Calcium: its role in alpha 1-adrenergic stimulation of atrial natriuretic peptide secretion.

alpha 1-Adrenergic agonists increase atrial natriuretic peptide (ANP) secretion. The mechanism of alpha 1-adrenergic-stimulated secretion is not known. In this study we examine the calcium dependency of alpha 1-agonist-stimulated ANP secretion. Isolated superfused rat left atria paced at 2 Hz were used for study. Superfusion with 10 microM phenylephrine increased ANP secretion by 2-fold. Lowering the superfusate calcium concentration from 1.8 to 0.2 mM totally negated the secretory response to phenylephrine. To determine whether this reflected a reduction in calcium influx, reduced calcium release from the sarcoplasmic reticulum (SR), or both, atria were superfused with 1 microM ryanodine, an inhibitor of SR calcium release. Ryanodine had no effect on phenylephrine-stimulated ANP secretion. Atria were superfused with 10 microM nitrendipine to determine whether calcium influx through voltage-dependent calcium channels was a mechanism of calcium entry for stimulation. Nitrendipine inhibited phenylephrine-stimulated ANP secretion by 49% without interfering with alpha 1-adrenergic antagonist receptor binding. This finding was supported by the observation that phenylephrine-stimulated secretion was 52% lower in nonbeating atria. alpha 1-Adrenergic agonists have been reported to enhance Na-H antiporter activity. To determine whether the resulting rise in intracellular sodium may alter Na-Ca exchange to raise intracellular calcium levels, atria were superfused with the Na-H antiporter inhibitor, 5-(N,N-hexamethylene)amiloride. Superfusion with 25 microM 5-(N,N-hexamethylene)amiloride did not inhibit phenylephrine-stimulated ANP secretion. Lastly, the calcium dependency of the maintenance of an established response to phenylephrine was examined. Atria were superfused with phenylephrine in buffer containing 1.8 mM calcium for 45 min, followed by superfusion with phenylephrine in 0.2 mM calcium for 30 min. There was no fall in phenylephrine-stimulated secretion by atria superfused in 0.2 mM calcium. In contrast, addition of the alpha 1-adrenergic antagonist phentolamine induced an immediate fall in phenylephrine-stimulated ANP secretion. We conclude that 1) calcium influx is necessary to initiate alpha 1-agonist-stimulated ANP secretion; 2) calcium release from the SR does not play a role in alpha 1-agonist-stimulated secretion; 3) calcium entry through L-type calcium channels is responsible for half of the calcium influx; 4) enhanced Na-H antiporter activity does not play a role in alpha 1-agonist-stimulated secretion; and 5) maintenance of alpha 1-agonist-stimulated secretion is not dependent on calcium influx.     

Stimulation of calcium flows induced by endothelin in cultured smooth muscle cells

Endothelin is a potent vasoconstrictor peptide isolated from the conditioned medium of porcine aortic endothelial cells. The action of endothelin is thought to be associated with calcium entry via calcium potential channels (Yanagisawa et. al. Nature 1988; 38:411-415). The present study was designed to determine the effect of endothelin on calcium fluxes (influx and efflux) on rat aortic smooth muscle cells in culture. The unidirectional influx of calcium was measured 15, 45, 75 and 105 seconds after the addition of trace amounts of 45Ca++ (5 microCi/ml) to the cells incubated with or without endothelin. Endothelin (50nM) stimulated calcium influx from a basal level of 312 +/- 17 to 537 +/- 12 pmol/mn/10(6) cells. This stimulation was dose-dependent with an EC50 value of about 10 nM. When cells were preincubated with calcium antagonists (nifedipine, dilttiazem, D600, nicardipine and flunarizine) at a final concentration of 1 microM, the endothelin-stimulated calcium influx was not modified. The unidirectional efflux of calcium was measured after an overload of cells with 45Ca++ (5 microCi/ml) for 18 hours, over 10 seconds intervals. In the first 30 seconds after the addition of endothelin (100 nM), the amount of 45Ca++ released was 3 times that in the absence of the peptide. The effect of endothelin was concentration dependent and similar to those observed with other vasoconstrictor peptides (vasopressin and angiotensin II). The results indicate that endothelin does not directly act on voltage-dependent calcium channels. The endothelin-stimulated calcium efflux suggests a mobilization of calcium from intracellular store sites followed by extrusion through an activation of a specific receptor-dependent calcium channel.     

