Cardiotonic action of [8]-gingerol, an activator of the Ca++-pumping adenosine triphosphatase of sarcoplasmic reticulum, in guinea pig atrial muscle

[8]-Gingerol (gingerol), a component of ginger, produced a concentration-dependent positive inotropic effect on guinea pig isolated left atria at concentrations of 1 X 10(-6) to 3 X 10(-5) M. Gingerol also exhibited positive inotropic and chronotropic effects on guinea pig right atria. The gingerol-induced inotropic effect was abolished by ryanodine, but was little affected by propranolol, chlorpheniramine, cimetidine, tetrodotoxin, diltiazem or reserpine. The time to peak tension and relaxation time within a single contraction were shortened by gingerol (1 X 10(-5) M) as well as isoproterenol, whereas they were prolonged by BAY K 8644. In guinea pig isolated atrial cells, gingerol (3 X 10(-6) M) caused an increase in the degree and the rate of longitudinal contractions. In guinea pig left atria, gingerol (1 X 10(-6) to 3 X 10(-5) M) gave little influence on the action potential, although it increased the contractile force of the atria. The whole-cell patch-clamp experiments showed that the slow inward current was little affected by gingerol (1 X 10(-6) to 3 X 10(-5) M) in voltage-clamped guinea pig cardiac myocytes. The measurement of extravesicular Ca++ concentration using a Ca++ electrode indicated that gingerol (3 X 10(-6) to 3 X 10(-5) M) accelerated the Ca++ uptake of fragmented sarcoplasmic reticulum (SR) prepared from canine cardiac muscle in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative behavioral, neurochemical and pharmacological activities of dihydropyridine calcium channel activating drugs.

The behavioral (deficits in motor function in mice), neurochemical (affinity for mouse brain membrane dihydropyridine receptors, effects on neurotransmitter/metabolite levels in mice) and pharmacologic (effect on the contractile activity of guinea pig ileal longitudinal smooth muscle) properties of the calcium channel activators (+/-)-BAY K 8644, (+/-)-202-791 (and their corresponding channel activating and antagonist enantiomers) and CGP-28392 were investigated and compared. The calcium channel activating enantiomers (-)-S-BAY K 8644, (+)-S-202-791 and (+/-)-BAY K 8644, (+/-)-202-791 and CGP-28392 produced a dose-dependent impairment of rotarod ability and decreases in motor activity in mice with the following order of potency: (-)-S-BAY K 8644 greater than (+/-)-BAY K 8644 much greater than (+)-S-202-791 greater than (+/-)-202-791 = CGP-28392. The calcium channel antagonists (+)-R-BAY K 8644 and (-)-R-202-791 were behaviorally inactive but blocked the behavioral effects of (-)-S-BAY K 8644. The binding of dihydropyridine calcium channel activator and antagonist enantiomers to mouse brain membranes was described by both one and two site models. (-)-S-BAY K 8644, (+/-)-BAY K 8644, (+)-S-202-791 and CGP-28392 produced contractions in partially depolarized (15 mM K+) strips of guinea pig ileal longitudinal smooth muscle which differed in the degree of maximum contraction obtained. (+)-R-BAY K 8644 and (-)-R-202-791 inhibited potassium-induced contractions (80 mM K+) in guinea pig ileal longitudinal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

TMB-8 as a pharmacologic tool in guinea pig myocardial tissues. I. Effects of TMB-8 on force of contraction and on action potential parameters in atrial and papillary muscles.

The compound 8-)N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) had been introduced as an intracellular Ca++ antagonist. We have studied the effects of TMB-8 on electrical and mechanical activity of isolated cardiac tissues in order to estimate its spectrum of action in heart muscle. In spontaneously beating right atria of the guinea pig, TMB-8 (1-100 microM) had a negative chronotropic effect. In left atria, TMB-8 (1-100 microM) induced a frequency-dependent biphasic inotropic effect: A transient increase in force of contraction was followed by a sustained decrease; the latter could be antagonized partially by an increase in [Ca++]o. TMB-8 prolonged the time-to-peak force. At high concentrations of TMB-8 (greater than 10 microM), the electrical stimulation threshold was elevated. TMB-8 (20 microM) competitively inhibited the positive inotropic effect of Bay K 8644 and reduced the magnitude of the positive inotropic and/or chronotropic effects of veratridine, (-)-isoproterenol, forskolin, histamine and (-)-phenylephrine. TMB-8 (30 microM) prolonged the action potential duration (APD) [in particular at 90% of repolarization (APD90)] and the refractory period, and decreased the AP amplitude and Vmax. In right ventricular papillary muscles, TMB-8 (30 microM) shortened the APD (APD20 = APD50 greater than APD90) and the refractory period but hardly affected the AP amplitude and Vmax. The resting membrane potential remained unchanged in both tissues. These findings suggest that in addition to interference with the Ca++ release from the sarcoplasmic reticulum, TMB-8 also affects the membrane conductances for cations.     

