Receptor subtypes mediating the inotropic effects and Ca(2+) signaling induced by endothelin-1 through crosstalk with norepinephrine in canine ventricular myocardium

In canine ventricular myocardium, endothelin-1 (ET-1) alone induced only a weak transient negative inotropic effect (NIE). However, ET-1 induced a marked sustained positive inotropic effect (PIE) subsequent to a transient NIE in the presence of norepinephrine (NE) at low concentrations (0.1 - 1 nM) and elicited a pronounced sustained NIE in the presence of NE at high concentrations (around 100 nM). Thus, the extent of beta-adrenoceptor stimulation induced by NE played a crucial role in determining the characteristics of the inotropic effects of ET-1. The characteristics of ET receptor subtypes involved in contractile regulation and Ca(2+) signaling induced by ET-1 were determined. The ET-1-induced transient NIE and decrease in Ca(2+) transients were abolished by the selective ET(A)-receptor antagonist FR319317, but not by the selective ET(B)-receptor antagonist BQ-788. The sustained PIE and the increase in Ca(2+) transients induced by ET-1 were abolished by FR319317, but not inhibited by BQ-788. In contrast, the sustained NIE of ET-1 was abolished by the non-selective ET antagonist TAK-044, markedly attenuated by FR319317, and partially inhibited by BQ-788. ET-1 alone elicited a PIE in the presence of BQ-788, which indicates that the activation of ET(B)-receptors counteracts the development of the PIE of ET-1. The current findings indicate that both ET(A) and ET(B) receptors are involved in the regulation of Ca(2+) signaling and contractility in canine ventricular myocardium.     

Wortmannin Inhibits the Increase in Myofilament Ca2+ Sensitivity Induced by Cross-Talk of Endothelin-1 With Norepinephrine in Canine Ventricular Myocardium

Endothelin-1 (ET-1) modulates cardiac contractility by cross-talk with norepinephrine (NE) in canine ventricular myocardium. The present experiments were performed to investigate the influence of wortmannin that has inhibitory action on phosphatidylinositol 3-kinase (PI3-K) (IC₅₀ = 3 nM) and myosin light chain kinase (MLCK) (IC₅₀ = 200 nM) on Ca²⁺ signaling and the inotropic effects of ET-1 induced by cross-talk with NE. Experiments were carried out in isolated canine ventricular trabeculae and indo-1/AM–loaded single ventricular cardiomyocytes. ET-1 alone elicited a transient small negative inotropic effect (NIE). In the presence of NE at low (1 – 10 nM) and high (100 nM) concentrations, ET-1 induced a long-lasting positive inotropic effect (PIE) or a marked sustained NIE, respectively. Wortmannin up to 300 nM did not affect the contractility; and at 1 μM and higher, it decreased the basal contraction without suppressing Ca²⁺ transients. Wortmannin (1 μM) inhibited the long-lasting PIE of ET-1 without affecting the ET-1–induced increase in Ca²⁺ transients. Wortmannin at the same concentration did not affect the ET-1–induced transient and sustained NIE and the PIE mediated by β-adrenoceptor stimulation. These results imply that wortmannin exerts selective inhibitory action on the increase in myofilament Ca²⁺ sensitivity induced by cross-talk of ET-1 with NE probably through an inhibition of MLCK in canine ventricular myocardium.     

On the inotropic actions of arginine vasopressin in ventricular muscle of the guinea pig heart

Arginine vasopressin (AVP) (0.03-3 microM) produced transient negative responses that were followed by slowly developing positive inotropic responses in guinea pig papillary muscles. The inotropic responses were antagonized by OPC-21268 (10 microM), but not by OPC-31260, and suppressed by neomycin (3 mM). The negative inotropic response was hardly affected by staurosporine (10 nM) and H-7 (10 microM). AVP (1 microM) elicited a transient decrease followed by a slowly developing increase in I(Ca). The latter increase in I(Ca) was not elicited in the presence of staurosporine (1 nM). These results indicate that AVP stimulates V1 receptors that couple to phosphoinositide hydrolysis and thereby increases intracellular Ca2+ and activates protein kinase C.     