Endothelin increases atrial natriuretic peptide production in cultured rat diencephalic neurons.

The vasoactive peptides atrial natriuretic peptide (ANP) and endothelin have been localized to several areas of the extravascular brain where they are produced in neurons, often overlapping in distribution. Since endothelin has been found to increase ANP secretion from the heart, we examined a possible regulation by endothelin of the secretion and production of ANP in fetal diencephalic cultures of neurons from gestational day 16 rats. Endothelin produced a concentration-related increase in the secretion of ANP. The ability of endothelin to induce ANP secretion was dependent upon calcium, since added nifedipine, a calcium channel blocker, significantly reversed the effects of this vasoconstrictor peptide. ANP 103-126 was the predominant form secreted, as determined by HPLC followed by RIA. The production of this peptide, assessed by specific mRNA expression, was increased more than 2-fold, in dose-related fashion. These studies provide the first evidence that endothelin regulates ANP gene expression in the brain. Additionally, our secretion studies from neurons are consistent with the known stimulatory effects of endothelin on ANP release from the heart. The regulation of ANP production and secretion by the vasoconstrictor peptide endothelin is one level at which ANP and endothelin might interact in the in vivo brain.     

Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action

Calcitonin gene-related peptide (CGRP) is present in nerve fibers within atrial tissue, raising the possibility that CGRP release may influence atrial natriuretic peptide (ANP) secretion. We, therefore, examined the effect of CGRP on immunoreactive ANP (ANP-IR) secretion. Isolated rat left atria paced at 2 Hz were superfused with 0.1 microM CGRP. A biphasic 2-fold increase in ANP-IR secretion occurred in response to CGRP. We next examined the mechanism of CGRP-stimulated secretion. The biphasic ANP-IR secretory response to CGRP was similar to that induced by superfusion with the beta-adrenergic agonist isoproterenol and (Bu)2cAMP, but distinct from that of the non-cAMP dependent stimuli phenylephrine, ouabain, and Bay K 8644. Superfusion with 0.1 microM CGRP for 4 min with 100 microM isobutylmethylxanthine increased atrial cAMP content from 4.29 +/- 1.21 to 10.32 +/- 2.14 pmol/mg atrial weight (P less than 0.001). Atria were next superfused with methacholine, an inhibitor of adenylyl cyclase activation. The addition of 0.1 microM isoproterenol or 0.1 microM CGRP to the superfusate containing 10 microM methacholine failed to stimulate ANP-IR secretion and lowered cAMP accumulation by 70%. Superfusion with 10 microM atropine negated the inhibitory effects of methacholine. We conclude that 1) CGRP stimulates ANP-IR secretion in vitro; and 2) CGRP-stimulated secretion appears to be mediated by cAMP. Thus, ANP-IR secretion may be modulated by atrial nerve fibers containing CGRP in vivo.     

Effects of atrial natriuretic peptide on endothelin-induced vasoconstriction and intracellular calcium mobilization.

Mechanisms for the antagonistic action of atrial natriuretic peptide (ANP) toward endothelin were studied. We examined the effects of ANP on endothelin-induced vasoconstriction in isolated perfused kidney and the elevation of intracellular free calcium concentration using Fura-2 in cultured rat vascular smooth muscle cells (VSMC). Endothelin produced an increase in renal vascular resistance in isolated perfused kidney (2 x 10(-10) mol/l, +109%; P less than 0.01). ANP completely abolished renal vasoconstriction induced by endothelin. Endothelin produced a biphasic elevation in intracellular calcium concentration in the VSMC. Ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetr aac etic acid (EGTA; 3 mmol/l) or nicardipine (10(-8) mol/l) decreased the sustained elevation of intracellular calcium concentration by 10(-8) mol/l endothelin to the basal level (endothelin, +15% versus EGTA + endothelin, -1%; P less than 0.05; endothelin, +18% versus nicardipine + endothelin, +5%; P less than 0.01). Pretreatment with 10(-6) mol/l ANP suppressed neither the peak phase of intracellular calcium concentration elevation (endothelin, 10(-8) mol/l, +35% versus endothelin + ANP, +31%; not significant) nor the sustained phase of intracellular calcium concentration elevation (endothelin, 10(-8) mol/l, +21% versus endothelin + ANP, +16%; not significant). ANP markedly increased the production of cyclic 3',5'-guanosine monophosphate (cGMP) in the VSMC (ANP, 10(-6) mol/l, +1100%; P less than 0.01). However, endothelin did not influence cGMP production in the presence or absence of ANP. ANP may antagonize the vasoconstrictive action of endothelin through an effect on the steps subsequent to the mobilization of intracellular calcium in the pathway of vascular smooth muscle contraction.     

Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats

Intravenous (bolus) administration of endothelin results in a transient fall in blood pressure that is accentuated in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto normotensive rats (WKY). In attempting to discern possible mechanisms underlying this depressor response, we examined the ability of endothelin to release atrial natriuretic factor (ANF) from isolated, spontaneously contracting atria from SHR and WKY. Isolated right atria were suspended under 3.0 g of resting force in tissue baths with the amount of immunoreactive ANF (irANF) released after exposure to endothelin assessed by radioimmunoassay. Endothelin (10(-8) and 10(-7) M) caused a concentration-dependent increase (1.5-4.5-fold) in the release of irANF, which was significantly greater in atria of SHR compared with WKY. The greater release of irANF in atria of SHR versus WKY was not related to tissue weight or changes in contractile rate or force induced by endothelin. Therefore, endothelin appears to cause a direct release of irANF from rat right atria in vitro. As found for the depressor response in vivo, endothelin is more efficacious in the hypertensive compared with the normotensive atrial preparation. Release of ANF may be important in the hypotensive response to endothelin in vivo.     

Inhibition of atrial wall stretch-induced cardiac hormone secretion by lavendustin A, a potent tyrosine kinase inhibitor.

The cellular processes linking mechanical wall stretch to atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secretion from the heart are unclear. In the present study, a paced perfused rat heart preparation was used to study the signaling mechanisms of atrial wall stretch-induced secretion of ANP and BNP. Vehicle or drugs were infused into the perfusate for 40 min and right atrial wall stretch was superimposed for 10 min after 25-min drug infusions by elevating the level of the pulmonary artery cannula tip. Lavendustin A, a potent inhibitor of protein tyrosine kinases, at the concentrations of 0.5 and 1.3 microM decreased atrial wall stretch-induced ANP secretion (53% and 68%, respectively, P < 0.001) in the perfused rat heart preparation, whereas no difference in the hemodynamic variables (heart rate, contractile force and perfusion pressure) were noted between groups. Lavendustin A also completely abolished the wall stretch-induced secretion of BNP. Several other protein kinase inhibitors including staurosporine (protein kinase C inhibitor), ML-9 (myosin light chain kinase inhibitor), KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) and H-89 (protein kinase A inhibitor) had no significant effect on atrial wall stretch-stimulated ANP secretion. In a separate series of experiments, in which the right atria were stretched for 2 h, administration of lavendustin A (1 microM) but not staurosporine (30 nM) significantly decreased sustained wall stretch-induced ANP secretion. Okadaic acid, a potent protein phosphatase A2 (PPA2) and PP1 inhibitor, at the concentration of 100 nM had no effect on basal ANP secretion but significantly accelerated the ANP secretory response to atrial wall stretch (P < 0.05). In conclusion, the findings that inhibitors of protein tyrosine kinase and protein phosphatase selectively modulated atrial wall stretch-induced ANP secretion suggest a new mechanism involving endogenous protein tyrosine activity in the regulation of natriuretic peptide exocytosis from cardiac myocytes.     