Differential effects of somatostatin on atrial and ventricular contractile responses in guinea pig heart: influence of pretreatment with islet-activating protein

The effects of somatostatin on the contractile response of guinea pig cardiac preparations were investigated and compared with those of carbachol and adenosine. Somatostatin produced a concentration-dependent negative inotropic effect in the left atria, which was accompanied by a decrease in action potential duration. The maximum decrease in contractility which was obtained at 3 x 10(-6) M was around 40% of the predrug control values and far less than those produced by carbachol and adenosine. Somatostatin failed to produce inotropic effect on the papillary muscle and did not influence the spontaneously beating rate of the right atria. In the papillary muscles, however, somatostatin inhibited the positive inotropic effect of isoproterenol in a concentration-dependent manner as did carbachol and adenosine. In addition, somatostatin caused a significant inhibition of the isoproterenol-induced increase in cyclic AMP levels without affecting the basal level of cyclic AMP. In the papillary muscle, the inhibitory effect of somatostatin on the positive inotropic response to isoproterenol was significantly attenuated by pretreatment with islet-activating protein, and was significantly antagonized by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. These results suggest that somatostatin receptors in guinea pig ventricular muscles are coupled with adenylate cyclase via islet-activating protein-sensitive GTP-binding protein, whereas the negative inotropic effect of somatostatin in the left atria might be mediated by a subtype of somatostatin receptors which is different from that in the ventricle.     

Cardiac histamine receptors: differences between left and right atria and right ventricle.

Histamine can stiumulate the heart by directly interacting with cardiac histamine receptors. In the present study we have investigated the cardiac effects of histamine, 4-methylhistamine (a specific H2-receptor agonist) and 2-pyridylethylamine PEA, a specific H1-receptor agonist] on spontaneously beating right atria and electrically driven left atria and right ventricle strips of the guinea pig. Left atria were driven at 1 Hz and right ventricle strips at 2.5 Hz and at twice the threshold voltage. Histamine and PEA produced a dose-dependent positive inotropic effect on the left atria. The dose-response curves were shifted to the right in a parallel fashion by promethazine (3 x 10(-6)M)+... Burimamide did not affect either dose-response curve. Histamine and 4-methylhistamine had a positive chromnotropic effect on right atria and a positive inotropic effect on right ventricle strips.     

Cardiac adenylate cyclase activity, positive chronotropic and inotropic effects of forskolin analogs with either low, medium or high binding site affinity

A series of in vitro studies were conducted examining the adenylate cyclase stimulation, positive chronotropic and inotropic effects of forskolin and nine analogs which exhibited a range of [3H]forskolin binding site affinities (K1) from 0.020 to 3.174 microM. A significant (P less than .001) linear correlation (r = 0.94) was found between binding site affinity and adenylate cyclase stimulation (EC50) for forskolin and the nine structural analogs. Adenylate cyclase activity was also significantly correlated with the positive chronotropic and inotropic effects of these substances on isolated guinea pig atria. Compounds with K1 values between 0.020 and 1.136 microM produced concentration-dependent increases in heart rate and contractile force in isolated spontaneous and electrically paced guinea pig atria, respectively. In contrast, an analog with a K1 of 3.174 microM caused significant (P less than .05) negative chronotropic and inotropic effects at concentrations above 10 microM. The optimal separation between positive inotropic and chronotropic activity was found with compounds displaying potent [3H]forskolin binding site affinity but moderate adenylate cyclase stimulation, i.e., K1 and EC50 values of approximately 0.05 to 0.10 and 3 microM, respectively. The results of this study show that the forskolin analog, P87-7692 [7-desacetyl-7-(O-propionyl)-hydroxyl amino-carbonyl-forskolin], has marked activity with a wide separation between positive inotropic (248 +/- 41%) and chronotropic effects (43 +/- 13%) at 6.2 microM and may serve as a prototype for a forskolin-based cardiotonic.     

