disease showed a reduction in their ability to bind human thrombin. Thrombin binding studies in the high affinity range showed 1,500 sites for the Bernard-Soulier platelets as against 4,000 for normal controls. However, the dissociation constant was the same for both normals and patients (4.4 nM) indicating identical affinity for thrombin at the available sites. In the low affinity range, the Bernard-Soulier platelets showed 8,800 thrombin binding sites as against 24,000 for the controls, but again with identical values of Kd (37 nM). In addition, platelets from these Bernard-Soulier patients showed a decreased rate of aggregation with thrombin at both optimal (300 mU/ml) and suboptimal (60 and 120 mU/ml) thrombin concentrations. The decreased amount of thrombin which can bind to Bernard-Soulier platelets and the decrease in thrombin-induced aggregation may partly explain the hemostatic defect in these patients. In addition, the identical ratios of high affinity and low affinity binding sites in normals and in patients (0.37 and 0.36, and 0.36, respectively) supports the idea of a single class of binding sites for thrombin on the platelet surface.
G A Jamieson, T Okumura