Isolated superfused juxtaglomerular cells from rat kidney: a model for study of renin secretion.

Freshly isolated rat juxtaglomerular cells (JGC) were superfused to study renin secretion rate (RSR) at the cellular level. Effluates from the superfusion chamber collected in 20-min intervals showed a time-dependent decline in RSR from 85.5 +/- 32 to 4.0 +/- 2.4 ng ANG I. ml-1. h-1. mg protein-1. min-1 within 100 min of collection (mean +/- SE, n = no. of JGC preparations/superfusion chambers = 9/18). Addition of adenosine deaminase type II (ADA II, 3 U/1.4 mg protein) to the superfusion medium increased RSR more than fourfold to 402 +/- 100 ng in the first collection period, which dropped to 237.5 +/- 67 ng ANG I. ml-1. h-1. mg protein-1. min-1 (n = 9/18) within 100 min. This ADA II effect was rapid in onset and fully reversible. When the purified ADA type VII, with a 40-fold higher specific activity, was added to the superfusate, RSR was increased only by 96 +/- 17.8% compared with controls. This ADA VII (5 U/30 microgram) effect could be mimicked by the selective adenosine A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-6) mol/l). Since albumin stimulated RSR in a concentration-dependent fashion, to an extent similar to that of ADA II, we assume that the ADA II effect was largely unspecific in nature. We conclude that 1) superfusion of isolated JGC from rats is suitable for investigations of renin secretion at the cellular level, 2) the increase in RSR by ADA II appears to be only in part due to deamination of endogenously generated adenosine, and 3) albumin in the superfusate induces a similar stimulatory effect as ADA II.

Potassium diet as a determinant for the renal response to systemic potassium channel modulation in anesthetized rats.

The role of potassium intake in the response of kidney function and plasma renin activity (PRA) to systemic application of U37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydro chloride), a putative blocker of ATP-sensitive potassium channels (K(ATP)), and P1075 (N-cyano-N'-(1,1-dimethylpropyl)-N"-pyridylguanidine), an opener of K(ATP) channels, was studied in the anesthetized rat. It was found that under normal potassium diet (0.7% K), U37883A (15 mg/kg, i.v.) increased urinary flow rate (UV) and sodium excretion (UNaV), decreased urinary potassium excretion (UKV), and significantly diminished heart rate (HR) without affecting mean arterial blood pressure (MAP) or glomerular filtration rate (GFR). P1075 (10 microg/kg, i.v.) lowered UV, UNaV and UKV, at least in part due to the fall in MAP and GFR. PRA was diminished by U37883A and increased by P1075. Variation in potassium diet (0.04 or 2% K) left the response in MAP, HR or GFR to both potassium channel modulators essentially unchanged. The reduction in renal excretion rates to P1075 also appeared unaffected, further supporting a predominant role of the change in MAP and GFR in this response. Variation in potassium diet, however, elicited the following alterations: (1) under both low and high potassium diet U37883A did no longer cause a significant natriuresis; (2) U37883A elicited a significant kaliuresis under high potassium diet, whereas potassium excretion remained essentially unchanged on very low levels under low potassium diet; (3) the increase in PRA to P1075 was blunted under low potassium diet. Additional experiments provided evidence that P1075 releases renin from freshly isolated juxtaglomerular cells of rats on normal but not on low potassium diet. In summary, systemic potassium channel modulation employing U37883A or P1075, respectively, exerts distinct effects on blood pressure and heart rate independent of potassium diet. In contrast, potassium diet appears to be a determinant for the concomitant responses in plasma renin activity and renal sodium and potassium excretion.

Effect of KATP channel blocker U37883A on renal function in experimental diabetes mellitus in rats.

An increase in glomerular filtration rate (GFR) in early diabetes mellitus is considered a risk factor for the development of diabetic nephropathy. Insulin deficiency may increase the activity of ATP-sensitive potassium channels (KATP), which could promote afferent arteriolar vasodilation und thus contribute to glomerular hyperfiltration in early diabetes mellitus. To further elucidate this hypothesis we performed renal clearance experiments in anesthetized rats at 2 and 6 weeks after onset of streptozotocin-induced insulin-treated diabetes mellitus and studied the acute effect of the putative KATP channel blocker 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydr ochloride (U37883A) on renal function. In control rats, application of U37883A (1.5 mg/kg i.v. bolus plus 1.5 mg/kg/hr) induced a significant reduction in heart rate, but did not affect or even slightly increased mean arterial blood pressure. Furthermore, U37883A did not significantly affect renal vascular resistance, renal blood flow or GFR, but caused an eukaliuretic diuresis and natriuresis and lowered plasma renin activity. Diabetic rats at both 2 or 6 weeks after streptozotocin exhibited essentially an identical response to U37883A; in particular, RVR and glomerular hyperfiltration remained unchanged. These results show that in both control and diabetic rats, the renal excretory function, renin secretion and pace setting in the heart were sensitiv to U37883A, implying a functional contribution of KATP channel activity. However, in both control and diabetic rats, renal vascular resistance, renal blood flow, or GFR were not altered by U37883A. These results argue against a substantial role for KATP channels in the basal control of renal hemodynamics in both nondiabetic and diabetic rats.

