Showing papers in "American Journal of Physiology in 1987"

Role of neutrophils in ischemia-reperfusion-induced microvascular injury

Abstract: Recent studies indicate that polymorphonuclear neutrophils (PMNs) infiltrate the intestinal mucosa during ischemia and after reperfusion. To determine whether PMNs mediate the increased microvascular permeability produced by ischemia-reperfusion (I/R) we treated cats with either saline, antineutrophil serum (ANS), or a monoclonal antibody specific for the beta-chain of the CD18 complex (MoAb 60.3) that prevents neutrophil adherence and extravasation. Intestinal microvascular permeability to plasma proteins was measured in control preparations (0.08 +/- 0.007), in preparations subjected to 1 h of ischemia then reperfusion (I/R, 0.32 +/- 0.02), I/R preparations treated with ANS (0.13 +/- 0.01), and I/R preparations treated with MoAb (0.12 +/- 0.003). Our results indicate that both PMN depletion (to less than 10% control) and prevention of PMN adherence significantly attenuate the increased microvascular permeability induced by I/R. These findings, coupled to previous results obtained from this model, support the hypothesis that neutrophils, which accumulate in the mucosa in response to xanthine oxidase activation, mediate the oxyradical-dependent injury produced by reperfusion of the ischemic bowel.

Cadmium inhibition of Ca2+ uptake in rainbow trout gills

Abstract: The effects of cadmium (Cd2+) on calcium (Ca2+) transport in the gills of rainbow trout (Salmo gairdneri) were studied. The gill epithelium of freshwater fish represents a model for a Ca2+-transporting tight epithelium. Unidirectional Ca2+ fluxes in the gills were estimated in an isolated saline-perfused head preparation. Ca2+ influx was not affected when up to 10 microM Cd were added to the ventilatory water at the start of flux determinations (in vitro exposure). However, after 16 h in vivo preexposure of the fish to 0.1 microM Cd in the water, a 79% inhibition of Ca2+ influx was observed. Ca2+ efflux was not affected when up to 10 microM Cd were added to the ventilatory water during the flux determination. Ca2+ efflux in fish preexposed to 0.1 microM Cd for 16 h was also not affected; a preexposure to 1 microM Cd, however, resulted in a 173% increase in Ca2+ efflux rates. Tracer retention in the gill tissue indicated that both Ca2+ and Cd2+ enter the gill epithelium via a lanthanum (La3+)-inhibitable pathway. It is concluded that Cd2+ readily enters the branchial epithelial cells, similarly as Ca2+ does via La3+-sensitive apical Ca2+ channels. The inhibitory action of Cd2+ on transepithelial Ca2+ influx seems to result from an inhibition of the basolateral Ca2+ transport, occurring after a critical intracellular Cd2+ concentration has been reached.

Formation and release of purine catabolites during hypoperfusion, anoxia, and ischemia.

Abstract: To answer some of the as yet unresolved questions about the formation, metabolism, and release of purine catabolites in hypoxic myocardium, we compared their release from isolated rabbit hearts during hypoperfusion, anoxia, and after ischemia, with and without nucleoside-transport inhibition Results provide evidence to suggest the following Besides the supply-to-demand ratio for O2, other factors may affect the formation of adenosine The myocyte is the major source of purine catabolites Adenosine is not produced within the myocyte Once in the interstitium, adenosine is (largely) taken up in the endothelial cells, where it is catabolized to inosine and hypoxanthine and released that way into the lumen Some of the adenosine can reach the lumen unchanged through clefts Nucleoside transport inhibition prevents the escape through the endothelial cells and thus the formation and release of inosine and hypoxanthine As a consequence, more adenosine will accumulate in the interstitium and more will reach the lumen through the clefts The pathway, as proposed, explains the long known paradox of increased extracellular levels of adenosine after inhibition of nucleoside transport

Role of adenosine in regulation of cerebral blood flow: effects of theophylline during normoxia and hypoxia.

