Species differences in the positive inotropic response to DPI 201-106, a novel cardiotonic agent

The positive inotropic activity of the novel cardiotonic DPI 201-106 was investigated in rat and guinea pig isolated hearts. For comparative purposes, the adenylate cyclase stimulant forskolin and the sodium channel agonist veratridine were also evaluated in both species. DPI 201-106 and veratridine produced greater inotropic effects in rat hearts than in guinea pig hearts, whereas forskolin produced comparable effects. In both species the inotropic response to DPI 201-106 and veratridine, but not forskolin, was reversed by the sodium channel antagonist tetrodotoxin. These results confirm that the positive inotropic effect of DPI 201-106 is due to stimulation of the sodium channel and demonstrate for the first time that species differences exist in the inotropic response to this novel cardiotonic drug.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma. Role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B4 (LTB4), leukotriene D4 (LTD4) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15 microM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB4, LTD4 and histamine. An antagonist of calmodulin, trifluoperazine (1-200 microM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC50 of this compound for inhibition of histamine was much lower (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

Maitotoxin, a potent, general activator of phosphoinositide breakdown

Maitotoxin (MTX), a potent marine toxin, elicits a calcium-dependent activation of cells that can be inhibited by calcium channel blockers like nifedipine. MTX also stimulates phosphoinositide breakdown in smooth muscle cells, NCB-20 cells and PC12 cells through a nifedipine-insensitive mechanism. We now report that MTX stimulates phosphoinositide breakdown in a wide variety of cells, and appears to represent the first general activator of this second messenger-generating system. MTX-induced stimulation of phosphoinositide breakdown is dependent in every cell line on the presence of extracellular calcium. In differentiated HL60 cells, in which a chemotactic peptide (fMLP) activates phosphoinositide breakdown via a pertussis toxin-sensitive mechanism, MTX-induced stimulation is not affected by pertussis toxin treatment. A phorbol ester has no effect on the response to MTX. Thus, MTX stimulates phosphoinositide breakdown through a calcium-dependent mechanism that at least in three cell lines (PC12, NCB20 and HL60) is not mediated by a pathway that involves a pertussis toxin-sensitive guanine nucleotide-binding protein.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B₄ (LTB₄), leukotriene D₄ (LTD₄) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15uM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB₄, LTD₄ and histamine. An antagonist of calmodulin, triflouperazine (1–200 uM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC₅₀ of this compound for inhibition of histamine was much lower (2–3uM) than the IC₅₀ for inhibition of leukotrienes (75 uM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compunds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.     

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.     

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.     

Role of G-proteins in T cell activation: non-hydrolysable GTP analogues induce early ornithine decarboxylase activity in human T lymphocytes.

Rapid activation of ornithine decarboxylase is one of the earliest recognized events during induction of a mitogenic response in human T lymphocytes. Here we show that the non-hydrolysable GTP analogues guanine-5-(gamma-thio)trisphosphate and guanylyl-5-imidodiphosphate, introduced into human T cells by means of a transient membrane permeabilization technique, can replace an external mitogenic ligand, such as concanavalin A, in inducing early ornithine decarboxylase activity. Neomycin inhibits this rapid activation at concentrations known to bind to phosphoinositides. One of the two compounds formed in polyphosphoinositide breakdown, inositol-1,4,5-trisphosphate, also induces ornithine decarboxylase activity. The other, diacylglycerol, apparently does not, since the phorbol ester, tetradecanoyl phorbol acetate, which is thought to mimic the action of diacylglycerols, does not alter basal ornithine decarboxylase activity in T cells until several hours after administration. These findings indicate that guanine nucleotide-binding regulatory (G-) protein(s) participates in the transduction of the mitogenic signal. The intracellular target system for this G-protein may include phosphoinositide breakdown and generation of inositoltrisphosphate, which might be involved in the early activation of ornithine decarboxylase.     

Effect of ouabain on mechanical and electrical properties of rat and guinea pig hearts at 180 C.

The effects of ouabain 10(-6) M on rat and guinea pig hearts have been studied at 18 degrees C, in order to reduce almost fully both the Na+, K+-dependent ATPase activity and the ouabain induced inhibition of this enzyme. In isolated guinea pig hearts the positive inotropic response to ouabain obtained at 32 degrees C disappeared at 18 degrees C. On the contrary, the contractile strength of rat hearts was slightly reduced by ouabain and in the same manner at both temperatures. Current and voltage clamp experiments carried out at 18 degrees C in ventricular fibres revealed that ouabain 10(-6) M decreased both the action potential overshoot and the fast sodium current in rat and guinea pig, by reduction of the membrane sodium conductance. Ouabain did not change the calcium current in guinea pig preparations, whereas in rat heart muscle this current was reduced. The effects of ouabain on both the action potential plateau and outward repolarizing current indicated some inconsistencies from preparation to preparation and cannot therefore be considered as significant. The persistence of the ouabain induced alterations of g Na (in rat and guinea pig) and calcium current (in rat) at 18 degrees C supports the hypothesis of two ouabain cell receptors in heart muscle.     

