Receptors for atrial natriuretic peptide are decreased in the kidney of rats with streptozotocin-induced diabetes mellitus.

To determine whether decreased renal responsiveness to atrial natriuretic peptide (ANP) in diabetes is mediated by alterations in the renal ANP receptor, ANP receptor density and affinity were measured 17-20 d after streptozotocin injection and compared with values in vehicle-treated controls and streptozotocin-treated rats made euglycemic with insulin. Plasma ANP concentration was significantly greater in hyperglycemic diabetic rats than in control or euglycemic diabetic rats. Both in glomeruli and inner medulla, ANP receptor dissociation constant did not differ among the three study groups, whereas the maximum binding capacity was decreased significantly in hyperglycemic diabetics in comparison with controls and euglycemic diabetics. Glomerular clearance receptors were also decreased significantly in hyperglycemic diabetic rats in comparison with control and euglycemic diabetic rats. To determine whether the decreased number of renal ANP receptors in diabetic rats was associated with a decreased biological response, we measured ANP-dependent cyclic GMP (cGMP) accumulation by isolated glomeruli and inner medullary collecting duct cells in vitro. cGMP accumulation was significantly less in hyperglycemic diabetic rats than in controls or euglycemic diabetic rats both in the presence or absence of the phosphodiesterase inhibitor zaprinast. cGMP phosphodiesterase activity in inner medullary collecting duct cells obtained from control and hyperglycemic diabetic rats did not differ. Thus, the decreased number of biologically active ANP receptors in the kidneys of diabetic rats is accompanied by decreased biological responsiveness in vitro and provides a potential explanation for the reduction in renal sensitivity to ANP in this condition.     

Chronic caffeine ingestion sensitizes the A1 adenosine receptor-adenylate cyclase system in rat cerebral cortex.

Caffeine consumption causes significant physiologic effects due to its antagonism of adenosine receptors. The A1 adenosine receptor is coupled in an inhibitory manner to adenylate cyclase. To study the effects of chronic caffeine ingestion, rats were provided with 0.1% caffeine drinking solution for 28 d. The A1 adenosine receptor agonist radioligand [3H]phenylisopropyladenosine identifies two affinity states in control rat cerebral cortex membranes with a high affinity dissociation constant (KH) of 0.40 +/- 0.08 nM and low affinity dissociation constant (KL) of 13.7 +/- 3.9 nM, with 33% of the receptors in the high affinity state. In membranes from caffeine-treated animals, all of the A1 receptors are shifted to the high affinity state with a dissociation constant (KD) of 0.59 +/- 0.06 nM. Guanylyl-imidodiphosphate (10(-4) M) decreases binding by 43% in control membrane, with no change in KH or KL, while membrane binding in caffeine-treated animals decreases by 45% with a threefold shift in KD to 1.5 +/- 0.3 nM. Concomitant with the enhanced high affinity A1 receptor state and increased sensitivity to guanine nucleotides, membranes from treated animals show a 35% enhancement in (-)-N6-(R-phenylisopropyl)adenosine-mediated inhibition of adenylate cyclase compared with controls (P less than 0.03). Photoaffinity crosslinking the receptors with [125I]N6-2-(3-iodo-4-aminophenyl)ethyladenosine reveals that A1 receptors from both groups migrate as Mr 38,000 proteins. beta-adrenergic receptor binding with [125I]iodocyanopindolol shows a decrease in the number of beta-receptors from 233 +/- 7 fmol/mg protein in control membranes to 190 +/- 10 fmol/mg protein in treated membranes (P = 0.01). These data indicate that the adenosine receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system and downregulation of beta-adrenergic receptors, and provides a molecular mechanism for the caffeine withdrawal syndrome.     