The immunoreactivity of glial fibrillary acidic protein in mesangial cells and podocytes of the glomeruli of rat kidney in vivo and in culture.

In this study the presence of glial fibrillary acidic protein (GFAP) in kidney is for the first time demonstrated in cryostat sections and cultures of isolated glomerular explants derived from rat kidneys. In double immunolabelling analysis of adult rat kidney sections using antiserum against GFAP and monoclonal antibody (mAb) against vimentin or desmin, the presence of immunoreactivity for GFAP could be observed in the glomerulus of the kidney and vascular cells situated in the peritubular space which expressed vimentin and desmin. Labelling of the sections with absorbed antiserum against GFAP completely abolished the staining in all these cells. The mAb against GFAP, clone GF12.24 which is known to label GFAP both in neural and non-neural cells, recognised its antigen only in the cells located in glomeruli. The investigations performed on early 2- or 3-day-old cultures from glomerular explants revealed different patterns of staining for GFAP in mesangial cells and podocytes: weak filamentous in mesangial cells and a strong non-filamentous perinuclear pattern in podocytes. Due to prominent perinuclear expression in podocytes GFAP may be considered as a marker of these cells. A different pattern of distribution of immunoreactivity for GFAP in podocytes and mesangial cells might be due to function-related posttranslational modifications of GFAP resulting in assembly or disassembly of GFAP filaments. The different pattern of staining for GFAP in the podocytes and mesangial cells, cells which exert a different influence on the capillaries of the glomeruli, suggests a role for GFAP in regulation of the tension and permeability of vascular walls. Previous investigations and present studies hint at GFAP as being a general marker of perivascular cells.

The possible role of histamine-mediated cAMP formation as a link between H2-receptor stimulation and ultrastructural changes in guinea-pig oxyntic cells.

Guinea-pig oxyntic cell tubulin has been isolated and in vitro aggregation has been studied. The spontaneous assembly of isolated tubulin was significantly accelerated and increased by 1 mmol/l GTP. Histamine and forskolin increased tubulin polymerization only when detergent dispersed oxyntic cells or crude membranes were added. The forskolin response occurred very rapidly with an EC50 of approximately 30 mumol/l and did not require GTP. Histamine promoted tubulin aggregation with an EC50 of about 5 mumol/l only in the presence of GTP. Ranitidine completely inhibited the effects of histamine. From these data it is suggested that, in the oxyntic cell, histamine H2-receptor activated adenylate cyclase and the corresponding increase in cAMP play a role in eliciting characteristic ultrastructural changes by initiating formation of microtubules as a first step in the cascade of events leading to an increase in the secretory surface area.

The effects of nicotine on basal and stimulated gastric secretions in the conscious cat and in isolated guinea pig gastric mucosal cells.

Intravenous (i.v.) administration of nicotine in conscious cats significantly stimulated basal gastric acid output. The effect was completely blocked by atropine and ranitidine. Submaximally stimulated gastric acid secretion was not further increased by nicotine. In isolated guinea pig parietal cells nicotine significantly increased basal acid secretion by about 20% and potentiated the response to maximally effective concentrations of histamine but had no influence on the carbachol response. In isolated parietal cells stimulated either by nicotine, histamine or both, atropine pretreatment increased or inhibited the acid response in concentration-dependent manner. From these data, it is concluded that nicotine had direct stimulatory effects on isolated parietal cells and potentiated the histamine mediated response in the isolated cell preparation but not in the intact animal model.

Are prostaglandins involved in somatostatin mediated inhibition of gastric secretion in the cat?

The role of prostaglandins in somatostatin mediated gastric inhibitory effects has been investigated in conscious cats. The effect of somatostatin on pentagastrin-, insulin- and histamine plus bethanechol-stimulated gastric acid and pepsin secretion was determined with and without indomethacin pretreatment. Somatostatin significantly inhibited acid and pepsin secretion and this effect was not diminished by cyclo-oxygenase inhibition. It is concluded that there is no evidence that endogenous prostaglandins mediate the inhibitory effects of somatostatin on gastric acid and pepsin secretion in the cat.