Amiloride: relationship between cardiac effects and inhibition of Na+/Ca2+ exchange

The potassium sparing diuretic amiloride at concentrations ranging between 0.1-0.8 mM inhibited the Na+/Ca2+ exchange in sarcolemmal vesicles isolated from beef heart. The rate of exchange activity was 50% reduced by 0.35 mM amiloride. In spontaneously beating atria isolated from normal and reserpinized guinea-pigs, amiloride produced a concentration-dependent positive inotropic effect and negative chronotropic effect (EC50 = 0.7 mM). Amiloride protected spontaneously beating atria and left atria driven at 1 Hz from digitalis cardiotoxicity assessed in terms of a raised end-diastolic tension. It is suggested that the positive inotropic effect, negative chronotropic effect of amiloride and heart protection against digitalis toxicity are related to the observed inhibition of sarcolemmal Na+/Ca2+ exchange activity.     

Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943.

1. The effects of endothelin-1 (ET-1) on intracellular Ca2+ ion level and cell contraction were simultaneously investigated in rabbit ventricular cardiac myocytes loaded with indo-1/A1. The role of Na+/Ca2+ exchange in ET-1-induced positive inotropic effect (PIE) was examined by using KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate), a selective inhibitor of reverse mode Na+/Ca2+ exchange. 2. ET-1 at 0.3 pM - 1 nM increased cell contraction and Ca2+ transient (CaT) with EC50 values of 2.9 pM and 1.2 pM, respectively, and the increase in amplitude of CaT was much smaller relative to the PIE: ET-1 at 1 nM increased peak cell shortening by 237%, while it increased peak CaT by 167%. For a given level of PIE, ET-1-induced increase in CaT was much smaller than that induced by elevation of [Ca2+]o and by isoprenaline. Therefore, ET-1 shifted the relationship between peak CaT and cell shortening to the left relative to the relationship for increase in [Ca2+]o, an indication that ET-1 increased myofibrillar Ca2+ sensitivity. 3. KB-R7943 at 0.1 microM and higher inhibited contraction and CaT induced by 0.1 nM ET-1 and at 0.3 microM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1-0.3 microM, KB-R7943 neither inhibited baseline contraction and CaT nor the isoprenaline-induced response, although at 1 microM and higher it had a significant inhibitory action on these responses. 4. These results indicate that in rabbit ventricular myocytes both increases in CaT and myofibrillar Ca2+ sensitivity contribute to the ET-induced PIE, and the activation of reverse mode Na+/Ca2+ exchange may play a crucial role in increase in CaT induced by ET-1 in rabbit ventricular cardiac myocytes.     

Effects of amiloride and ouabain on contractile state, Ca and Na fluxes, and Na content in cultured chick heart cells

Amiloride has been reported to reduce the positive inotropic and toxic actions of cardiac glycosides in patients as well as in experimental animals. To investigate the mechanism by which amiloride interacts with glycosides at the cellular level, we examined the effect of amiloride and ouabain on cellular Na content and uptake, Ca flux via Na-Ca exchange, and contractile state. Amiloride (1 mM) reduced cellular Na content by 16% (p less than 0.05) under normal conditions and by 45% in the presence of 1 mM ouabain compared to respective control values observed in the absence of amiloride. Amiloride (1 mM) reduced the initial rate of 45Ca uptake by 40% in ouabain (1 mM)-treated cells. This reduction of 45Ca uptake could be mimicked by lowering cellular Na content by 42%. Amiloride (1 mM) did not alter significantly the initial rate of 24Na uptake under normal conditions but reduced it by 32% in the presence of 3 microM ouabain. Amiloride (1 mM) produced a transient increase followed by a gradual decrease in the amplitude of cell motion over 60 min to 10% of control level. At other concentrations between 0.1 and 3 mM, amiloride produced negative inotropic effects only. Amiloride increased the concentration of ouabain needed to produce rhythm disturbances and contracture, and reduced Na-free contracture amplitude by 18%. These results are consistent with the view that amiloride antagonizes the arrhythmogenic effects of ouabain by inhibiting the glycoside-induced elevation in cellular Na content and, consequently, the increases in [Ca]i that occur via Na-dependent pathways. The reduced cellular Na content appears to be due to decreased Na influx via Na-H exchange.     