Atrial natriuretic peptide inhibits angiotensin II-induced contraction of isolated glomeruli and cultured glomerular mesangial cells of rats: the role of calcium

We examined the contractile effect of angiotensin II on isolated rat glomeruli and cultured glomerular mesangial cells, as well as the possible involvement of calcium in this contraction. In addition, the effect of rat atrial natriuretic peptide (ANP) on angiotensin II-induced contraction was studied. Computer-assisted image analysis was used to measure glomerular cross-sectional area and planar mesangial cell surface area in several experimental conditions. Angiotensin II induced a decrease in glomerular cross-sectional area and planar mesangial cell surface area that was both time and dose dependent. This effect was partially blocked by preincubation with verapamil or TMB-8, the latter compound being more effective. Incubation with ANP prevented angiotensin II-induced reduction of glomerular cross-sectional area and planar mesangial cell surface area and reversed contraction in mesangial cells. Incubation with TMB-8 impaired the effect of ANP. These data suggest that angiotensin II could exert part of its physiologic effects in the kidney through calcium-dependent mesangial and glomerular contraction. ANP inhibited this contraction, possibly explaining some of its renal effects.     

Endothelin (ET)-3 stimulates cyclic guanosine 3',5'-monophosphate production via ETB receptor by producing nitric oxide in isolated rat glomerulus, and in cultured rat mesangial cells.

We investigated the effects of endothelins on receptor-mediated cyclic nucleotide metabolism in rat glomerulus, inner medullary collecting duct (IMCD), and also in cultured rat glomerular mesangial cells. Endothelin (ET)-3 dose-dependently stimulated cGMP accumulation in glomerulus, which was higher than that of ET-1 or ET-2. ETB receptor agonist IRL 1620 produced cGMP in a dose-dependent manner, mimicking the effect of ET-3. ETA receptor antagonist BQ123-Na did not inhibit ET-3- or IRL 1620-stimulated cGMP generation. NG-monomethyl-L-arginine (L-NMMA) significantly inhibited ET-3- or IRL 1620-induced cGMP production, suggesting that ET-3- or IRL 1620-stimulated cGMP generation was mediated through nitric oxide (NO). Intracellular Ca chelator BAPTA/AM and calmodulin antagonist W-7, but not Ca channel blocker nicardipine, significantly inhibited ET-3- or IRL 1620-induced cGMP generation. In cultured rat mesangial cells, ET-3 stimulated cGMP generation through NO in the presence of fetal calf serum, which was not inhibited by addition of BQ123-Na. In IMCD, ET-3 had no stimulative effect on cGMP generation. We conclude that ET-3 stimulates NO-induced cGMP generation through ETB receptor in glomerulus. This effect seems to be mediated through intracellular Ca/calmodulin, but not through Ca influx via L-type Ca channel. Mesangial cells can be a source of NO coupled to ETB receptor activation in glomerulus. From these results, mesangial ETB receptor may work to counteract the vasoconstrictive effect of endothelin caused via ETA receptor in glomerulus.     

Transforming growth factor-beta. Murine glomerular receptors and responses of isolated glomerular cells.

Proliferation of resident glomerular cells and the accumulation of mesangial matrix are histologic abnormalities which are observed in the course of many progressive glomerular diseases. We explored the potential regulatory effects of transforming growth factor-beta (TGF-beta) on these processes. We found that cultured mouse glomerular endothelial, mesangial, and epithelial cells as well as isolated intact rat glomeruli possess high-affinity receptors for TGF-beta. We also found that, although TGF-beta consistently inhibited the proliferation of glomerular endothelial and epithelial cells, it acted as a bifunctional regulator of mesangial cell proliferation. TGF-beta significantly increased the production of collagen and fibronectin by glomerular mesangial cells whereas only fibronectin production was augmented in glomerular epithelial cells. The presence of TGF-beta receptors on intact glomeruli and on each glomerular cell type and the demonstrated responsiveness of these cells to TGF-beta combine to suggest that potentially important interactions may occur between resident glomerular cells and TGF-beta in vivo.     

Mechanism of sodium modulation of glomerular angiotensin receptors in the rat.