Abstract: We studied the effects of the methylxanthine theophylline, an adenosine receptor blocker, on cerebral circulation. Cerebral blood flow (CBF) was measured by the retroglenoid outflow and microsphere techniques, and pial circulation changes were observed through a closed cranial window. Intraperitoneal administration of theophylline in normoxic animals resulted in a biphasic response of pial vessels and CBF. At low concentrations (0.05 mumol/g) of theophylline, pial vessel diameter and CBF decreased, whereas vasodilatation and hyperemia were observed at higher levels. After intraperitoneal administration of either 0.05 or 0.2 mumol/g, hypoxic hyperemia was attenuated both during short (c. 30 s) and sustained (c. 2-3 min) hypoxia, as was hypoxic pial arteriolar vasodilatation. These actions of theophylline appear to be due to adenosine receptor blockade, since micromolar concentrations were achieved in cerebrospinal fluid (CSF), and no increases in adenosine 3',5'-cyclic monophosphate concentrations in brain were noted. Moreover, theophylline (either intraperitoneal or topical) blocked pial vasodilatation caused by topically applied adenosine, but had little effect on hypercarbic hyperemia or pial vasodilatation induced by topically applied acetylcholine. The results of these studies suggest that adenosine is involved in the maintenance of resting cerebral vascular tone and has a paramount role in the regulation of CBF during hypoxia.

Volume regulatory loss of Na, Cl, and K from rat brain during acute hyponatremia

Abstract: This study quantitatively evaluates the contribution of tissue Na, Cl, and K loss to brain volume regulation during acute dilutional hyponatremia (DH) and examines the mechanism of Na loss. DH was produced in pentobarbital sodium-anesthetized rats by intraperitoneal infusion of distilled water and brain water and electrolytes analyzed 30 min, 1 h, 3 h, 4 h, or 6 h later. The rate of Na and Cl loss was greatest during the first 30 min of DH (0.43 and 0.47 meq X kg tissue dry wt-1 X min-1, respectively). Net loss of Na and Cl was maximal after 3 h of DH. K loss was slower, achieving significance after 3 h. Electrolyte loss was sufficient to account for observed brain volume regulation after three or more hours of DH. Measurements of 22Na influx and efflux across the blood-brain barrier showed that barrier permeability to Na is unchanged during DH. Analysis of results using a two-compartment model of plasma-brain exchange suggests that loss of brain Na during DH does not result solely from a shift of electrolyte across the blood-brain barrier to plasma, and thus provides indirect evidence for an additional pathway for Na loss, presumably via cerebrospinal fluid.

Blunted natriuresis to atrial natriuretic peptide in chronic sodium-retaining disorders.

Abstract: Renal responses to atrial natriuretic peptide were examined in conscious dogs with congestive heart failure (tricuspid insufficiency) and in conscious rats with nephrotic syndrome (adriamycin). Heart-failure dogs displayed elevated atrial pressure and heart weights, blunted natriuresis to a saline load, and ascites. Nephrotic rats displayed proteinuria, hypoproteinemia, sodium retention, and ascites. In control animals, atrial natriuretic peptide increased absolute and fractional urine flow rate and urinary sodium excretion. Although atrial natriuretic peptide increased absolute and fractional urine flow rate and urinary sodium excretion in conscious heart-failure dogs and nephrotic rats, the responses were markedly blunted. In heart-failure dogs, infusion of atrial natriuretic peptide increased plasma concentrations of norepinephrine and epinephrine. In nephrotic rats, renal denervation reversed the blunted diuretic and natriuretic responses to atrial natriuretic peptide. Mean arterial pressure, glomerular filtration rate, and p-aminohippurate clearance were affected similarly by atrial natriuretic peptide in heart-failure dogs or nephrotic rats vs. control animals. Conscious congestive heart-failure dogs and conscious nephrotic rats have blunted diuretic and natriuretic responses to atrial natriuretic peptide.

Temporal relation between energy metabolism and myocardial function during ischemia and reperfusion.

Abstract: The purpose of the present investigation was to study the relation between energy metabolism and contractile function in the isovolumic guinea pig heart. 31P nuclear magnetic resonance spectroscopy...

Stretch-activated potassium channels in renal proximal tubule.

Abstract: A short open-time potassium (K) channel that has previously been identified in the basolateral membrane of Necturus proximal tubule (17) is activated by membrane stretch. Application of between 12 and 20 cmH2O negative pressure to the patch pipette reversibly increases mean number of open basolateral K channels (NP0) by a factor of 5.3 +/- 2 in cell-attached patches (n = 4) and a factor of 13.7 +/- 5 in excised patches (n = 8). This stretch activation does not alter channel selectivity or conductance and depends on neither the direction of K current nor the orientation of the patch ("inside-out" vs. "outside-out"). The increase in NP0 occurs within seconds after applying negative pressure to the patch and is proportional to applied negative pressure. Stretch activation of the basolateral potassium channel may play an important role in proximal tubule cell volume regulation. For example, if swelling stretches the basolateral membrane, the resulting increase in NP0 could restore cell volume by loss of K (with an accompanying anion) followed by osmotic exit of water.

