Effect of ATP and amiloride on ANF binding and stimulation of cyclic GMP accumulation in rat glomerular membranes

We investigated ANF binding and stimulation of cGMP accumulation in isolated rat glomerular membranes in the presence and absence of amiloride and ATP. Amiloride enhanced high affinity binding of ANF without affecting its stimulation of cGMP. In contrast ATP decreased binding and decreased basal cGMP accumulation without affecting the ability of ANF to stimulate cGMP. These data indicate that ANF binding and stimulation of cGMP accumulation can be regulated independently supporting further the concept of receptor heterogeneity in renal glomerular membranes.     

An activator of protein kinase C (phorbol dibutyrate) attenuates atrial-natriuretic-factor-stimulated cyclic GMP accumulation in smooth-muscle cells.

Rat thoracic aortic smooth-muscle cells (A-10; A.T.C.C. CRL 1476) displays a high density of vasopressin and atrial-natriuretic-factor (ANF) receptors and a low density of beta-adrenergic receptors. ANF stimulates cGMP (cyclic GMP) accumulation in a time- and dose-dependent fashion. Pretreatment of these cells with phorbol dibutyrate (PDBu), a known activator of protein kinase C, attenuated ANF-stimulated cGMP accumulation without affecting basal cGMP concentrations. This effect was concentration-dependent and was observed as early as 2 min after treatment. 4 alpha-Phorbol 12, 13-didecanoate (alpha PDD), which does not activate protein kinase C, did not inhibit the cGMP accumulation. PDBu pretreatment did not affect the density and affinity of ANF receptors. These data suggest that PDBu, presumably via activation of protein kinase C, might stimulate phosphorylation of a key regulatory protein in the ANF/cGMP pathway.     

The opposing effects of calmodulin, adenosine 5'-triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells.

In the present study, we investigated the effects of calmodulin, adenosine 5'-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) (a protein kinase C activator) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA (activated protein kinase C) inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.     

Endothelin inhibits the atrial natriuretic factor stimulated cGMP production by activating the protein kinase C in rat aortic smooth muscle cells.

Preincubation of rat thoracic aortic smooth muscle cells with endothelin inhibits the atrial natriuretic factor (ANF)-induced cGMP accumulation in these cells in a concentration dependent manner. The maximal inhibition of 64% was afforded by 1 x 10(-6) M endothelin and the half maximal inhibition (IC50) was achieved with 1 x 10(-9) M endothelin. Endothelin (1 x 10(-6) M) also increased the plasma membrane bound protein kinase C (PKC) activity by 4 fold. Hormone-dependent increase in PKC activity was limited to plasma membranes only and some decrease in cytosolic PKC activity was observed. However, phorbol 12-myristate 13-acetate (PMA) (1 x 10(-6)M) provoked a total loss of cytosolic PKC activity and a net gain in membranous PKC activity indicative of the translocation of the enzyme. Pretreatment of these cells with H-7, a PKC inhibitor, released the endothelin and PMA-mediated attenuation of ANF-stimulated cGMP formation. These results suggest that PKC is involved in the regulation of ANF-induced cGMP accumulation and that the vasoconstrictor activity of endothelin might involve inhibition of the vasorelaxant activity of ANF through the inhibition of cGMP accumulation in smooth muscle cells (SMCs) of the rat aorta.     

Atrial natriuretic factor receptor on cultured Leydig tumor cells: ligand binding and photoaffinity labeling