Functional impairment of renal afferent arteriolar voltage-gated calcium channels in rats with diabetes mellitus.

Experiments were performed to test the hypothesis that diabetes mellitus is associated with impaired afferent arteriolar responsiveness to opening of voltage-gated calcium channels. Diabetes was induced by injection of streptozocin (65 mg/kg, i.v.) and insulin was administered via an osmotic minipump to achieve moderate hyperglycemia. Sham rats received vehicle treatments. 2 wk later, the in vitro blood-perfused juxtamedullary nephron technique was used to allow videomicroscopic measurement of afferent arteriolar contractile responses to increasing bath concentrations of either Bay K 8644 or K+. Baseline afferent arteriolar diameter in kidneys from diabetic rats (26.4+/-1.2 microm) exceeded that of Sham rats (19.7+/-1.0 microm). Bay K 8644 evoked concentration-dependent reductions in afferent diameter in both groups of kidneys; however, arterioles from Sham rats responded to 1 nM Bay K 8644 while 100 nM Bay K 8644 was required to contract arterioles from diabetic rats. The EC50 for K+-induced reductions in afferent arteriolar diameter was greater in diabetic kidneys (40+/-4 mM) than in kidneys from Sham rats (28+/-4 mM; P < 0.05). In afferent arterioles isolated by microdissection from Sham rats and loaded with fura 2, increasing bath [K+] from 5 to 40 mM evoked a 98+/-12 nM increase in intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i responses to 40 mM K+ were suppressed in afferent arterioles from diabetic rats (delta = 63+/-5 nM), but were normalized by decreasing bath glucose concentration from 20 to 5 mM. These observations indicate that the early stage of insulin-dependent diabetes mellitus is associated with a functional defect in afferent arteriolar L-type calcium channels, an effect which may contribute to suppressed afferent arteriolar vasoconstrictor responsiveness and promote glomerular hyperfiltration.     

Pharmacological analysis of the cardiac actions of xamoterol, a beta adrenoceptor antagonist with partial agonistic activity, in guinea pig heart: evidence for involvement of adenylate cyclase system in its cardiac stimulant actions

The pharmacological effects of xamoterol, a beta adrenoceptor antagonist with partial agonistic activity, were examined in guinea pig cardiac preparations and compared with those of isoproterenol to assess possible mechanisms of its cardiac stimulant actions. Xamoterol produced a positive inotropic effect in the papillary muscles and a positive chronotropic effect in the spontaneously beating right atria in a concentration-dependent manner. The maximum inotropic and chronotropic effects of xamoterol were about 33 and 35% of those of isoproterenol, respectively. Although xamoterol failed to produce a consistent increase in contractile force in the left atria, the positive inotropic effect of the agent was observed clearly in preparations obtained from reserpine-pretreated animals. The positive inotropic and chronotropic effects of xamoterol were antagonized by atenolol, but not by ICI 118,551. On the other hand, xamoterol antagonized competitively the positive inotropic and chronotropic responses to isoproterenol. In papillary muscles the increases in contractile force induced by xamoterol and isoproterenol were depressed markedly in the presence of carbachol or adenosine. In all of left atria, right atria and papillary muscles obtained from reserpine-pretreated animals, xamoterol caused a significant elevation in cyclic AMP levels, while inhibiting the isoproterenol-induced increase in cyclic AMP levels. Computer-assisted analysis of concentration-response curves for the inhibition by xamoterol of the binding of [125I]iodocyanopindolol in the membranes from guinea pig ventricles showed the existence of the 5'-guanylylimidodiphosphate sensitive, highly affinity site of beta adrenoceptors for xamoterol, suggesting that xamoterol may induce the formation of a ternary complex with the beta adrenoceptor and a stimulatory guanine nucleotide regulatory protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of a new Ca(2+) sensitizer