Specific binding of 125I-angiotensin to high affinity glomerular receptors varies directly with the level of dietary sodium. To investigate the mechanism of sodium regulation of glomerular angiotensin receptors, groups of Sprague-Dawley rats were maintained on one of three levels of sodium intake for at least 5 d: high sodium (7.39 meq/24 h), moderate sodium (0.88 meq/24 h), and low sodium diets (0.01 meq/24 h). An additional group was given low sodium diet with daily injections of furosemide (1 mg/kg i.p.). To dissociate the effects of dietary sodium from those of circulating angiotensin II levels on glomerular receptor regulation, a fifth group was placed on high sodium diet and given a continuous infusion of angiotensin via an implanted minipump (100 ng/min) for 21 d. There was a strong negative correlation (r = -0.98, P less than 0.01) between plasma angiotensin II and glomerular angiotensin receptor density. Dietary sodium, potassium, or water consumption did not correlate with angiotensin II receptor concentration. The affinity constant did not vary in any of the groups (2.33 +/- 0.30 X 10(8) M-1). The time course of sodium regulation of glomerular angiotensin II receptors was studied in rats switched from a moderate sodium to either a high sodium diet or a low sodium diet plus furosemide. Receptor density was unchanged at 24 h, varied directly with sodium intake for 1-5 d when induction was maximal, and remained constant for at least 21 d. The time course of receptor regulation closely paralleled changes in plasma angiotensin II. Additional studies were undertaken to demonstrate that glomerular angiotensin II receptors are down-regulated by circulating hormone. Rats maintained on moderate sodium intake were killed 2 min after the induction of anesthesia with pentobarbital (50 mg/kg i.p.) or by rapid decapitation. Despite a 50-fold elevation of plasma angiotensin II in anesthetized rats (424 +/- 154 vs. 8.6 +/- 1.0 pg/ml, P less than 0.001) angiotensin receptor density was unchanged (anesthetized, 1,016 +/- 126 vs. unanesthetized, 1,290 +/- 84 fmol/mg). The infusion of angiotensin II (100 mg/min) for 15 min or 2 h into anesthetized rats maintained on moderate sodium intake resulted in a 50% reduction in specific angiotensin binding that could not be reversed by the dissociation of endogenous angiotensin. These data are compatible with modulation of receptor density by circulating hormone and can not be accounted for by prior receptor occupancy.     

Preclinical pharmacology of a new serotonergic receptor antagonist, LY281067

The preclinical pharmacologic activity of LY281067 shows it to be a potent and highly selective serotonergic (5-HT2) receptor antagonist. Based upon binding studies with 5-HT2 receptors in brain cortical membranes and block of 5-HT-induced contractions in the rat jugular vein, LY281067 showed high affinity at 5-HT2 receptors with a dissociation constant of approximately 1 nM. Furthermore, LY281067 was a highly selective 5-HT2 receptor antagonist without appreciably binding to 5-HT1, D1 or D2 receptors or interacting with histamine (H1), cholinergic, beta adrenergic or alpha-1 adrenergic receptors in smooth muscle. LY281067 had modest affinity at alpha-2 receptors with a dissociation constant of approximately 100 nM. Oral bioavailability of LY281067 in spontaneously hypertensive rats was excellent with an oral to i.v. dose ratio approximating 4, based upon blockade of pressor responses to 5-HT as an in vivo estimate of 5-HT2 receptor antagonist activity. Furthermore, LY281067 blocked quipazine-induced increase in serum corticosterone concentration, an increase thought to be mediated by activation of central 5-HT receptors. After oral administration to rats, LY281067 antagonized vascular 5-HT2 receptors with a relatively long duration of action (greater than 6 hr), an observation likely to be related to plasma concentrations of both the parent and an active metabolite. Lastly, LY281067 was relatively nontoxic, possessing a therapeutic index of approximately 1000 after oral administration to rats. In summary, LY281067 is a potent and highly selective, orally active 5-HT2 receptor antagonist with a relatively long duration and wide margin of therapeutic safety.     

Localization and regulation of renal receptors for angiotensin II and atrial natriuretic peptide.