The adrenal capsule alters the response of zona glomerulosa cells to atrial natriuretic peptide

Atrial natriuretic peptide (ANP) is a potent inhibitor of potassium-stimulated aldosterone secretion. In the present study, we observed rat alpha ANP to inhibit aldosterone secretion stimulated by 10 mM potassium with an IC50 of 0.15 +/- 0.02 nM (mean +/- SE) in dispersed rat adrenal glomerulosa cells. However, when rat adrenal capsules, which contain the zona glomerulosa, were superfused in vitro, ANP had no effect on aldosterone secretion. Superfusion with 10 mM potassium increased aldosterone secretion 3- to 4-fold above baseline. Addition of 10 nM ANP to the superfusate did not lower potassium-stimulated aldosterone secretion. When this same ANP-containing superfusate was incubated with dispersed adrenal glomerulosa cells, potassium-stimulated aldosterone secretion was inhibited by 90%, proving sustained biological potency of the superfused ANP. Incubation of [125I]iodo-ANP with adrenal capsules for 60 min resulted in 83% degradation of [125I]iodo-ANP, whereas no detectable degradation was observed with dispersed adrenal glomerulosa cells. Removal of blood from the adrenal capsules or culturing the capsules for 48 h did not render them responsive to superfused ANP. In contrast, superfusion of 0.1 mM cycloheximide inhibited potassium-stimulated aldosterone secretion by 90%. These results suggest that the adrenal capsule contains an ANP-degrading enzyme(s). This enzyme may be produced by adrenal glomerulosa cells. The local existence of a degrading enzyme for ANP may allow the zone glomerulosa to regulate its response to ANP.     

Mechanisms of atrial natriuretic peptide secretion from the atrium

Since the discovery of atrial natriuretic peptide (ANP) more than 20 years ago, numerous studies have focused on the mechanisms regulating ANP secretion. From a physiological standpoint, the most important factor governing ANP secretion is mechanical stretching of the atria, which normally occurs when extracellular fluid volume or blood volume is elevated. In addition, the ability of several vasoconstrictors to increase ANP secretion can be traced to their indirect effects on atrial stretch via increases in cardiac preload or afterload. Whether vasoconstrictors such as angiotensin II and vasopressin have a direct positive or negative effect on ANP secretion has not been determined with certainty. Two paracrine factors derived from endothelial cells play important roles in modulating ANP secretion. Endothelin, a potent vasoconstrictor, stimulates ANP secretion and augments stretch induced ANP secretion. The dramatic increase in ANP release produced by cardiac ischemia appears to be mediated in part by endothelin. Nitric oxide (NO), an important vasodilator, is also produced by endothelial cells and inhibits ANP secretion acting through cyclic GMP as an intracellular messenger. Several recent studies have helped to define the cellular mechanism contributing to regulation of ANP secretion including stretch-activated ion channels, prostaglandins, cytochrome P450, G proteins and cell calcium. A number of steps in the cellular transduction of the ANP signal remain to be resolved. The release of ANP in disease states such as myocardial infarction and heart failure appears to be related to both mechanical and cellular events.     

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.     

Mechanism of cholinergic stimulation of aldosterone secretion in bovine adrenal glomerulosa cells

The effect of cholinergic stimulation on aldosterone secretion was examined in bovine adrenal glomerulosa cells. Both acetylcholine and carbachol stimulated aldosterone secretion in a dose-dependent manner. Acetylcholine-induced secretion was inhibited by atropine but not by hexamethonium, suggesting that cholinergic agonists act on muscarinic receptors. The mechanisms of cholinergic agonist action were compared with those of angiotensin II. Like angiotensin II, carbachol generated calcium signal in glomerulosa cells. When [3H]inositol-labeled cells were stimulated by carbachol, there was an immediate increase in [3H]inositol trisphosphate, followed by a relatively slow increase in [3H]inositol bisphosphate. Carbachol increased the cytosolic concentration of calcium transiently but not intracellular cAMP. Carbachol caused a rapid 3-fold increase in 45Ca fractional efflux ratio in 45Ca-prelabeled cells both in the presence and absence of extracellular calcium. Carbachol also increased calcium influx; however, carbachol-induced influx was smaller than that of angiotensin II. In a perifusion system, the time course of carbachol-induced aldosterone secretion was biphasic. However, when calcium influx was increased to a value similar to that in angiotensin II-treated cells by combination of carbachol and BAY K-8644, this combination induced a monophasic and sustained secretory pattern. These results indicate that muscarinic cholinergic agonists stimulate aldosterone secretion via the calcium messenger system, and the biphasic secretory response to cholinergic agonist is due to a smaller increase in calcium influx.     