Atrial natriuretic factor (ANF) is a peptide hormone discovered recently from the heart atrium that possesses potent natriuretic and vasorelaxant activities. Recently we found that ANF markedly stimulates intracellular cGMP and almost completely inhibits cAMP accumulation in testicular interstitial tumor cells [Pandey, K. N., Kovacs, W. J., & Inagami, T. (1985) Biochem. Biophys. Res. Commun. 133, 800-806]. These actions of ANF suggest the presence of ANF receptors in testicular interstitial cells. In this study, cultured murine Leydig tumor cells have been shown to contain specific binding sites for ANF. Saturation binding studies indicated a single class of binding sites with a Kd of 5 X 10(-9) M at a density of 2 X 10(6) sites/cell. The binding of mono[125I]iodo-ANF (125I-ANF) was competed by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensin I, bovine luteinizing hormone, and human chorionic gonadotropin were not able to compete against 125I-ANF. The binding of 125I-ANF was rapid, reaching maximum levels in 15 min at 4 degrees C. At 37 degrees C, the cell-bound 125I label was quickly decreased. Pretreatment of cells with NH4Cl, chloroquine, or NaN3 resulted in significant increases in maximum levels of the cell-bound 125I radioactivity. A photoaffinity reagent for ANF receptor was prepared by reacting ANF with succinimido 4-azidobenzoate, and resultant 4-azidobenzoyl- (AZB-) ANF was purified by high-performance liquid chromatography (HPLC). AZB-ANF was radioiodinated by use of chloramine T and purified again by HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative regulation of atrial natriuretic factor receptor coupled membrane guanylate cyclase by phorbol ester. Potential protein kinase C regulation of cyclic GMP signal in isolated adrenocortical carcinoma cells of rat

Rat adrenocortical carcinoma cells possess a high density of atrial natriuretic factor (ANF) receptors which are coupled with membrane guanylate cyclase and corticosterone production. Herein we show that pretreatment of these cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, attenuates the ANF-stimulated cyclic GMP accumulation in a dose-dependent manner. The half maximum inhibitory concentration of PMA was 10(-10) M. When these cells were incubated with PMA in the presence of 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine, a protein kinase C inhibitor, the PMA-mediated attenuation of ANF-stimulated cyclic GMP formation is blocked. These results suggest that protein kinase C negatively regulates the ANF-receptor coupled membrane guanylate cyclase system in these cells.     

Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

Lack of inhibition by atrial natriuretic factor on cyclic AMP levels in single nephron segments and the glomerulus

Recently an inhibitory effect of atrial natriuretic factor (ANF) on the adenylate cyclase system has been reported in vascular tissue. In seeking similar affects in renal tissue, we studied the effect of ANF on cyclic AMP levels in single nephron segments and in glomeruli from the rat. Individual nephron segments or glomeruli were incubated in the presence of a phosphodiesterase inhibitor, with or without parathyroid hormone (PTH) or arginine vasopressin (AVP) and varying concentrations of ANF at 37 degrees C for 2 min. The capacity for alpha 2-adrenoceptor inhibition of adenylate cyclase was demonstrated in the proximal convoluted tubule, cortical collecting tubule and in glomeruli. Nevertheless, ANF could not inhibit cAMP formation in any of these nephron segments nor in the glomerulus. Thus, unlike the vasculature, ANF has no inhibitory effect on cAMP formation in these renal tissues.     

Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF.     

Dynamics of atrial natriuretic factor-guanylate cyclase receptors and receptor-ligand complexes in cultured glomerular mesangial and renomedullary interstitial cells.

The dynamics of the guanylate cyclase receptor of atrial natriuretic factor (GCA-ANF receptor) were investigated in cultured glomerular mesangial and renomedullary interstitial cells from the rat. In these cells, the GCA-ANF receptor did not mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 and did not undergo ligand-induced endocytosis. Glomerular mesangial cells were able, however, to mediate internalization and lysosomal hydrolysis of 125I-ANF1-28 via clearance ANF (C-ANF) receptors and to promote rapid receptor-mediated internalization and lysosomal hydrolysis of 125I-(Sar1) angiotensin II. Radioligand specifically bound to surface GCA-ANF receptors was rapidly dissociated at 37 degrees C (k(off) greater than 0.8 min-1), with a Q10(30-37 degrees C) greater than 6. The dissociation was markedly slower at subphysiological temperatures (Q10(4-30 degrees C), 2-3) or in the presence of 0.5 mM amiloride. The results demonstrate that the GCA-ANF receptor, contrary to C-ANF receptors and most other polypeptide hormone receptors, is a membrane resident protein that does not mediate internalization and lysosomal hydrolysis of ligand. The termination of the interaction of ANF with GCA-ANF receptors results from a physiological process that leads to rapid dissociation of receptor-ligand complexes. The unique dynamics of GCA-ANF receptor-ligand complexes are likely to contribute importantly to stimulus-response homeostasis of ANF.     