The benzimidazole molecule was modified to synthesize a Ca(2+) sensitizer devoid of additional effects associated with Ca(2+) overload. Newly synthesized compounds, termed 1, 2, 3, 4, and 5, were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Compound 3 resulted as the most effective positive inotropic agent, and experiments were performed to study its mechanism of action. In spontaneously beating atria, the inotropic effect of 3 was concentration-dependent (3.0 microM-0.3 mM). Compound 3 was more potent and more active than the structurally related Ca(2+) sensitizers sulmazole and caffeine, but unlike them it did not increase the heart rate. In electrically driven atria, the inotropic activity of 3 was well preserved and it was not inhibited by propranolol, prazosin, ranitidine, pyrilamine, carbachol, adenosine deaminase, or ruthenium red. At high concentrations (0.1-1.0 mM) 3 inhibited phosphodiesterase-III, whereas it did not affect Na(+)/K(+)-ATPase, sarcolemmal Ca(2+)-ATPase, Na(+)/Ca(2+) exchange carrier, or sarcoplasmic reticulum Ca(2+) pump activities of guinea pig heart. In skinned fibers obtained from guinea pig papillary muscle and skeletal soleus muscle, compound 3 (0.1 mM, 1 mM) shifted the pCa/tension relation curve to the left, with no effect on maximal tension and no signs of toxicity. Compound 3 did not influence the basal or raised tone of guinea pig isolated aorta rings, whose cells do not contain the contractile protein troponin. The present results indicate that the inotropic effect of compound 3 seems to be primarily sustained by sensitization of the contractile proteins to Ca(2+).     

Inhibitory effects of calcium channel agonists on renin release from rat kidney cortical slices

Inhibitory effects of calcium channel agonists such as Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] and CGP 28392 [ethyl-4-(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl- 5-oxofuro-(3,4-b)-pyridine-3-carboxylate] on renin release were investigated, using rat kidney cortical slices. Bay K 8644 or CGP 28392 alone had no effect on renin release from the slices, whereas both compounds produced a concentration-dependent inhibition of the release in the presence of 15 mM potassium. The Bay K 8644-induced inhibitory action was more effective and potent than that seen with CGP 28392. Bay K 8644 caused a leftward shift of the dose-response curve of the potassium-induced decrease in renin release. In contrast, the dose-response relationships of the release to norepinephrine and methoxamine were not affected by Bay K 8644. The combination of the maximum effective doses of calcium channel agonists and norepinephrine exerted an apparent additive effect on the release of renin. The inhibitory effects of Bay K 8644 and CGP 28392 were attenuated in the presence of decreased extracellular calcium concentrations. Nifedipine and verapamil elicited a blocking action on the inhibition of renin release by Bay K 8644 or CGP 28392, in a concentration-dependent manner. Calmodulin antagonists, such as trifluoperazine and calmidazolium suppressed significantly the decreasing effect of Bay K 8644 or CGP 28392 on renin release from the slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

o-Isothiocyanate dihydropyridine (oNCS-DHP), a long-acting, reversible inhibitor of the Ca++ channel

The binding characteristics and pharmacological properties of o-isothiocyanate dihydropyridine [oNCS-DHP; 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1, 4-dihydropyridine] were investigated in guinea pig heart and ileum. [3H]oNCS-DHP bound to a single population of high-affinity sites (Bmax = 107 fmol/mg of protein and Kd = 0.99 nM) in cardiac membranes, with a specificity characteristic of dihydropyridine receptors. After incubation of membranes with the tracer (0.5 nM), addition of excess nifedipine (1 microM) caused a dissociation of [3H]oNCS-DHP from its binding site. The reversibility of [3H]oNCS-DHP binding was confirmed by the lack of affinity labeling of cardiac membranes as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. oNCS-DHP inhibited the inward Ca++ current of isolated guinea pig cardiac myocytes as determined in voltage-clamp experiments. In isolated perfused guinea pig hearts, oNCS-DHP caused a concentration-dependent increase in coronary artery flow and a decrease in left ventricular pressure. The effects of the highest concentration (0.3 microM) were still near maximal after a 1-h washout. Suppression of K+ depolarization-induced contractures of isolated ileal longitudinal muscle strips by oNCS-DHP remained maximal even after 5 h of washout. In all of the three biological test systems investigated, the Ca++ channel activator Bay K 8644 caused a complete and rapid reversal of the inhibitory effects of oNCS-DHP. Thus, it can be concluded that oNCS-DHP does not bind irreversibly to Ca++ channel dihydropyridine receptors in guinea pig heart and ileum. However, the o-isothiocyanatophenyl substituent on the dihydropyridine molecule confers upon the compound a very long duration of Ca++ channel blocking activity.     

Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge.