Interleukin-1 decreases renal sodium reabsorption: possible mechanism of endotoxin-induced natriuresis.

Abstract: Administration of pyrogen or endotoxins such as Escherichia coli lipopolysaccharide can elicit a marked increase in urinary sodium excretion. This response occurs without any elevation in the filtered load of sodium and it does not appear to be prostaglandin mediated. The various effects produced by endotoxins appear to have interleukin-1 as a common mediator. In the present work, we have studied whether human recombinant interleukin-1 beta (hrIL-1) could affect the renal handling of sodium and thus, could be implicated in natriuretic response to pyrogens or endotoxins. We observed that hrIL-1 intravenously injected into conscious rats provokes a marked increase in sodium excretion. This natriuretic response was not associated with any increase in glomerular filtration rate (clearance of [3H]inulin), nor was it accompanied by significant changes in the urinary excretion of potassium, calcium, or inorganic phosphate. The only concomitant alteration was a decrease in urinary pH. Pretreatment with indomethacin abolished the effect of hrIL-1 on urinary pH but did not modify the natriuretic response. In conclusion, hrIL-1 elicits a selective decrease in tubular sodium reabsorption, which does not appear to involve a change in prostaglandin synthesis. This observation strongly suggests that interleukin-1 could be a key mediator in endotoxin-induced natriuresis.

Description of LV pressure-volume relations by time-varying elastance and source resistance.

Abstract: If the left ventricle (LV) behaves as a time-varying elastance [E(t)] that is independent of load, then definition of E(t) during normal ejecting beats should permit accurate prediction of LV pressure (LVP) during a maximally afterloaded (isovolumic) beat. We tested this hypothesis in six dogs preinstrumented to measure LVP and aortic flow (Q) and to determine LV volume (V) from three dimensions. LVP and V were varied by caval occlusions. These data were used to determine E(t) and minimal volume required to generate pressure (Vo) at 10-ms intervals during systole using a simple E(t) model, P(t) = E(t) [V(t)-Vo], where P(t) is LVP at any time after the onset of contraction, and V(t) is the LV volume at t. LVP was measured during isovolumic beats generated by sudden balloon occlusion of the ascending aorta. The simple E(t) model accurately predicted isovolumic LVP during the first 70 ms of systole (r = 0.99) and also the end-systolic LVP but underestimated LVP during midsystole by 48 +/- 5 (SD) mmHg (P less than 0.05). When a pressure-dependent source resistance (K = 0.0015 s/ml) was added to the model to reduce LVP in proportion to Q, such that P(t) = E(t) [V(t)-Vo] X [1 - KQ]), LVP during the isovolumic beat was accurately predicted throughout systole (r = 0.99). However, the time to develop peak isovolumic pressure was 22 +/- 7 ms less than predicted. Similar results were obtained during inotropic stimulation with dobutamine in five animals.

Effects of adenosine infusion into renal interstitium on renal hemodynamics.

Abstract: This study was designed to investigate the hemodynamic effects of exogenous adenosine in the interstitium of the rat kidney. Adenosine or its analogues were infused into the renal interstitium by means of chronically implanted capsules. Infusion of adenosine (bolus 0.5 mumol plus 0.1 mumol/min) decreased glomerular filtration rate (GFR) from 0.81 +/- 0.06 (mean +/- SE) to 0.37 +/- 0.06 ml/min while having no effect on renal blood flow (RBF). The metabolically stable analogue, 2-chloradenosine (2-ClAdo), (bolus 10 nmol plus 2 nmol/min) decreased GFR from 0.73 +/- 0.07 to 0.21 +/- 0.06 ml/min. Interstitial infusion of theophylline, an adenosine receptor antagonist, completely abolished the effects of adenosine and 2-ClAdo on GFR. The distribution of adenosine, when infused into the renal interstitium, was determined using radiolabeled 5'-(N-ethyl)-carboxamidoadenosine (NECA), a metabolically stable adenosine agonist. After continuous infusion, [3H]NECA was distributed throughout the kidney. The effects of NECA to reduce GFR were similar to those of adenosine and 2-ClAdo. We conclude that increased levels of adenosine in the renal interstitium markedly decrease GFR without affecting RBF in steady-state conditions. The marked effects of adenosine agonists during their infusion into the renal interstitium and the complete blockade of these effects by theophylline suggest an extracellular action of adenosine.