B lymphocyte receptors and polyphosphoinositide degradation

Resting B lymphocytes can be activated and induced to proliferate by antibodies against their antigen receptors (anti-lg). We demonstrate an early increase in the level of [3H]inositol trisphosphate in [3H]inositol-labeled murine B cells, which suggests breakdown of phosphatidylinositol bisphosphate by phospholipase C. In line with this, the level of [3H]1,2-diacylglycerol was also elevated after incubation of [3H]arachidonic-acid-labeled B cells with anti-Ig. Anti-lg also caused a rapid increase in the level of cytosolic Ca2+ in B cells. In contrast, two other polyclonal B cell activators, lipopolysaccharide and phorbol myristate acetate, failed to induce any of these effects. Our results suggest that anti-lg may induce B cell growth via phosphoinositide degradation and Ca2+ mobilization, and that phorbol myristate acetate, and possibly lipopolysaccharide, bypass these initial events.     

Early effects of beta-adrenergic and muscarinic secretagogues on lipid and phospholipid metabolism in guinea pig parotid acinar cells. Stimulation of 2,3-sn-diacylglycerol formation by isoproterenol

The early effects (0-120 s) of the beta-adrenergic secretagogue isoproterenol (2.10(-5) M) and the muscarinic secretagogue carbamoylcholine (2.10(-6) M) on various parameters of lipid and phospholipid metabolism were studied in isolated guinea pig parotid acinar cells. Both agonists enhanced within 10-20 s the incorporation of radioactive palmitate into the diacylglycerol, the triglyceride, and the phosphatidylinositol fractions but significantly diminished radioactive palmitate recovered in the acyl-CoA fraction. Carbamoylcholine decreased and isoproterenol increased the recovery of radioactive palmitate in the free fatty acid fraction. All changes had returned almost to control levels after 120 s. In cells prelabeled with [3H]arachidonate, carbamoylcholine exerted similar effects, whereas isoproterenol was almost ineffective. Both agonists stimulated the incorporation of radioactive glycerol into diacylglycerols 2-3-fold, while only carbamoylcholine stimulated the incorporation of [32P]phosphate into phosphatidylinositol and phosphatidate. Both agonists induced an increase in total diacylglycerols, carbamoylcholine being about twice as effective as isoproterenol. A lower concentration of carbamoylcholine (6.5.10(-7) M) had the same quantitative effect as 2.10(-5) M isoproterenol on the increase of total diacylglycerols. Even under these conditions carbamoylcholine, but not isoproterenol led to a significant translocation of protein kinase C from the soluble to the particulate fraction. Isoproterenol remained ineffective in this respect also when intracellular free calcium was increased with a calcium ionophore. This is explained by the finding that isoproterenol stimulates preferentially the formation of 2,3-sn-diacylglycerol, and carbamoylcholine preferentially stimulates the formation of 1,2-sn-diacylglycerol. The results indicate that in the guinea pig parotid acinar cell the two agonists do not only lead to activation of a triglyceride lipase (isoproterenol) or phosphoinositide-specific phospholipase(s) (carbamoylcholine), but also to a rapid change of flux through a number of other enzyme-catalyzed reactions involved in diacylglycerol turnover.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Activation of the C3b receptor: effect of diacylglycerols and calcium mobilization

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.     

Effect of thymol on calcium handling in mammalian ventricular myocardium

Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Maitotoxin stimulates phosphoinositide breakdown in neuroblastoma hybrid NCB-20 cells

1. Maitotoxin (MTX) was an extraordinarily potent stimulant of phosphoinositide breakdown in the neuroblastoma hybrid NCB-20 cells. 2. Maximal responses were obtained at 0.25-0.5 ng MTX/ml, and resulted in increased formation of [3H]inositol mono-, bis-, and trisphosphates. Increased formation of [3H]inositol bis- and trisphosphate was observed as early as 15 sec after the addition of MTX. 3. MTX-induced phosphoinositide breakdown in NCB-20 cells was not antagonized by organic (nifedipine, methoxyverapamil) or inorganic (Mn2+, Co2+, Cd2+) calcium channel blockers. However, the response on phosphoinositide breakdown was completely eliminated in the absence of extracellular calcium. 4. The results suggest that MTX either directly stimulates phosphoinositide breakdown in a calcium-dependent manner or acts indirectly through calcium channels insensitive to organic/inorganic calcium channel blockers.     

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay K8644 and inhibited by 8-bromo-cyclic GMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclic GMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.