Xestoquinone (XQN) isolated from the sea sponge Xestospongia sapra produced dose-dependent cardiotonic effects on guinea pig left and right atria. A direct action of XQN (1-30 microM) on the contractile machinery of cardiac myofilaments was demonstrated in chemically skinned fiber preparations from guinea pig papillary muscles. In atrial preparations, the XQN-induced inotropic effect was markedly inhibited by verapamil or nifedipine, but was not affected by practolol, chlorpheniramine, cimetidine, tetrodotoxin or reserpine. The Ca++ dependence curve for the contractile response of the atria was substantially shifted to the left by XQN (10 microM), and this XQN-induced shift was reversed by verapamil. The time-to-peak tension and relaxation times of the atrial contractions were shortened by XQN, and the action potential duration was markedly prolonged. Whole-cell patch clamp recordings in left atrial strips confirmed that XQN (30 microM) increased the slow inward current. However, there was a temporal dissociation between altered tension development and prolongation of the action potential duration. Cyclic AMP phosphodiesterase activity was inhibited and tissue cyclic AMP content of guinea pig left atria was increased by XQN (0.3-10 microM) in a concentration-dependent manner, but increases in cyclic AMP content did not occur in parallel with increases in contractile response. These observations suggest that an enhancement of intracellular cyclic AMP content and Ca++ influx across the cell membrane contribute to the late phase of XQN-caused cardiotonic responses, whereas the early phase may largely be elicited through direct activation of contractile elements. XQN may provide a novel leading compound for valuable cardiotonic agents.     

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activator-antagonist pairs in smooth muscle

The actions of the enantiomers of Bay K 8644 and 202-791 were studied in rat tail artery and guinea pig ileal longitudinal smooth muscle using pharmacologic and radioligand binding assays. (-)-(S)-Bay K 8644 (below 10(-7) M in rat tail artery and 3 X 10(-7) M in guinea pig ileum) and (+)-(S)-202-791 (below 10(-6) M) induced contractions and potentiated the responses to KCl depolarization in both smooth muscle preparations. In contrast, (+)-(R)-Bay K 8644 and (-)-(R)-202-791 inhibited the responses to KCl-induced depolarization. At higher concentrations, (-)-(S)-Bay K 8644 (10(-7) to 10(-6) M) and (+)-(S)-202-791 (10(-6) to 3 X 10(-5) M) in rat tail artery, and (-)-(S)-Bay K 8644 (3 X 10(-7) to 3 X 10(-6) M) in guinea pig ileal smooth muscle relaxed the tissues contracted maximally at lower concentrations of the same drug. Cross antagonism between 1,4-dihydropyridine activators was observed when (-)-(S)-Bay K 8644 (10(-7) to 10(-6) M) relaxed the maximum contraction in response to (+)-(S)-202-791. [3H]Nitrendipine bound in a tail arterial microsomal preparation to a single class of site, with KD of 3.63 X 10(-10) M and maximum binding of 552.7 fmol mg-1 protein. In both rat tail artery and guinea pig ileal smooth muscle, (-)-(S)-Bay K 8644, (+)-(R)-Bay K 8644, (+)-(S)-202-791 and (-)-(R)-202-791 inhibited specific [3H]nitrendipine binding competitively; the Kl values correlate well to the pharmacologic EC50 (for activators) or IC50 (for antagonists, measured against 80 mM KCl depolarization) values. The biphasic response to (-)-(S)-Bay K 8644 and (+)-(S)-202-791 suggests that the properties of Ca++ channel activation and antagonism may reside within a single 1,4-dihydropyridine molecule.     

Negative and positive inotropic responses to muscarinic agonists in guinea pig and rat atria in vitro

In the guinea pig atria, carbachol, acetylcholine and bethanechol elicited negative inotropic and positive inotropic effects. In the rat atria, a negative inotropic response occurred, but the positive inotropic response was small. The positive and negative inotropic responses to carbachol and bethanechol (but not acetylcholine) were unaffected by pretreating the animals with reserpine and were antagonised by pirenzepine with pKB values of 6.7. Pretreatment with pertussis toxin abolished the negative inotropic responses, but was without effect on the positive inotropic responses in the guinea pig. Pretreatment of rats with pertussis toxin abolished the negative inotropic response and enhanced the positive inotropic response. The positive inotropic response was attenuated by pretreatment with dietary lithium for 2 weeks, whereas no effect was observed on the negative inotropic response. Negative and positive inotropic responses to muscarinic agonists in these species are mediated directly through an M2 muscarinic receptor. The ability of dietary lithium to selectively inhibit the positive inotropic response may provide evidence for the involvement of inositol phospholipid hydrolysis in this effect.     