The anatomical distribution of receptors for angiotensin II (Ang II) and atrial natriuretic peptide (ANP) within the kidney has been investigated by in vitro autoradiography. Ang II and ANP receptor binding occurs together in several sites in the kidney, including renal vasculature, glomeruli, proximal convoluted tubule of the outer cortex, and the vasa recta bundles of the inner stripe of the outer medulla. However, in the glomeruli, Ang II receptor binding occurs predominantly in mesangial cells, while ANP receptors are localized mainly to the visceral epithelial cells. In the inner medulla, there is a moderate density of ANP receptors in marked contrast with Ang II binding which is not detected in this site. Both Ang II and ANP receptors are modulated by alterations in sodium and fluid intake, and the peptides themselves. The overlapping distribution of receptors for these two peptide hormones in several intrarenal sites may provide an anatomical basis for their physiological interaction to regulate renal hemodynamics and tubular reabsorption of sodium and water.     

D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin: a selective high affinity radioligand for delta opioid receptors with exceptional specific activity.

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 +/- 67 pM and a receptor density (Bmax) value of 36.4 +/- 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 +/- 0.88 X 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H DPDPE and 0.8 nM [3H] [D-Pen2,4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 +/- 0.117 X 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of delta opioid receptors.     

Dietary protein and glomerular response to subtotal nephrectomy in the rat

Renal tubules and glomeruli undergo hypertrophy after partial renal ablation. Glomerular and vascular scarring and progressive renal insufficiency may later occur. In the present studies, we have documented the filtration characteristics of glomeruli isolated from rats that had undergone subtotal nephrectomy and been fed a normal protein (NP) diet (24 gm protein per 100 gm chow) or a protein-restricted (PR) diet (6 gm protein per 100 gm chow) and compared these glomeruli to those of sham-operated (SHAM) rats maintained on a normal diet. Body weight of SHAM rats increased by 30% during the 28 weeks of the study. Weight of NP rats did not change significantly, and weight of PR rats decreased by about 25%. Seven weeks after surgery, the glomeruli of NP rats were significantly larger and the ultrafiltration coefficients (Kf) were higher than those of SHAM rats; glomeruli of PR rats were of normal size but also had increased Kf. Twenty-eight weeks after surgery, the glomeruli of rats in each group were larger than at 7 weeks. Kf was no longer elevated in NP rats. Glomerular capillary hydraulic conductivity (Lp) was equal in all groups at 8 days but was increased at 7 weeks and decreased 28 weeks after surgery in NP rats. We hypothesize that glomerular hypertrophy occurs in response to renal ablation and that subsequent changes in the capillary wall lead to diminished Lp. Protein restriction in this model prevents both glomerular hypertrophy and a decrease in Lp.     

P2Y receptors present in the native and isolated rat glomerulus

Extracellular ATP can mobilize intracellular calcium in rat glomeruli by interacting with P2Y receptors. However, the identity of the receptor subtypes involved is not known. In the present study, we have used RT-PCR to identify mRNAs for specific P2Y receptor subtypes expressed in the rat glomerulus: mRNA for P2Y1, P2Y2, P2Y4 and P2Y6 receptors was detected. Functional expression of P2Y1 and P2Y2/P2Y4, but not P2Y6, receptors in intact glomeruli was confirmed by measuring the relative stimulation of the inositol phosphate pathway induced by selective agonists of a particular receptor subtype. Finally, we have used available polyclonal antibodies to confirm the expression of P2Y1 and P2Y2 in the glomerulus, in mesangial cells and glomerular epithelial cells (podocytes), respectively; but we could not demonstrate P2Y4 or P2Y6 receptor expression by this means. In a separate series of experiments, we have examined the possibility that intra-renal sympathetic nerve terminals are a source of extracellular ATP and that this would be supported, though not excluded, by supersensitivity to ATP following denervation. Nucleotide-induced stimulation of the inositol phosphate pathway was measured in both control rats and rats that had been sympathectomized by intraperitoneal injection of 6-hydroxydopamine. The response to norepinephrine was measured as a positive control. In the sympathectomized rats, the effect of norepinephrine was significantly enhanced, whereas ATP-induced inositol phosphate production was unaffected, being similar in both groups of animals.     

Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein, n = 9) but had no effect on cAMP accumulation in epithelial cells. This effect was dose-dependent and time-dependent. Stimulation of cAMP accumulation occurred in the range of 5 X 10(-6) M-10(-4) M histamine with a half maximal stimulatory effect of 2 X 10(-5) M. Initial stimulation was noted by 30 s, and maximum stimulation was observed at 5 min. The H2 antagonist cimetidine (10(-4) M) abolished the stimulatory effect of histamine (10(-4) M), while equimolar concentrations of the H1 antagonist diphenhydramine had no significant effect on cAMP accumulation. Moreover, the specific H2 agonist dimaprit, but not the H1 agonist 2-pyridylethylamine, stimulated cAMP accumulation. Histamine had no effect on cAMP accumulation in epithelial cells or on cyclic guanosine monophosphate accumulation in epithelial or mesangial cells. Since the in vivo infusion of histamine reduces ultrafiltration coefficient and since mesangial cell contraction is thought to be responsible for the reduction in the ultrafiltration coefficient, we examined the effect of histamine on the contractile property of mesangial cells. Histamine (5 X 10(-6)-10(-4) M) contracted mesangial cells, and the H1 antagonist diphenhydramine (10(-4) M) but not the H2 antagonist cimetidine (10(-4) M) prevented histamine (10(-4) M) induced contraction. In addition, the H1 agonist 2-pyridylethylamine, but not the H2 agonist dimaprit, contracted mesangial cells. Histamine and its specific agonists and antagonists induced contraction of isolated glomeruli as assessed by glomerular planar surface area in a manner parallel to their effect on mesangial cells. Cinnarizine (10(-5) M), a Ca++ channel blocker, or Ca++, Mg++-free medium prevented histamine (10(-4) M) induced mesangial cell and glomerular contraction. Thus, histamine enhances cAMP accumulation specifically in mesangial cells via an H2 receptor. In contrast, histamine contracts mesangial cells and glomeruli via an H1 receptor, an effect that is dependent on extracellular Ca++ entry. These findings show that histamine potentially influences intraglomerular hemodynamics via effects on mesangial cell contraction. Moreover, our findings considered with the in vivo observation that histamine reduces kf via and H1 receptor provide further support of the hypothesis that mesangial cell contraction regulates the glomerular capillary surface area available for filtration. Our studies also show that this contractile effect of histamine is dependent on extracellular calcium. The presence of a cAMP system sensitive to histamine may have major implications in the pathogenesis of inflammatory glomerulopathies. Mesangial cells possess characteristics similar to circulating and tissue immune effector cells, including lysosomal enzyme release, oxygen radical production, and release of a number of immunomodulatory factors. Histamine and cAMP have been shown to modulate such characteristics of inflammatory cells. It is therefore conceivable that histamine, via its interaction with H2 receptors and subsequent generation cAMP, may have profound effects on such properties of mesangial cells, suggesting that this autacoid may modulate not only glomerular hemodynamics but also immune, inflammatory responses within the glomerulus.     

Modulation of Cyclic Nucleotides in Isolated Rat Glomeruli: ROLE OF HISTAMINE, CARBAMYLCHOLINE, PARATHYROID HORMONE, AND ANGIOTENSIN-II