Electrogenic Na/HCO3 cotransport across basolateral membrane of isolated perfused Necturus proximal tubule

Abstract: This study was undertaken to determine whether the proximal tubule of the mud puppy Necturus maculosus possesses a basolateral Na/HCO3 cotransporter. We examined the effects on basolateral membrane...

Cimetidine transport in isolated luminal membrane vesicles from rabbit kidney.

Abstract: Experiments were conducted to study the transport of the histamine H2-receptor antagonist, cimetidine, in luminal membrane vesicles prepared from rabbit renal cortex. Cimetidine accumulated in the vesicles with time. Cimetidine uptake was sensitive to changes in vesicle size, suggesting that the compound is transported into an osmotically reactive intravesicular space. Its rate of uptake could be described by both a saturable and a nonsaturable process. The Km was 4.6 +/- 4.0 microM and the Vmax was 6.8 +/- 2.3 pmol X s-1 X mg protein-1 (mean +/- SD, n = 4). N1-methylnicotinamide (NMN), cimetidine, cimetidine sulfoxide, and ranitidine inhibited the uptake of cimetidine. Cimetidine uptake in the presence of an outwardly directed proton gradient was enhanced in vesicles preloaded with a higher concentration of unlabeled cimetidine (2.4 X 10(-4) M). An outwardly directed proton gradient enhanced the uptake of cimetidine to values exceeding its equilibrium accumulation. Uptake stimulated in this way could be inhibited by the cation, NMN, the bases, ranitidine, and cimetidine sulfoxide, and interestingly, by the anion, probenecid. The effect of probenecid did not appear to be due to nonspecific effects on membrane binding, membrane potential, or vesicle size. These data are consistent with data obtained in isolated perfused proximal tubules, demonstrating that probenecid inhibits cimetidine transport. The data in this study suggest that the effect of probenecid on cimetidine transport specifically involves the transporter in the luminal membrane.

Intramyocardial blood volume change in first moments of cardiac arrest in anesthetized goats.

Abstract: The effect of cardiac relaxation on the intramyocardial blood volume was studied by measuring the integrated difference between arterial inflow and great cardiac venous outflow. In nine anesthetized goats, the left main coronary artery was perfused under constant pressure. The great cardiac vein was drained under pressure control. The venous flow signal was amplified so that the integrated intramyocardial blood volume was constant in the beating heart. With normal vasomotor tone, the mean change in vascular volume was 3.01 +/- 0.18 (SE) ml/100 g left ventricle (LV); 67% of the volume change was achieved in 1.60 +/- 0.09 s. For the fully dilated bed (adenosine infusion), the values were 4.13 +/- 0.33 ml/100 g and 0.96 +/- 0.06 s, respectively. The volume change could be correlated with the venous pressure during cardiac arrest (Pvd) and the change in mean left ventricular pressure after cardiac arrest (r = 0.95). The correlation improved when data were selected for Pvd less than 6 mmHg to r = 0.98. We assumed that the change in vascular transmural pressure can be approximated as half the mean left ventricular pressure change. The intramyocardial vascular compliance was then estimated as 0.104 +/- 0.012 and 0.146 +/- 0.028 ml X mmHg-1 X 100 g-1 for control and adenosine conditions, respectively. The long time constants excluded the large epicardial veins as the site of volume change; they were much longer than the duration of diastole in the beating heart. We conclude that the intramyocardial vascular compartment is capable of volume expansion on the order of 20% of its normal volume when myocardial compression by ventricular systole is suspended.

Effects of PDGF on inositol phosphates, Ca2+, and contraction of mesangial cells.