Mechanisms of atrial natriuretic peptide (ANP) secretion by rat hearts perfused in vitro--Ca2(+)-dependent signal transduction for ANP release by mechanical stretch

Effects of temperature, contraction frequency, and intraatrial pressure on immunoreactive ANP release were investigated in isolated rat hearts perfused in Langendorff or working mode. A reduction of temperature from 37 C to 27 degrees C caused a decrease of ANP release by 64% indicating its marked temperature-dependency (Q10 = 2.92). An increase of atrial contraction frequency from 300 to 500/min in Langendorff-perfused hearts did not cause a significant change in the ANP release. An elevation of left atrial filling pressure of working hearts from 8 to 18 and 28 cmH2O was associated with pressure-dependent, and reversible increase of the ANP release. This pressure-induced release of ANP was inhibited in a low calcium (50% Ca2+) medium or by nifedipine (10(-7) M). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7, 10(-7)M), a potent calmodulin inhibitor, or ryanodine (10(-8)M) had similar inhibitory action against the pressure-induced increase of ANP release. These results indicate that ANP secretion is primarily regulated by mechanical stretch or distension of the atrial wall, while the atrial contraction frequency is less important as a physiological stimulus for the secretion. The stretch-induced ANP secretion may require an influx of calcium through the voltage-dependent Ca2+ channels. It was also suggested that Ca2+ release from the sarcoplasmic reticulum leading to an activation of calcium-calmodulin kinase may be included in the intracellular processes of ANP release by mechanical stretch.     

Passive mechanical stretch releases atrial natriuretic peptide from rat ventricular myocardium.

Ventricular hypertrophy is characterized by augmentation of synthesis, storage, and release of atrial natriuretic peptide (ANP) from ventricular tissue, but the physiological stimulus for ANP release from ventricles is not known. We determined the effect of graded, passive myocardial stretch on ANP release in isolated, arrested, perfused heart preparations after removal of the atria in 13-20-month-old Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). By this age, ANP gene expression was increased in the hypertrophic ventricular cells of SHR, as reflected by elevated levels of immunoreactive ANP and ANP mRNA and the increased ANP secretion (SHR, 93 +/- 14 pg/ml, n = 22; WKY rats, 22 +/- 2 pg/ml, n = 20; p less than 0.001) from perfused ventricles after removal of the atria. The release of ANP from ventricles was examined at two levels of left ventricular pressure by increasing the volume of the intraventricular balloon for 10 minutes. Stretching of the ventricles produced a rapid but transient increase in ANP secretion. As left ventricular pressure rose from 0 to 14 and 26 mm Hg in WKY rats and from 0 to 13 and 27 mm Hg in SHR, increases in ANP release into the perfusate of 1.4 +/- 0.1-fold and 1.5 +/- 0.2-fold (p less than 0.05) in WKY rats and 1.1 +/- 0.1-fold and 1.6 +/- 0.2-fold (p less than 0.05) in SHR, respectively, were observed. There was a highly significant correlation between the left ventricular pressure level and the maximal concentration of ANP in the perfusate during stretching (p less than 0.001, r = 0.59, n = 42), as well as between the maximal ANP concentrations in perfusate during stretching and the ventricular weight/body weight ratios of the corresponding animals (r = 0.38, p less than 0.05, n = 42). High performance liquid chromatographic analysis revealed that the ventricles both before and during stretch primarily released the processed, active, 28-amino acid ANP-like peptide into the perfusate. These results indicate that stretching is a direct stimulus for ventricular ANP release and show that ANP is also a ventricular hormone.     