The effects of antimitotic and microfilament disrupting agents on functions of isolated guinea-pig parietal cells.

In isolated guinea-pig parietal cells pretreated for 60 min with the H2-antagonist ranitidine, the antimitotic agents colchicine and vinblastine, the microfilament-disrupting agent cytochalasin B resulted in a concentration-dependent inhibition of histamine-stimulated acid secretion up to 80%. Ranitidine reduced histamine binding to the membrane located H2-receptor. The anti-cytoskeletal agents inhibited the cellular histamine uptake but did not effect the histamine methyltransferase activity which was significantly reduced by ranitidine. The data suggest that cytoskeletal elements like microtubules and microfilaments are of very specific functional significance not only in the secretory process of the parietal cell but also for cellular transport mechanisms.

Evidence against prostaglandin-mediation of somatostatin-inhibition of gastric secretions.

The inhibitory activities of somatostatin and PGE2 against pentagastrin-stimulated gastric acid and pepsin secretions were investigated, with and without pretreatment with the cyclooxygenase inhibitor indomethacin, in conscious cats prepared with gastric fistulae. Somatostatin was a potent inhibitor of acid secretion in both vagus intact and vagotomized animals, and its effect was not diminished by indomethacin pretreatment. Somatostatin inhibition of pepsin secretion was diminished after indomethacin, but a similar effect was noted with exogenous PGE2, suggesting a mechanism unrelated to inhibition of prostaglandin synthesis. It is concluded that there is no evidence to implicate endogenous prostaglandins in somatostatin inhibition of feline gastric exocrine secretions.

Subclasses of muscarinic receptors in isolated gastric mucosal cells: receptor characterization and parietal cell function.

Muscarinic receptors were characterized in isolated intact chief and parietal cell enriched cell populations from canine and guinea-pig gastric mucosa by binding of tritiated N-methylscopolamine ([3H]NMS). Antagonist and agonist binding was studied by displacement of [3H]NMS with non-radioactive atropine, pirenzepine, pilocarpine and carbachol. Model analysis points to the existence of two binding sites in each of the two cell populations. The number of binding sites per cell was 1.7-1.8 times higher in parietal than in chief cell populations. Subclasses of muscarinic receptors as characterized by pirenzepine binding were compatible with the suggested A- and C- (high and low affinity) binding sites. The observation that in canine cells GMPPNP induced a conformational change of the high affinity binding site for pirenzepine could suggest that their proportion might depend on environmental factors. Binding parameters were related to specific parietal cell function as measured by aminopyrine accumulation as index for acid secretion. The carbachol effects depended on the calcium concentration and were competitively inhibited by pirenzepine. The physiological relevance of muscarinic receptor heterogeneity in gastric mucosal cells is unknown although the data support the hypothesis that involvement of muscarinic binding sites in calcium transport mechanisms connected with parietal cell function and possible conformational changes of the binding sites might be regulatory parameters in gastric secretory processes.

Histidine decarboxylase inhibition by O-methyl-3(+)catechin and gastric acid secretion in the cat.

In the conscious cat the histidine decarboxylase inhibitor O-methyl-3(+)catechin (Zy 15029) promoted a dose-dependent atropine-sensitive increase in basal acid output. Gastric acid secretion stimulated by food or insulin at different time intervals after pretreatment with Zy 15029 was dose and time dependently diminished up to 70% whereas acid output following pentagastrin stimulation was not reduced by doses effective against the former two stimuli. Only a high dose, which due to side effects has to be claimed as not tolerable in the cat, reduced acid output by about 40%, when application of Zy 15029 and stimulation were 90 min apart. It is suggested that in the cat gastric acid response following the three different stimuli was at least in part but to a variable extent mediated by endogenous histamine. Dose-dependent side effects of Zy 15029 might have been due to histidine decarboxylase inhibition in brain and changes in histaminergic neurotransmission.

Histamine uptake and metabolism in intact isolated parietal cells.

3H-Histamine binding, uptake and metabolism were investigated in intact isolated and enriched parietal cells from the dog and guinea pig. Histamine uptake was sodium dependent and followed by intracellular metabolism. The only metabolite that was detected and extracted from cytosol has been identified by TLC to N tau-methylhistamine. The histamine N-methyltransferase activity appeared to be sodium dependent and was inhibited by mepyramine and chlorpromazine, and also by higher concentrations (10(-4)--10(-3) mol/l) of cimetidine. Two blockers of the sodium channel, amiloride an aminoguanidine, also reduced the enzyme activity by an as yet unknown mechanism.

Binding studies with 3H-atropine in intact isolated guinea pig gastric mucosal cells.