Abstract: Platelet-derived growth factor (PDGF) is a potent mitogen and vasoactive polypeptide for aortic smooth muscle. Because contractile glomerular mesangial cells synthesize a PDGF-like molecule and may respond to PDGF released by infiltrating cells at the site of glomerular inflammation, we studied the effects of exogenous, highly purified PDGF on 1) contraction of cultured rat mesangial cells and 2) membrane phosphoinositide turnover and cytosolic free calcium ([Ca2+]i), as putative mechanisms of membrane signal transduction. PDGF, 10(-11) and 10(-10) M, contracted 56.1 +/- 5.2 and 72.9 +/- 6.4% of the cells, respectively, with an average decrease of cross-sectional area of 22.0 +/- 2.6 and 28.1 +/- 2.7% of basal, as assessed by image-analysis microscopy. PDGF also rapidly increased total water-soluble inositol phosphates, measured after anion-exchange chromatography on perchloric acid-extracted cells, and simultaneously raised [Ca2+]i, measured by the fluorescent intracellular probe fura-2, from basal levels of 83.1 +/- 6.8 to a peak of 229.4 +/- 20.0 nM. We conclude that PDGF stimulates contraction of rat mesangial cells via a phospholipase C-dependent pathway, with potential relevance to the control of glomerular hemodynamics and mesangial proliferation in immune-mediated glomerular disease.

Reconstructing the rate of appearance of subcutaneous insulin by deconvolution.

Abstract: In this paper a deconvolution scheme is presented to reconstruct the rate of appearance of subcutaneously injected insulin. Relevant aspects of experiment design are briefly described. Intravenous insulin kinetics are modeled to determine the impulse response of the system. The deconvolution problem is not ill conditioned and is solved using a least-squares method without imposing constraints on the input. An estimate of the error of the reconstructed input is provided. The reliability of the deconvolution scheme is tested by means of an independent validation study. Finally, the different sources of error that affect the method are discussed, and a figure of the global error is derived.

Faster ribosome synthesis induced by elevated aortic pressure in rat heart

Abstract: An increase in aortic pressure from 60 to 120 mmHg accelerated ribosomal protein synthesis in rat hearts during 1 or 2 h of labeling with 0.4 mM [3H]phenylalanine. When hearts were perfused with buffer that contained 20 mM glucose and normal plasma concentrations of 19 other amino acids without added insulin, ribosomal protein synthesis relative to the rate of total protein synthesis increased from approximately 0.22 to 0.36 and 0.30 as aortic pressure was raised from 60 to 120 mmHg during 1 or 2 h of labeling, respectively. With the addition of insulin, the relative rate of ribosomal protein synthesis averaged 0.33 at an aortic pressure of 60 mmHg and increased to 0.42 when aortic pressure was raised to 120 mmHg. These results indicate that elevation of aortic pressure has a preferential effect on synthesis of new ribosomes. This response appears to be an early and physiologically significant event in cardiac hypertrophy.

Electrolyte transport in a central core model of the renal medulla

Abstract: Transport of Na+, K+, Cl-, urea, and water is described in a central core model of the renal medulla. Equations for mass balance, Poiseuille flow, and the Nernst-Planck equation describe the continuous behavior of the system along the medullary axis and along the distal nephron; the Kedem and Katchalsky phenomenology describes passive transmural transport; active transmural transport obeys Michaelis-Menten kinetics. Numerical solution of the differential equations shows that to a close approximation any combination of active Na+ and active Cl- transport can generate the same concentration profiles but will generate very different potential profiles, and consequently, very different K+ absorption in thick ascending limb of Henle's loop. If a net transport stoichiometry of 2 Cl- ions to 1 Na+ ion is selected for the pumps, an active Cl- transport rate of approximately 10,000 peq.s-1.cm-2 gives K+ and Na+ concentrations in early distal nephron and a medullary osmolality profile in reasonable agreement with experimental data.

Influence of apolipoprotein E on soluble and heparin-immobilized hepatic lipase.

Abstract: The effect of human apolipoprotein E (apoE), either alone or in combination with apoC, on the lipolysis of a radiolabeled triglyceride emulsion was studied with hepatic lipase in solution and immobilized on heparin-Sepharose. The soluble hepatic lipase was inhibited, whereas the heparin-immobilized lipase was stimulated by apoE. This stimulation was attenuated by combining apoE with either apoC-II or C-III. The heparin-immobilized lipase demonstrated much less lipolysis of the zwitterionic phosphatidylcholine-stabilized triglyceride emulsion than did the soluble enzyme. This difference was less when the emulsion was stabilized by a nonionic detergent. apoE inhibited lipase activity when assayed under conditions (0.4 M NaCl) of bound enzyme and unbound substrate. Increasing the emulsion apoE content beyond optimum inhibited lipolysis by the immobilized enzyme. Kinetic analysis of phosphatidylcholine-stabilized triglyceride emulsions revealed a significant decrease in immobilized enzyme Km and an increase in Vmax when the emulsion was supplemented with apoE. Distributing the immobilized lipase in clustered aggregates produced more lipolysis than when the same enzyme content was uniformly bound.