The influence of resting tension on immunoreactive atrial natriuretic peptide secretion by rat atria superfused in vitro

Atrial natriuretic peptide is a potent diuretic hormone secreted by the atria in response to volume expansion. We examined the effect of resting tension on atrial natriuretic peptide secretion by rat atria superfused in vitro. Left atria were hooked between an electrode and force transducer and superfused with medium 199. The atria were studied at a pacing frequency of 0 or 3 Hz. Atrial natriuretic peptide content of the superfusate was measured by radioimmunoassay. In nonpaced and paced atria, increasing resting tension three- to five-fold caused immunoreactive atrial natriuretic peptide secretion to increase by 35 +/- 5% (mean +/- SEM, n = 6, p less than 0.01) and 30 +/- 3% (n = 4, p less than 0.01), respectively. Lowering resting tension by 50% in nonpaced and paced atria lowered immunoreactive atrial natriuretic peptide secretion by 30 +/- 3% (n = 7, p less than 0.01) and 24 +/- 3% (n = 6, p less than 0.01), respectively. To exclude the possibility that release of norepinephrine or acetylcholine from endogenous nerve endings was mediating this effect, the atria were superfused with the combination of propranolol 0.1 microM, phentolamine 1.0 microM, and atropine 10 microM. These concentrations of the antagonists were 125-fold or higher than their Kd for binding to their respective receptors. The antagonists did not block the rise in immunoreactive atrial natriuretic peptide secretion; neither did they inhibit an established rise in immunoreactive atrial natriuretic peptide secretion induced by increasing the resting tension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kir6.2-deficient mice are susceptible to stimulated ANP secretion: K(ATP) channel acts as a negative feedback mechanism?

OBJECTIVE: While atrial natriuretic peptide (ANP) has been shown to be released mainly from cardiac muscle cells in response to atrial distension, the regulatory mechanisms of ANP secretion are still not fully understood. We sought to determine whether the ATP-sensitive K+ (K(ATP)) channel modulates the secretion of ANP, using mice with homozygous knockout of the Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene. METHODS: K(ATP) channel currents were recorded from isolated mouse atrial cells with patch-clamp techniques. Plasma ANP concentrations in anesthetized mice and ANP content and secretion in isolated atrial preparations were determined by radioimmunoassay. Action potentials were recorded from the isolated atria. RESULTS: Exposure to 2,4-dinitrophenol (100 microM) evoked a glibenclamide-sensitive K(ATP) channel current in atrial cells from wild-type (WT) but not Kir6.2 knockout (Kir6.2 KO) mice. Although there were no significant differences in the basal plasma ANP levels between WT and Kir6.2 KO mice, volume expansion caused a significant elevation of plasma ANP concentration in Kir6.2 KO but not WT mice with accompanying hypotension. When isolated left atria were stretched, ANP secreted into the bath from Kir6.2 KO atria was significantly higher than that from WT atria. Furthermore, stretching the atria from WT but not Kir6.2 KO mice significantly shortened the action potential duration. A hypotonic stretch of the membrane induced the glibenclamide-sensitive K(ATP) channel current in atrial cells from WT but not Kir6.2 KO mice. CONCLUSIONS: Kir6.2 is essential for the function of K(ATP) channel in mouse atrial cells. Given that Kir6.2 KO mice are susceptible to stretch-induced secretion of ANP, our results suggest that K(ATP) channels may act as a negative feedback mechanism for the control of ANP secretion.     

Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na(+)-H+ exchanger.

Endothelin, a 21-amino acid vasoactive peptide, is among the most potent positively inotropic agents yet described in mammalian heart. Having demonstrated that endothelin's inotropic effect is due, in part, to an apparent sensitization of cardiac myofilaments to intracellular calcium, we determined whether this could be due to a rise in intracellular pH (pHi). In isolated adult rat ventricular cells loaded with the H(+)-selective fluorescent probe BCECF, 100 pM endothelin increased contractile amplitude to 190 +/- 26% of baseline and pHi by 0.08 +/- 0.02 (n = 8), whereas 1 nM endothelin increased pHi by 0.13 +/- 0.03 with little further increase in contractility. Amiloride (10(-4)M) prevented the increase in pHi in response to endothelin and reduced the inotropic response by 45%, although the inotropic effect could be readily restored by subsequent NH4Cl-induced alkalinization. Similarly, inhibitors of protein kinase C (H-7 and sphingosine) diminished or abolished the rise in pHi after endothelin superfusion while causing a decline in its inotropic effect comparable with that observed with amiloride. Pretreatment with pertussis toxin, which we have demonstrated results in complete ADP-ribosylation of the alpha-subunits of Go and Gi GTP-binding proteins and abolition of endothelin's positive inotropic effect, only partially reduced the intracellular alkalinization induced by the peptide, suggesting a complex signal transduction mechanism. Thus, the positive inotropic action of endothelin is due in part to stimulation of the sarcolemmal Na(+)-H+ exchanger by a protein kinase C-mediated pathway, resulting in a rise in pHi and sensitization of cardiac myofilaments to intracellular Ca2+.     