Effects of phorbol 12,13-diacetate on human isolated bronchus

Protein kinase C appears to be involved in the regulation of airway contractility. Phorbol 12,13-diacetate (PDA; 0.01-10 microM), a protein kinase C activator, produced a transient relaxation followed by a sustained contraction of human isolated bronchus. Different protein kinase C inhibitors (calphostin C, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7), nifedipine (NIF; 1 microM) or incubation with Ca(2+)-free medium, inhibited the spasmogenic response to phorbol, while ouabain (10 microM) suppressed only the initial relaxation. These results indicate that the initial relaxation, in response to PDA, is related to the activation of Na(+)/K(+)-ATPase, while the ensuing contraction depends on extracellular Ca(2+) entry.Incubation with PDA (1-5 microM) depressed the maximal relaxation to theophylline and caffeine obtained at 37 degrees C but augmented the spasmogenic responses to methylxanthines (10 mM) obtained in cooled preparations. These effects do not result apparently from increased extracellular entry of Ca(2+), but instead, from facilitation of the release of Ca(2+) from intracellular stores.     

Negative inotropic effect of endothelin-1 in the mouse right ventricle

Effects of endothelin-1 on the contraction and cytosolic Ca(2+) concentrations (?Ca(2+)(i)) of the mouse right ventricle were investigated. Endothelin-1 (1-300 nM) elicited a negative inotropic effect in a concentration-dependent manner. The endothelin-1-induced negative inotropy was antagonized by a selective endothelin ET(A) receptor antagonist, BQ-123 (cyclo ?Asp-Pro-Val-Leu-Trp-; 3, 10 microM). Endothelin-1 reduced the peak amplitudes of both the ?Ca(2+)(i) transient and contraction without changing inward Ca(2+) current. The relationship between peak amplitude of ?Ca(2+)(i) and peak force generated by changing the extracellular Ca(2+) concentration (?Ca(2+)(o)) was not affected by endothelin-1. In addition, the trajectory of the ?Ca(2+)(i)-contraction phase plane diagram obtained at 2 mM ?Ca(2+)(o) in the absence of endothelin-1 was superimposable on that obtained at 4 mM ?Ca(2+)(o) in the presence of endothelin-1 (300 nM). Endothelin-1 (300 nM) translocated protein kinase C from cytosol to membrane, suggesting activation of protein kinase C. Further, a selective protein kinase C inhibitor, bisindolylmaleimide I (10 microM), inhibited the endothelin-1-induced negative inotropy. These results suggest that endothelin-1 elicits negative inotropy by reducing the amplitude of the ?Ca(2+)(i) transient without changing inward Ca(2+) current through the activation of the endothelin ET(A) receptor followed by protein kinase C activation in the mouse right ventricle.     

Pharmacological characteristics of the positive inotropic effect of angiotensin II in the rabbit ventricular myocardium.

1. In order to elucidate the mechanism underlying the positive inotropic effect (PIE) of angiotensin II (AII), we measured changes in phosphoinositide hydrolysis and contractile force induced by AII in the rabbit ventricular myocardium. 2. AII (1.0 nM-3 microM) produced a PIE in a concentration-dependent manner in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM), the maximal response being about 40% of that to isoprenaline and the EC50 30 nM. 3. The PIE of AII was associated with a concentration-dependent increase in the total duration of contraction; the time to peak force and the relaxation time were prolonged. 4. AII (10 nM-30 microM) elicited an accumulation of [3H]-inositol monophosphate in a concentration-dependent manner in rabbit ventricular slices prelabelled with myo-[3H]-inositol. 5. The PIE and the accumulation of [3H]-inositol monophosphate induced by AII were inhibited by a non-selective AII receptor antagonist, saralasin (10 nM-1 microM) and by a selective AT1 receptor antagonist, losartan (10 nM-1 microM), but not a selective AT2 receptor antagonist, PD 123319 (1 microM). 6. A tumour-promoting phorbol ester, phorbol 12,13-dibutyrate (PDBu, 10-100 nM), inhibited the AII-induced PIE and [3H]-inositol monophosphate accumulation in a concentration-dependent manner. 7. These results suggest that AII exerts a PIE through activation of AT1 receptors and subsequent acceleration of phosphoinositide hydrolysis. Activation of protein kinase C by PDBu may inhibit the AII-induced stimulation of phosphoinositide hydrolysis and thereby the PIE of AII in the rabbit ventricular myocardium.     