Because glomerular functions are modulated by numerous humoral agents, probably acting through cyclic nucleotides, the effects of some polypeptide hormones and biogenic amines on cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) were studied in glomeruli isolated from rat renal cortex. Glomeruli and cortical tubules were prepared by a combination of sieving and density-gradient centrifugation. Under basal conditions, the contents of cAMP and cGMP in glomeruli were significantly higher than in tubules and unfractionated renal cortical tissue.Histamine caused a striking increase in cAMP in glomeruli (+Delta% 675+/-87) and, to a lesser degree, increased cAMP in tubules (+Delta% 103+/-25) or in tissue slices. This stimulation was dose-dependent in the range of 1 muM-1 mM histamine. Metiamide (an H(2)-antagonist), but not pyrilamine (an H(1)-antagonist) blocked the effect of histamine on cAMP, which indicates that histamine causes its effect via interaction with H(2) receptors. Histamine caused less extensive increases in cGMP in both glomeruli and tubules. Carbamylcholine caused a marked increase in cGMP in glomeruli (+Delta 295+/-7) and a much lower increase in tubules (+Delta% 70+/-20); these effects were blocked by atropine. Parathyroid hormone (1 mug/ml) increased cAMP and, to a much lesser degree, also cGMP in glomeruli. In tubules, parathyroid hormone caused much more extensive increases in cAMP than in glomeruli; no changes, or rather a small decline in cGMP, was observed. Angiotensin-II (2 muM) markedly lowered cAMP in glomeruli (-Delta% -45+/-8) and in tubules (-Delta% 33+/-7) but had no effect on cGMP. Bradykinin (20 muM) did not consistently influence either cAMP or cGMP in glomeruli or tubules.Present results demonstrate that cAMP and cGMP metabolism in glomeruli are controlled independently by humoral agents known to alter glomerular functions in vivo. Our findings are consistent with the view that histamine and cholinergic agents generated and (or) released locally in glomeruli or in their vicinity may play important roles as mediators of immunopathological injury of glomeruli, and that these effects are mediated by cAMP and (or) cGMP through interaction with H(2) receptors and muscarinic receptors. Likewise, our results suggest that the effects of angiotensin-II and parathyroid hormone on glomerular dynamics may be mediated by cyclic nucleotides.Thus, we surmise that extrarenal as well as intrarenal humoral agents may play an important role in the pathology and physiology of glomeruli through mediation of either cAMP, cGMP, or both.     

Ameliorative effects of dietary protein restriction in chronic aminonucleoside nephrosis

Because many investigators have demonstrated the efficacy of dietary protein restriction in various experimental models of glomerular injury that progress to focal and segmental glomerulosclerosis (FSGS), we used this dietary maneuver in chronic aminonucleoside nephrosis. This model of glomerular disease, which uses a single intravenous injection of the puromycin aminonucleoside (PAN), slowly progresses over 18 weeks with mesangial cell proliferation and FSGS as the pathologic hallmarks. We performed renal functional and histopathologic studies in 21 rats with chronic aminonucleoside nephrosis. Group 1 rats (n = 10) were fed a standard rodent diet containing 23.4% protein, whereas group 2 animals (n = 11) were maintained with a 6% protein diet replete with electrolytes, minerals, and vitamins. In those animals subjected to dietary protein restriction, proteinuria was significantly reduced at 14, 28, 84, and 126 days after PAN administration. In rats maintained with the 6% protein diet at 126 days after PAN delivery, there was also a significant reduction in the percent of glomeruli with segmental areas of glomerulosclerosis or hyalinosis and mesangial cell proliferation. We conclude that the renal functional and histologic consequences of chronic aminonucleoside nephrosis can be blunted by dietary protein restriction.     

Contraction of Cultured Rat Glomerular Cells of Apparent Mesangial Origin after Stimulation with Angiotensin II and Arginine Vasopressin

Studies to identify the physiological role of glomerular mesangial cells were undertaken using homogeneous cultures of rat glomerular cells of apparent mesangial origin (MS). Cultured MS cells were treated with arginine vasopressin (AVP), angiotensin II (AGII), prostaglandin E(2), and parathyroid hormone. AVP (0.1 nM) and AGII (1 nM) stimulated contraction of MS cells in vitro that was complete by 2 min at 37 degrees C or 10 min at 23 degrees C as observed by phase contrast and electron microscopy. Relaxation recurred 15 min after hormonal addition at 23 degrees C. Similar experiments in cloned rat glomerular epithelial cells or "renin"-producing cells did not demonstrate a contractile response. The contraction of MS cells was independent of cyclic AMP (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP) production, even when cyclic nucleotides were measured as early as 30 s after hormonal stimulation. To demonstrate that contraction was a function of hormone-receptor interaction, binding of [(3)H](8-lysine)vasopressin was studied. Specific binding for 1.6 and 5 nM hormone was both time- and dose-dependent. The estimated apparent affinity was 10 nM. In late MS cell passages (>16th) that no longer demonstrated hormone-stimulated contraction, no specific binding of [(3)H](8-lysine)vasopressin was observed.Incubations were modified to optimize the conditions for detecting the effect of hormones on cell cyclic nucleotide content. A supramaximal concentration of AVP (200 nM) increased the cAMP content of MS cells twofold in the presence of a phosphodiesterase inhibitor. Similar experiments with prostaglandin E(2) (1 mug/ml) led to a 1.5-6-fold increase in MS cell cAMP content, but no effect on contraction was observed. Neither hormone altered cGMP content. These data are further support for the independence of contraction and cyclic nucleotide production.Our studies suggest that MS cells are the equivalent of smooth muscle cells in the glomerulus and that their contraction may be important in control of glomerular filtration.     