Atrial natriuretic factor activates membrane-bound guanylate cyclase of chief cells

The present study examined the effect of atrial natriuretic factor (ANF) on cGMP generation by dispersed chief cells from guinea pig stomach. ANF caused a rapid dose-dependent increase in cGMP, a 7-fold increase in cGMP caused by 1 microM ANF, with or without 3-isobutyl-1-methylxanthine present. Methylene blue reduced cGMP in response to nitroprusside but not ANF. Guanylate cyclase activity of a chief cell membrane fraction doubled in response to ANF, but was not affected by nitroprusside. ANF had no effect on guanylate cyclase activity of the soluble fraction of lysed chief cells. Dose-response curves for whole cell cGMP production and membrane guanylate cyclase activity in response to ANF were closely related. These data indicate that ANF increases chief cell cGMP production by activating particulate guanylate cyclase, providing functional evidence that chief cells possess surface membrane receptors for ANF.     

The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase

We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no change of cGMP in the proximal tubules, a 2-fold increase over basal levels in the thick loop of Henle and a 3-fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50-fold being evident at 1-2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action.     

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cyclic GMP in atrial natriuretic factor stimulation of Na+,K+,Cl- cotransport in vascular smooth muscle cells

Atrial natriuretic factor (ANF) has been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an increase in intracellular cyclic GMP (cGMP) content. We have recently demonstrated that a prominent Na+,K+,Cl- cotransport system is present in VSMC and that a permeable cGMP analog (8-bromo-cGMP) stimulates activity of the cotransporter. We have also shown that the ANF peptide, rat atriopeptin III, stimulates Na+,K+,Cl- cotransport and elevates intracellular cGMP levels in VSMC. In the present study, we tested the hypothesis that ANF stimulation of Na+,K+,Cl- cotransport occurs via an increase in cGMP levels. When the quinolinedione, 6-anilo-5,8-quinolinedione (LY83583) (10 microM), was used to block formation of cGMP in VSMC from primary cultures of rat thoracic aorta, it was found that both basal and rat atriopeptin III (100 nM)-stimulated Na+,K+,Cl- cotransport were significantly inhibited. The effect of LY83583 was dose-dependent and the half-maximal inhibitory concentration was 0.5 microM. LY83583 also inhibited cotransport in the presence of a maximal concentration of 8-bromo-cGMP. However, this inhibition was not seen in cells also treated with 2-O-propoxyphenyl-8-azapurin-6-one (M&B 22,948), an inhibitor of cGMP phosphodiesterase. M&B 22,948 alone also increased levels of cotransport. Since inhibition of cGMP formation blocks ANF-stimulated Na+,K+,Cl- cotransport and inhibition of cGMP breakdown enhances Na+, K+, Cl- cotransport, we conclude that ANF stimulation of Na+,K+,Cl- cotransport in VSMC is mediated via increase in intracellular cGMP levels.     

Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor

The addition of ANF to Percoll-purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF-stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration-dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF-stimulated guanylate cyclase was enhanced by non-phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF-induced regulation of membrane-bound guanylate cyclase.     

Atrial natriuretic factor-induced cGMP accumulation in rat anterior pituitary cells in culture is not coupled to hormonal secretion

While atrial natriuretic factor (ANF) does not influence ACTH secretion, it was reported to have a marked stimulatory effect on the intracellular accumulation of cGMP in rat anterior pituitary cells in culture. Since many biological actions of ANF appear coupled to its excitatory action on target cell guanylate cyclase, the current study was designed to characterize the ANF-induced cGMP response in anterior pituitary with a view to determining whether the nucleotide plays a regulatory role in the secretory function of this gland. A 3 min exposure of cells in primary culture to 300 nM ANF (99-126) or 100 microM sodium nitroprusside (SNP), a stimulator of guanylate cyclase, causes maximal 10- and 3-fold elevations of cGMP levels, respectively. Following a progressive decrease, 6- and 2-fold increases over basal cGMP levels are still observed after 180 min of incubation with ANF (99-126) and SNP, respectively. The half-maximal stimulation of cGMP accumulation induced by a 10 min exposure to ANF (99-126), or rat atriopeptin II (ANF 103-125) is observed at 9 +/- 2 and 125 +/- 22 nM, respectively. ANF fragments (99-109) and (111-126), as well as human cardiodilatin (hANF 1-16), do not alter cGMP levels. Basal and ANF-induced cGMP levels are at least 10-fold higher in cell populations enriched in gonadotrophs compared to gonadotroph-impoverished preparations. A 3 h incubation of cells with ANF (0.1-1000 nM), however, fails to modify spontaneous or LHRH-induced LH secretion. Similarly, ANF does not alter spontaneous release of GH, TSH or PRL. The data suggest indirectly that gonadotrophs represent a principal site at which ANF acts to stimulate cGMP synthesis, but that the nucleotide is not a specific regulator of the LH secretory process; nor is it generally involved as a second messenger in the secretory function of any cell type of the anterior pituitary gland.     