Sulfate-bicarbonate exchange in brush-border membranes from rat renal cortex.

Abstract: Under Na+-free conditions, SO4(2-) uptake by rat renal brush-border membrane (BBM) vesicles could be driven by imposition of a HCO3- gradient (in greater than out). The initial rate of SO4(2-) uptake was stimulated 10-fold, and peak overshoot exceeded equilibrium uptake by 2-3 times. Cl-, SCN-, NO3-, I-, and OH- were able to substitute for HCO3-. Divalent anions, including SO4(2-) itself, were less effective as counterions. Similarly, in the absence of Na+,SO4(2-) efflux was stimulated most effectively by the monovalent ions. HCO3- -SO4(2-) exchange was cis-inhibited by disulfonic stilbenes [4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)], phloretin, Hg, and S2O3(2-). HCO3- driven SO4(2-) uptake was saturable, with an apparent Km of 0.4 mM for SO4(2-). Simultaneous imposition of Na+ (out greater than in) and HCO3- (in greater than out) gradients produced approximately additive stimulation of SO4(2-) uptake. The HCO3- -driven component of SO4(2-) uptake, but not the component driven by Na+, was inhibited by SITS. Finally, Na+-driven SO4(2-) accumulation could be reduced by imposing an out greater than in HCO3- gradient, conditions accelerating exchange driven SO4(2-) efflux. These findings indicate the presence of separate Na+-SO4(2-) cotransport and SO4(2-)-anion exchange pathways in the same BBM vesicles.

Regulation of twitch tension in sheep cardiac Purkinje fibers during calcium overload.

Abstract: The dependence of twitch tension on the interval (delta t) between depolarizations was examined in voltage-clamped sheep cardiac Purkinje fibers under conditions of calcium overload. During the development of calcium overload (produced by sodium-pump inhibition), twitch amplitude changes from a monotonic function of delta t to an oscillatory one. We investigated the cellular processes underlying this oscillatory relationship. Measurable calcium current was blocked by D 600 (25 microM), but neither the twitch nor the oscillatory dependence of twitch tension on delta t was abolished. Caffeine (2 mM), applied to modify sarcoplasmic reticulum function, decreased the oscillatory period of the twitch/interval relationship as it increased the frequency of spontaneous fluctuations of resting tension. Our results suggest that the oscillatory relationship between twitch amplitude and delta t is not caused by changes in the calcium current per se but rather by fluctuations in the amount of releasable calcium in the sarcoplasmic reticulum. Additionally, we conclude that the calcium current may not be a necessary prerequisite for depolarization to trigger calcium release from the sarcoplasmic reticulum under conditions of calcium overload.

Na-K pump site density and ouabain binding affinity in cultured chick heart cells.

Abstract: The possible existence of multiple [3H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [3H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s-1 [corrected]. Maximal specific [3H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [3H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [3H]ouabain and 125I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [3H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells.

Gastrin receptor characterization: affinity cross-linking of the gastrin receptor on canine gastric parietal cells.

Abstract: We applied affinity cross-linking methods to label the gastrin receptor on isolated canine gastric parietal cells in order to elucidate the nature of its chemical structure. 125I-labeled Leu15-gastrin and 125I-labeled gastrin2(-17) bound to intact parietal cells and their membranes with equal affinity, and half-maximal inhibition of binding was obtained at an incubation concentration of 3.2 X 10(-10) M unlabeled gastrin. 125I-gastrin2(-17) was cross-linked to plasma membranes or intact parietal cells by incubation in disuccinimidyl suberate. The membrane pellets were solubilized with or without dithiothreitol and applied to electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels. Autoradiograms revealed a band of labeling at Mr 76,000 and labeling of this band was inhibited in a dose-dependent fashion by addition of unlabeled gastrin to the incubation mixture. Dithiothreitol in concentrations as high as 100 mM did not alter the electrophoretic mobility of the labeled band. After taking into account the molecular weight of 125I-gastrin2(-17), our results suggest that the gastrin receptor on parietal cells is a single protein of Mr 74,000 without disulfide-linked subunits.

Bicarbonate transport in collecting tubules from outer stripe of outer medulla of rabbit kidneys.