Selective downregulation of rat renal clearance receptors for atrial natriuretic peptide by chronic high-salt intake: study on isolated membranes using 125I-labelled c-atrial natriuretic peptide-(4-23).

1. 125I-labelled c-atrial natriuretic peptide-(4-23) was used as radioligand for the direct quantification of atrial natriuretic peptide clearance receptors, and 125I-labelled rat atrial natriuretic peptide-(1-28) was used for the determination of total and biologically active atrial natriuretic peptide receptors, in renal glomerular and papillary membrane preparations of chronically salt-loaded and control rats. 2. The membrane preparation technique included acid-washing (pH 5), and receptor binding was assessed by saturation studies at 4 degrees C for 3 h. Chronic salt loading revealed a 35% decrease in clearance receptor number in the glomerular membrane and a 42% decrease in the papillary membranes. The absolute decrease in clearance receptor number was almost identical with the observed reduction in absolute number of total atrial natriuretic peptide receptors. Biologically active receptors were not affected by chronic salt loading. 3. Selective downregulation of clearance receptors for atrial natriuretic peptide may reflect an important role for the rat renal atrial natriuretic peptide system in long-term fluid and electrolyte regulation.     

The role of mononuclear leukocytes in the pathogenesis of glomerulonephritis

To specify the role of mononuclear (MN) leukocytes in the development of mesangial cell proliferation, which is one of the main manifestations of glomerulonephritis, cell cultures of renal glomeruli of rats with nephrotoxic serum (NTS) nephritis were examined for the intensity of mesangial cell proliferation, the MN-leukocyte count, and for IL-1 production. The amount of mesangial cells and the intensity of their proliferation in the cultures of glomerular cells from rats with NTS nephritis were much higher than in intact rat cultures. At the same time the glomerular cultures from rats with NTS nephritis demonstrated an increase in the MN leukocyte count together with enhancement of IL-1 production. The treatment with prednisolone averted accumulation of MN leukocytes by the glomerular cultures and noticeably reduced mesangial cell proliferation. The supernatant liquid of cultures of peripheral blood MN leukocytes from patients with active nephritis suppressed human fibroblast proliferation, exerting no such action on mesangial cell culture. During the treatment with prednisolone, the supernatant liquid produced a reverse effect on fibroblast and mesangial cell cultures, which was associated with the clinical improvement of the health status. It is assumed that mesangial cell proliferation seen in nephritis may be related to infiltration of the glomeruli by MN leukocytes and to elevated production by them of IL-1 and that the therapeutic action of prednisolone may be determined by suppression of these processes.     

Identification and characterization of functional angiotensin II receptors on cultured heart myocytes