Phorbol esters enhance stretch-induced atrial natriuretic peptide secretion.

Stretching of atrial myocytes stimulates atrial natriuretic peptide (ANP) secretion, but the cellular processes linking mechanical distention to ANP release are unknown. We studied whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion using the modified perfused rat heart preparation that enabled stepwise distention of the right atrium as an experimental model for stretch-stimulated ANP release. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate immunoreactive ANP (IR-ANP) concentration (from 8.3 +/- 1.1 ng/5 min to 13.9 +/- 2.0 ng/5 min, P less than 0.05, n = 14). During stretch, a slight inotropic response was observed, while heart rate and perfusion pressure remained unchanged. Increase in right atrial pressure in the presence of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, resulted in a significantly greater increase in the perfusate IR-ANP concentration than after vehicle infusion. The calculated ANP increase corresponding to the 2 mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.84-fold when 10 nM TPA was infused (P less than 0.05). Infusion of TPA at a dose of 24 nM further increased the stretch-induced ANP release by causing 2.22-fold (P less than 0.01) increase in IR-ANP secretion. As judged by gel filtration chromatography, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester. Additionally, a phorbol ester analog, 4 alpha-phorbol 12,13-didecanoate, which is incapable of binding to and activating protein kinase C, was inactive as an ANP secretagogue. In contrast, drugs known to increase the concentration of intracellular Ca2+ in myocytes, Bay K8644 (3 and 6 microns) and forskolin (0.14 microM), significantly inhibited the stretch-stimulated ANP release. This study shows that phorbol ester enhances atrial stretch-stimulated ANP secretion from the isolated perfused heart, suggesting that protein kinase C activity is positively coupled to the stretch-induced ANP release. The results further demonstrate the negative effect of increase in intracellular Ca2+ on stretch-induced ANP release.     

Effects of endothelin upon blood pressure in normotensive rabbits and in perinephritis hypertension.

AIMS: The effects of endothelin upon blood pressure were investigated in normotensive and hypertensive rabbits. METHODS: Endothelin was injected intravenously into conscious animals and blood pressure was monitored. Groups were pretreated with vehicle, calcium antagonists, indomethacin to block prostaglandin release, or NG-nitro-L-arginine methyl ester (L-NAME) to block endothelium-derived relaxing factor (EDRF) production in order to study the mechanisms of action of endothelin in normotensive and hypertensive animals. RESULTS: Intravenous endothelin caused a rapid depressor response lasting less than 1 min followed by a prolonged pressor response. Calcium antagonists attenuated this pressor response. Hypertensive animals showed a greater sensitivity to calcium antagonists than normotensives. High concentrations of calcium antagonists abolished the pressor response, revealing a more prolonged depressor response lasting up to 5 min. Indomethacin pretreatment caused an apparent dose-related increase in pressor responses in all animals. L-NAME pretreatment enhanced responses in normotensives but caused no change or a decrease in these responses in hypertensive animals. Neither calcium antagonists, indomethacin or L-NAME modified the initial depressor response to endothelin. However when given together with nifedipine infusion, which abolished the pressor response, indomethacin and L-NAME decreased the duration of the depressor response. CONCLUSIONS: In conscious rabbits extracellular calcium influx is important in mediating pressor responses to endothelin. In normotensive rabbits endothelin apparently causes release of prostaglandin and EDRF modifying responses. In hypertensive rabbits, a role for prostaglandins but not EDRF was observed in modulating responses to endothelin. Thus, the measured response to endothelin is the sum of a number of effects, the relative importance of which may be altered in pathological conditions.     

Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms

The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.