Differential inotropic effects of endothelin-1, angiotensin II, and phenylephrine induced by crosstalk with cAMP-mediated signaling process in dog ventricular myocardium

Endothelin-1 (ET-1), angiotensin II (Ang II), and phenylephrine, an alpha1-adrenoceptor agonist, share the common signaling process, resulting in activation of Gq protein-coupled receptor (GqPCR) to activate the hydrolysis of phosphoinositide (PI). They do not elicit any inotropic effect in isolated dog ventricular muscle. In the presence of forskolin or IBMX (3-isobutyl-1-methylxanthine), ET-1 produced a dual effect, that is, a positive inotropic effect (PIE) and/or a negative inotropic effect (NIE) depending on concentrations of forskolin or IBMX present simultaneously with ET-1. Phenylephrine produced a definite PIE and Ang II induced a small and transient PIE in the presence of forskolin or IBMX, but they did not elicit a NIE. Facilitation of Ca2+ influx via L-type Ca2+ channel may play a crucial role in the crosstalk because GqPCR agonists produced, likewise a PIE in the presence of Bay k 8644. GqPCR agonists failed to induce a PIE in the presence of dihydroouabain or elevated [Ca2+]o. These findings indicate that the accumulation of cAMP or activation of L-type Ca2+ channels markedly modulates the inotropic response to GqPCR agonists in a manner that leads to a PIE in dog ventricular myocardium. In addition, ET-1, but not Ang II or phenylephrine, activates the signal transduction process that results in a NIE.     

Dual effects of tetrandrine on cytosolic calcium in human leukaemic HL-60 cells: intracellular calcium release and calcium entry blockade.

1. Tetrandrine (TET, a Ca2+ antagonist of Chinese herbal origin) and thapsigargin (TSG, an endoplasmic reticulum Ca2+ pump inhibitor) concentration-dependently mobilized Ca2+ from intracellular stores of HL-60 cells, with EC50 values of 20 microM and 0.8 nM, respectively. After intracellular Ca2+ release by 30 nM TSG, there was no more discharge of Ca2+ by TET (100 microM), and vice versa. 2. Pretreatments with 100 nM rauwolscine (alpha 2-adrenoceptor antagonist), 100 nM prazosin (alpha 1-adrenoceptor antagonist), 10 nM phorbol myristate acetate (PMA, a protein kinase C activator) or 100 nM staurosporine (a protein kinase C inhibitor) had no effect on 100 microM TET-induced intracellular Ca2+ release. 3. After intracellular Ca2+ release by 30 nM TSG in Ca(2+)-free medium, readmission of Ca2+ caused a substantial and sustained extracellular Ca2+ entry. The latter was almost completely inhibited by 100 microM TET (IC50 of 20 microM) added just before Ca2+ readmission. In Ca(2+)-containing medium, 30 nM TSG caused a sustained phase of cytosolic Ca2+ elevation, which could be abolished by 100 microM TET. TET was also demonstrated to retard basal entry of extracellular Mn2+ and completely inhibit TSG-stimulated extracellular Mn2+ entry. 4. TSG-induced extracellular Ca2+ entry was insensitive to the L-type Ca2+ channel blocker, nifedipine (1 microM), but was completely inhibited by the non-selective Ca2+ channel blocker La3+ (300 microM). Depolarization with 100 mM KCl did not raise the cytosolic Ca2+ level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of bosentan (Ro 47-0203), an ETA-, ETB-receptor antagonist, on regional haemodynamic responses to endothelins in conscious rats.

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.     

Effects of amiloride in guinea-pig and rat left atrial contraction as affected by frequency of stimulation and [Ca2+]0-[Na+]0 ratio: role of Na+/Ca2+ exchange.