3H-atropine binding was studied in isolated intact guinea pig gastric mucosal cells and has been shown to be saturable. Scatchard plots of the binding system calculated under the assumption of only one binding site revealed KD values for the parietal cell enriched population of 1.25 x 10(-6) mol/l and 1.38 x 10(-6) mol/l for the nonparietal cells. The results show that in gastric mucosa both parietal and nonparietal cells contain cholinergic receptors.

[In-patient treatment of peptic ulcer with cimetidine. I. Effect on duodenal ulcer healing (author's transl)]. / Cimetidin in der stationären Behandlung des peptischen Ulkus. I. Wirkung auf die Heilung des Ulcus duodeni.

The effect of 300 mg cimetidine q.i.d. on ulcer healing was studied in a controlled double-blind clinical trial of 71 in-patients with duodenal ulcer. Healing occurred in 48.5% of patients in the cimetidine group after two weeks, and in 20.6% in the placebo group (P less than 0.05). The healing rate was 88% in the cimetidine group at four weeks, 79.4% in the placebo group. Only during the first day was ulcer pain significantly reduced in the cimetidine-treated patients. Neither basal nor pentagastrin-stimulated acid and pepsin secretions were affected by 17-day administration of cimetidine. The drug had to be withdrawn in two patients because of elevated serum-creatinine levels. There was no other untoward effect.

[In-patient treatment of peptic ulcer with cimetidine. II. Controlled double-blind trial on gastric ulcer patients (author's transl)]. / Cimetidin in der stationären Behandlung des peptischen Ulkus. II. Doppelblindstudie bei Ulcus ventriculi.

In a controlled double-blind clinical trial of 39 in-patients with gastric ulcer the effect of cimetidine on ulcer healing, ulcer pain and pentagastrin-stimulated acid and pepsin secretion was measured. A faster healing rate in the cimetidine group was statistically not significant. Cimetidine had no effect on ulcer pain and pentagastrin-stimulated acid and pepsin secretion. There were no serious untoward reactions.

The effect of anaesthesia on pentagastrin stimulated gastric acid secretion in the cat.

1. The effect of Saffan (alphaxalone and alphadolone acetate) and Nembutal (pentobarbitone) anaesthesia on gastric acid secretion stimulated by I.V. bolus injections of pentagastrin was investigated in the gastric fistula cat. The effect of Saffan anaesthesia was also tested on I.V. near-maximal and sub-maximal infusions of pentagastrin. 2. Neither anaesthetic reduced the acid secreted in response to bolus injections of pentagastrin. Increased acid secretion with the anaesthetic was noted at some doses of pentagastrin in both intact and vagotomized animals. 3. Saffan anaesthesia had no effect on near-maximal gastric acid secretion stimulated by pentagastrin 8 microgram kg-1 hr-1 but increased the response to pentagastrin 1 microgram kg-1 hr-1. 4. It is suggested that the differences in the acid secretory responses of the anaesthetized acutely prepared animal compared with the conscious animal are a consequence of the surgical procedures rather than the state of consciousness.

Comparison of the inhibition of histamine- and pentagastrin-stimulated gastric acid secretion by somatostatin in the cat.

The effect of cyclic somatostatin on pentagastrin- and histamine-stimulated gastric acid secretion in conscious cats was studied. Evidence is produced that somatostatin competitively inhibits pentagastrin-stimulated gastric acid secretion whereas it inhibits histamine-stimulated secretion by a mechanism which is not competitive in nature. The vagus nerves seem to be involved in the mode of action of somatostatin as the inhibitory effects are greater in vagotomized than in vagus intact animals.

Comparison of the effect of somatostatin on gastrointestinal function in the conscious and anaesthetized cat and on the isolated cat pancreas.

1. Somatostatin, 10 microgram kg-1 hr-1, inhibited gastric acid and pepsin secretion stimulated by pentagastrin, 8 microgram kg-1 hr-1, in conscious and anaesthetized cats with chronically implanted gastric fistulae. In the acutely surgically prepared anaesthetized cat, Somatostatin inhibited pepsin secretion but produced little inhibition of gastric acid secretion or mucosal blood flow. 2. Secretin stimulated pancreatic juice volume was not significantly reduced in acutely prepared anaesthetized cats, but there was a limited reduction of cholecystokinin-pancreozymin stimulated pancreatic amylase secretion and gall bladder contraction. 3. Somatostatin had neither stimulatory nor inhibitory effects on electrolyte and amylase secretion in the isolated saline-perfused cat pancreas. 4. The results suggest that some of the effects of Somatostatin may depend on the interaction on the target cell of other factors, nervous or humoral which may vary in different experimental preparations.