Effects of maitotoxin on atrial natriuretic factor-mediated accumulation of cyclic GMP in PC12 cells

Maitotoxin (MTX) activates calcium channels and stimulates phosphoinositide breakdown in pheochromocytoma PC12 cells, while having no effect on basal levels of the cyclic nucleotides cAMP and cGMP. Atrial natriuretic factor (ANF) induces a dose-dependent accumulation of cGMP in PC12 cells through the activation of a membrane bound guanylate cyclase. Effects of ANF on cGMP are independent of extracellular concentrations of calcium. Since agents that activate phosphoinositide breakdown can indirectly affect cyclic nucleotide formation, the effects of MTX on ANF-mediated accumulation of cGMP was studied. MTX induces a dose-dependent inhibition of ANF-mediated accumulation of cGMP. The inhibition by MTX requires the presence of extracellular calcium, but is unaffected by the calcium channel blocker nifedipine. The inhibitory effect of MTX is not mimicked by the calcium ionophore ionomycin. A phorbol ester, PMA, which stimulates protein kinase C, also inhibits ANF-mediated accumulation of cGMP. Sodium nitroprusside induces large accumulations of cGMP in PC12 cells through the stimulation of a soluble guanylate cyclase. Neither MTX nor PMA inhibit nitroprusside-mediated accumulation of cGMP. The results indicate that in PC12 cells, protein kinase C activation, either directly with PMA, and indirectly with MTX through phosphoinositide breakdown and formation of diacylglycerol, leads to inhibition of ANF-mediated, but not nitroprusside-mediated accumulation of cGMP.     

High-density lipoprotein binding to guinea-pig hepatic membranes. Comparison of guinea-pig and human ligands

Human and guinea-pig apo-E-free HDL were incubated with isolated guinea-pig hepatic membranes at 4 and 37 degrees C to determine the effects of temperature and heterologous systems on the equilibrium parameters of HDL binding. Receptor mediated HDL binding was highest at 37 degrees C for both lipoproteins. At 4 degrees C, a higher affinity constant (Kd) was observed when guinea-pig HDL was the ligand relative to human HDL; in contrast, both HDL preparations had similar Kd values at 37 degrees C. Calculated binding and receptor number (Bmax) were higher at both temperatures when guinea-pig HDL was the ligand. These results demonstrate a significant species difference in HDL binding to hepatic membrane which is partially temperature-dependent.     

Effects of atrial natriuretic factor,nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney

Summary In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 M) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 M) and ANF (0.01 M) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 M) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

Stereoselective accumulation of the beta-receptor blocking drug atenolol by human platelets.

We have studied the stereochemistry of accumulation of the hydrophilic beta-adrenoceptor antagonist rac-atenolol by human platelets in vitro. The accumulation was slow, not reaching equilibrium until 90 min at 37 degrees C. The uptake was temperature dependent with the accumulation at 37 degrees C being 3-4 times greater than at 4 degrees C. The accumulation was also stereoselective at 37 degrees C, favoring the active (-)-enantiomer over the (+)-enantiomer by 2.3-fold. Reserpine, but not desipramine, inhibited the platelet accumulation of rac-atenolol enantiospecifically. This uptake profile is different from the platelet uptake of lipophilic beta-blockers, which is characterized by nonspecific membrane binding, but similar to the carrier-mediated accumulation of the neurotransmitter norepinephrine by storage granules within the platelet.