1. The effect of amiloride (0.5 mM) on guinea-pig and rat left atria driven at various rates of stimulation and different [Ca2+]0-[Na+]0 ratios has been studied. 2. Amiloride elicited a positive inotropic response in guinea-pig left atria driven at 0.1 Hz, 0.5 Hz and 1 Hz when [Ca2+]0 was 3.6 mM, 1.8 mM and 0.9 mM respectively but not when [Ca2+]0 was 2.7 mM at 0.1 Hz, 0.9 mM at 0.5 Hz and 0.45 mM at 1 Hz. 3. A positive inotropic response was obtained in guinea-pig left atria driven at 0.1 Hz and 1 Hz when [Ca2+]0-[Na+]0(2) was increased respectively from 8 x 10(-5) to 16 x 10(-5) and from 2 x 10(-5) to 8 x 10(-5). The positive inotropic effect was evident only when the ratio was increased by increasing [Ca2+]0 and not by decreasing [Na+]0. 4. In the presence of amiloride, the force of contraction of guinea-pig left atria decreased instead of increasing, when the rate of stimulation was lowered from 1 Hz to 0.01 Hz. Amiloride inhibited the post-rest potentiation. 5. In rat left atria amiloride was devoid of any effect in all the above-mentioned experimental conditions. 6. It is suggested that the pattern of cardiac actions of amiloride can be explained by the inhibition of the Na+/Ca2+ exchange system.     

Suppression of positive inotropic and toxic effects of cardiac glycosides by amiloride

Effects of amiloride on the inotropic and toxic actions of cardiac glycosides were examined using left atrial muscle isolated from guinea pig heart. Preincubation of atrial muscle with amiloride significantly decreased the maximum positive inotropic effect of dihydrodigoxin but failed to reduce that of isoproterenol. Amiloride prevented the contracture and significantly reduced the incidence of arrhythmias induced by 2 microM digoxin. Similar experiments examining 5 microM digoxin-induced arrhythmias showed that amiloride increased both the time required to produce arrhythmias and the fractional occupancy of sarcolemmal Na,K-ATPase by digoxin at the onset of arrhythmias. The antagonism of cardiac glycoside actions was best observed during the decline in developed tension elicited by amiloride subsequent to its initial positive inotropic effect. Amiloride had no effect on binding site concentration for ATP-dependent [3H]ouabain binding but decreased affinity of the binding sites for ouabain in membrane preparations obtained from guinea pig heart. Furthermore, amiloride inhibited Na,K-ATPase activity and increased the IC50 value for ouabain inhibition of the enzyme. These results indicate that amiloride antagonizes the positive inotropic and toxic effects of cardiac glycosides. Possible mechanisms for the antagonism include inhibition of sarcolemmal Na+/Ca2+ or Na+/H+ exchange.     

Staurosporine, a protein kinase C inhibitor, inhibits atrial natriuretic peptide secretion induced by sarafotoxin, endothelin and phorbol ester.

We studied the effects of two peptides of the endothelin/sarafotoxin family, sarafotoxin-b (SRTX-b) and endothelin (ET-1), as well as the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on immunoreactive atrial natriuretic peptide (IR-ANP) release and on haemodynamic parameters (perfusion pressure, heart rate and contractile force) in isolated perfused rat hearts in order to examine the role of intracellular signals in the regulation of ANP secretion. Infusion of SRTX-b at doses of 0.9 and 2.7 nM for 30 min caused a gradual, dose-dependent increase in IR-ANP release and a more rapid coronary vasoconstriction similar to the infusions of ET-1 (2.7 nM) or TPA (46 nM), known to activate protein kinase C in heart cells. A transient inotropic response with a later decrease in contractile force was observed after infusion of each agent. SRTX-b and TPA produced a sustained chronotropic effect, while ET-1 did not significantly affect the heart rate. Infusion of 100 nM of staurosporine, a potent inhibitor of protein kinase C, did not affect basal IR-ANP release into the perfusion fluid but slightly decreased perfusion pressure, heart rate and contractile force. When infused together with SRTX-b, ET-1 or TPA, staurosporine significantly inhibited the ANP secretion, coronary vasoconstriction and changes in cardiac function induced by the peptides or phorbol ester. This study shows that SRTX-b stimulates ANP release with a potency similar to that of ET-1 and that the kinetics of their effects on ANP secretion resemble those of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influences of sodium on the contraction induced by oxytocin in rat testicular capsule

1. The effect of tetrodotoxin (5 microM), monensin (10 microM) and the replacement of Na+ by choline (choline medium) on the contractions of the rat testicular capsule induced by oxytocin (50 and 200 nM) have been studied. 2. The sodium channel blocker tetrodotoxin did not modify the oxytocin contraction. 3. The sodium ionophore monensin produces contraction of rat testicular capsule and reduces the oxytocin-induced contraction. The monensin contraction is inhibited by amiloride (0.1 mM). 4. Replacement of Na+ by choline increases the contraction induced by oxytocin and KCl (60 mM) but inhibits that induced by noradrenaline (3 microM). 5. The increase of contraction due to oxytocin in choline medium is inhibited by amiloride (50 microM and 1 mM) and when calcium is suppressed of the incubation medium.     