Cultured neonatal rat myocytes have been investigated as a potential system to study the molecular mechanism of angiotensin II (All) stimulation of heart contractile behavior. Intact cultured cells and the membranes from these cells bind [125I] All in a biphasic manner. The data were analyzed as two classes of binding sites on membrane preparations: Kd1 = 0.65 nM; maximum binding (Bmax1) = 245 fmol/mg of protein and Kd2 = 5.57 nM, Bmax2 = 720 fmol/mg of protein. The receptor on intact cells is specific as All peptide analogs were potent at inhibiting the binding of [125I]All. An All antagonist, [125I]Sar1,Leu8-All, recognized a single class of high affinity receptors on intact cells: Kd = 0.63 nM, Bmax = 318 fmol/mg of protein, or 45,300 sites per cell. Guanine nucleotides regulated the binding of All to membranes by shifting the receptor from a high affinity to a low affinity form without changing Kd2. Conversely, these nucleotides altered the high affinity binding of [125I]Sar1,Leu8-All by increasing Kd without changing Bmax. All was found to stimulate the contractile frequency of spontaneously beating myocytes with maximal effects (a 50% stimulation) observed at 5 nM hormone. The responses were reversible with half-maximal effects observed at doses around 1 nM. The antagonist, Sar1,Ala8-All, could inhibit the chronotropic stimulatory responses. It is concluded that these cultured myocytes express an All receptor system with properties consistent with a true hormone receptor system. Furthermore, studies on the pharmacology of the chronotropic responses of these cells to All demonstrate that these receptors are functional.     

Cyclic GMP accumulation by atriopeptins in cultured rat renal and vascular smooth muscle cells

To evaluate the role of cyclic guanosine monophosphate (cGMP) in the vascular and renal action of atrial natriuretic peptide (ANP), we compared the effects of atriopeptins (APs) on cGMP accumulation in cultured cells from rat mesenteric vascular smooth muscle (VSM), glomerular mesangium (GM) and renal papillary collecting tubule (RPCT), and also evaluated the relationship between renal sodium or water excretion and urinary cGMP in AP-infused rats. Both AP I and AP III increased intracellular cGMP levels dose-dependently in all types of cells, while they did not affect intracellular cAMP levels or prostaglandin synthesis. AP III was 100 times more potent than AP I. The magnitude change in cGMP levels was largest in GM cells. The sensitivity of VSM and GM cells to AP III were greater than that of RPCT cells. There were significant positive relationships between urinary excretion of sodium or water and that of cGMP levels in AP-infused rats. These results may suggest that GM and VSM cells are the principal targets for ANP to stimulate cGMP synthesis and, possibly, to exert the renal sodium and water excretion, and also support the hypothesis that cGMP mediates the cellular action of ANP.     

Simultaneous measurement of cholecystokinin-stimulated amylase release and cholecystokinin receptor binding in rat pancreatic acini.

In the past, isolated-dispersed pancreatic acini have been used to examine either cholecystokinin-stimulated amylase release or pancreatic acinar cholecystokinin receptors. We have developed and validated a method for simultaneous measurement of synthetic cholecystokinin octapeptide-stimulated (CCK8-stimulated) pancreatic amylase release and cholecystokinin receptors. After an 18-hour fast, rats were killed and their pancreatic acini isolated. Three-milliliter aliquots of acinar suspension were incubated for 60 minutes in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Ringer buffer containing graded doses of CCK8 and a constant amount (7 pmol/L) of iodine 125-labeled CCK8 (by the Bolton-Hunter method) ([125I]BH-CCK8). A 1 ml sample was removed from each flask for determination of amylase release, and the remaining 2 ml were used to determine cholecystokinin receptor capacities and affinities. The median effective dose for amylase release was 16 pmol/L, and release was maximal at 100 pmol/L CCK8 plus 7 pmol/L [125I]BH-CCK8, a dose that released 28% +/- 3% of total cellular amylase content. High affinity (equilibrium dissociation constant of high-affinity receptors = 58 +/- 8 pmol/L, receptor density of high-affinity receptors = 4 +/- 1 fmol/mg protein) and low affinity (equilibrium dissociation constant of low-affinity receptors = 7 +/- 2 nmol/L, receptor density of low-affinity receptors = 313 +/- 108 fmol/mg protein) cholecystokinin receptors were measured. The results demonstrate that CCK8-stimulated amylase release and cholecystokinin receptor binding in pancreatic acini can be measured concurrently and that the parameters of amylase release and cholecystokinin receptor binding are strikingly similar to those previously observed.(ABSTRACT TRUNCATED AT 250 WORDS)