Abstract: The purpose of this study is to characterize the features of bicarbonate (total CO2) transport in isolated perfused collecting tubules obtained from the outer stripe of the outer medulla (OMCTos) of rabbit kidneys. Under control conditions (25 mM HCO3- in the perfusate and bath), all OMCTos studied absorbed total CO2 at a mean rate of 8.61 +/- 0.44 pmol.mm-1.min-1. Ouabain (10(-4) M in the bath) did not affect the rate of total CO2 absorption (JtCO2). Addition of the diethylstilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a concentration of 10(-4) M or replacement of bath chloride by gluconate reduced JtCO2 by approximately 50%, whereas replacement of luminal chloride increased JtCO2 by 40%. The carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide in concentrations of 10(-4) M had little effect on JtCO2. In a concentration of 10(-3) M, acetazolamide reduced JtCO2 by only 31%. OMCTos obtained from rabbits with ammonium chloride-induced metabolic acidosis did not have increased rates of total CO2 absorption compared with the control, but treatment of animals with mineralocorticoids increased JtCO2. These results indicate that OMCTos are capable of significant bicarbonate absorption in vitro. This absorption 1) is independent of sodium transport, 2) appears to require, at least in large part, HCO3- or OH- -Cl- exchange across the basolateral cell membrane of acid-secreting cells, 3) is much more resistant to inhibition by carbonic anhydrase inhibitors than reported previously for other rabbit nephron segments, and 4) is stimulated by prior mineralocorticoid treatment of animals but not by prior metabolic acidosis in vivo.

Supersensitivity to NE alters renal function of chronically denervated rat kidneys

Abstract: The effect of norepinephrine (NE) infusion (10, 100, and 330 ng/min, iv) on renal blood flow (RBF), glomerular filtration rate (GFR), urine flow, and sodium excretion was studied during ganglionic blockade in Inactin-anesthetized Wistar rats with one kidney innervated and the contralateral kidney denervated 7-10 days before the experiment. During the NE infusions, steady-state mean arterial pressure was 73 +/- 3, 91 +/- 5, and 117 +/- 2 mmHg, whereas plasma NE concentration averaged 3.9 +/- 1.2, 26.4 +/- 3.2, and 78.1 +/- 4.8 pmol/ml, respectively. At the lowest dose, RBF and GFR were decreased significantly in both kidneys but were significantly lower in the denervated kidneys than in the innervated kidneys. Urine flow and total and fractional sodium excretion increased significantly from the innervated kidneys but not from the denervated kidneys during the 100 and 330 ng/min infusion of NE. When renal perfusion pressure was controlled at the level found after ganglionic blockade, RBF and GFR decreased significantly in both kidneys but to a greater extent in the denervated kidneys at all doses of NE. Urine flow and total and fractional sodium excretion decreased significantly from the denervated kidneys at all doses of NE but decreased from the innervated kidneys only at the highest dose. These results indicate that in chronically denervated kidneys both tubular and vascular responses to NE are altered. The data support the conclusion that denervation supersensitivity can significantly alter renal responses to increased plasma concentration of NE.

Renal synthesis and urinary excretion of eicosanoids during pregnancy in rats

Abstract: We tested whether vasodilatory prostaglandins (PGs) mediate the adaptation of the maternal renal circulation to pregnancy by assessing production of PGs by kidney tissues in vitro. We reasoned that if PGs are crucial, then local synthetic rates of these hormones would most likely be increased. Glomeruli harvested from gravid rats of gestational days 6, 12, and 20 did not demonstrate greater basal or stimulated syntheses of PGF2 alpha, PGE2, or 6-keto-PGF1 alpha than those from virgin controls. Production of PGE2 and 6-keto-PGF1 alpha by renal cortical slices obtained from pregnant rats was not greater than that of virgins either. Urine 24-h excretory rates of PGE2 and 6-keto-PGF1 alpha, but not PGF2 alpha, were significantly increased during pregnancy (all P less than 0.05 from prepregnant levels). During midgestation, when urinary excretion of PGE2 and 6-keto-PGF1 alpha was greatest, medullary syntheses of these PGs were found to be comparable between virgin and gravid animals. The present study, which fails to show augmented synthesis of PGs by renal tissues derived from gravid rats, is consistent with our previous investigation in which cyclooxygenase inhibition did not reduce the gestational increase of renal hemodynamics or restore the attenuated renal pressor responsiveness to exogenous angiotensin II. Taken together, we conclude that in rats, PGs most likely do not mediate the alterations in the renal circulation observed during pregnancy.