Veratridine activates a silent sodium channel in rat isolated aorta.

To investigate the existence of silent Na+ channels, isolated rat aorta was treated with veratridine (0.1 mM) and the resulting Ca2+ uptake was determined. After 30-min incubation the total tissue uptake of Ca2+ and Ca2+ uptake increased from 2.325 +/- 0.017 to 2.614 +/- 0.080 nmol/mg wet weight (ww) and from 162.6 +/- 9.7 to 218.1 +/- 13.0 pmol/mg ww, respectively. The veratridine-induced Ca2+ uptake was blocked by tetrodotoxin (1 microM; to 17 +/- 5%) but not altered by amiloride (10 microM-1 mM). Activation of Na+/Ca2+ exchange by Na+ removal increased Ca2+ uptake from 74.2 +/- 4.5 to 97.3 +/- 5.3 pmol/mg ww, which was suppressed by amiloride (10 microM-1 mM). Nifedipine (10 nM) and verapamil (0.1 microM) at concentrations at which depolarization-induced Ca2+ uptake was diminished did not attenuate veratridine-induced Ca2+ uptake. Phenytoin at 0.1 mM reduced the Ca2+ uptake induced by veratridine or by depolarization. R 56865 (0.1 microM) and R 59494 (1 microM), novel anti-ischemic compounds inhibiting slowly inactivating Na+ channels, suppressed the veratridine-induced but not the depolarization-induced Ca2+ uptake. Guanidinium uptake was increased by veratridine (0.1 mM) from 371.2 +/- 7.2 to 574.8 +/- 45.9 pmol/mg ww. These results suggest that the rat aorta possesses a Na+ channel which is electrically silent under normal conditions but could be activated by veratridine.     

Diamide: positive inotropic effect in isolated atria and inhibition of Na+/Ca2+ exchange in cardiomyocytes

The influence of frequency of stimulation and external calcium on the positive inotropic response of guinea-pig left atria to diamide and the inhibitory action on Na+/Ca2+ exchange activity of rat cardiomyocytes by this oxidant of sulphhydryl groups have been investigated. Diamide (50-500 microM) induces a concentration-dependent positive inotropic effect which is more pronounced when atria are driven at 1.0 Hz rather than at 0.5 and 0.1 Hz, and are bathed in 2.72 mM rather than in 1.36 mM external calcium. A decrease in the positive inotropic effect at 35 degrees C with respect to 29 degrees C is also observed. In addition, diamide in positive inotropic concentrations (100-300 microM) significantly reduces Na+/Ca2+ exchange activity and cytoplasmic glutathione levels in adult rat cardiomyocytes. The thiol reducing agent dithiothreitol either reverses or prevents diamide effects both in isolated atria and cardiomyocytes, suggesting that the actions of diamide are correlated to its property to oxidize sulphhydryl groups to disulphides. In view of the functional importance of Na+/Ca2+ exchange in myocardial contractility, it is proposed that diamide may increase the heart force of contraction by an inhibition of the sarcolemmal Na+/Ca2+ exchange activity.     

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP.

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester potentiates alpha 1-adrenoceptor-mediated positive inotropic effect in rat papillary muscle

Tumor-promoting phorbol esters may alter alpha 1-adrenoceptor-mediated cardiac response by stimulating protein kinase C activity. We investigated the effect of phorbol-12,13-dibutyrate (PDBu) on the positive inotropic effect (PIE) in rat left ventricular papillary muscle. PDBu (1-100 nM) potentiated the phenylephrine (PE)-induced PIE in a dose- and time-dependent manner. The PIE induced by PE and PDBu was abolished by pretreatment with 3 x 10(-7) M prazosin. PDBu also enhanced PE-induced slow responses 2- to 3-fold. These results suggest that PDBu enhances alpha 1-adrenoceptor-mediated PIE by potentiating slow Ca2+ channels, presumably through the activation of protein kinase C.