Intramural nerve-mediated inotropic responses of left atria of rats and guinea pigs: demonstration of alpha adrenergic and nonadrenergic noncholinergic responses in guinea pigs

The effects of transmural nerve stimulation (TNS) on contractile responses of rat and guinea pig atria were analyzed pharmacologically. Isolated left atria were electrically driven through AgAgCl field electrodes and TNS was performed by brief introduction of defined stimulation patterns through the same electrodes. Step elevations in stimulating voltage induced biphasic inotropic responses in the left atria of both species: an initial negative component which was usually overwhelmed by a subsequent positive one. The transient negative inotropic response was induced by parasympathetic cholinergic nerve excitation, inasmuch as it was abolished by atropine. In the left atrium of the rat, the TNS-induced positive inotropic response was due exclusively to adrenergic nerve excitation through activation of beta-1 adrenoceptors. In contrast, analysis of the time course of responses in guinea pig left atria after nerve stimulation at 10 Hz revealed a positive inotropic response consisting of two phases; rapid and delayed phases were superimposed upon each other. The rapid phase was reduced by atenolol, a beta-1 antagonist, and attenuated further by prazosin, an alpha-1 antagonist. In the presence of both atenolol and prazosin, TNS of guinea pig left atria still induced a positive inotropic response but it had a slow onset and decay. This is termed the delayed phase response. TNS induced a similar delayed inotropic response in atria from surgically sympathectomized or reserpine-pretreated guinea pigs, from which catecholamine-fluorescence nerves and responses to tyramine were absent. These results demonstrate that TNS excitated adrenergic, cholinergic and nonadrenergic noncholinergic nerves in guinea pig left atria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catecholamine-mediated positive inotropic effect of simple quinones is related to superoxide anion generation.

In guinea pig and rat cardiac tissue, redox cycling benzoquinones (2,5-dimethyl-p-benzoquinone and duroquinone) and naphthoquinones (menadione and 2,3-dimethoxy-1,4-naphthoquinone) generated superoxide anion (O2-.) both through one- and two-electron reductions, the generation being significantly greater in guinea pig than in rat tissue. In electrically driven left atria isolated from guinea pig and rat, menadione and 2,5-dimethyl-p-benzoquinone but not duroquinone caused a concentration-dependent positive inotropic effect. Unlike guinea pig, 2,3-dimethoxy-1,4-naphthoquinone had no effect in rat tissue. Naphthoquinones and 2,5-dimethyl-p-benzoquinone were more active in guinea pig than in rat tissue, their effect being dependent on the release of catecholamines from adrenergic stores. A linear relationship (r = 0.90) between the amount of O2-. generated by benzo- and naphthoquinones in guinea pig and rat heart and the extent of catecholamine-dependent positive inotropic effect was evident. An amount of O2-. higher than 600 nmol/g of tissue per min was calculated to be necessary to determine the catecholamine-mediated increase in contractility. Lipid peroxidation was not involved in quinone-induced catecholamine release.     

Ca²⁺ channel activators reveal differential L-type Ca²⁺ channel pharmacology between native and stem cell-derived cardiomyocytes

Human stem cell-derived cardiomyocytes provide new models for studying the ion channel pharmacology of human cardiac cells for both drug discovery and safety pharmacology purposes. However, detailed pharmacological characterization of ion channels in stem cell-derived cardiomyocytes is lacking. Therefore, we used patch-clamp electrophysiology to perform a pharmacological survey of the L-type Ca2? channel in induced pluripotent and embryonic stem cell-derived cardiomyocytes and compared the results with native guinea pig ventricular cells. Six structurally distinct antagonists [nifedipine, verapamil, diltiazem, lidoflazine, bepridil, and 2-[(cis-2-phenylcyclopentyl)imino]-azacyclotridecane hydrochloride (MDL 12330)] and two structurally distinct activators [methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate (Bay K8644) and 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176)] were used. The IC?? values for the six antagonists showed little variability between the three cell types. However, whereas Bay K8644 produced robust increases in Ca2? channel current in guinea pig myocytes, it failed to enhance current in the two stem cell lines. Furthermore, Ca2? channel current kinetics after addition of Bay K8644 differed in the stem cell-derived cardiomyocytes compared with native cells. FPL 64176 produced consistently large increases in Ca2? channel current in guinea pig myocytes but had a variable effect on current amplitude in the stem cell-derived myocytes. The effects of FPL 64176 on current kinetics were similar in all three cell types. We conclude that, in the stem cell-derived myocytes tested, L-type Ca2? channel antagonist pharmacology is preserved, but the pharmacology of activators is altered. The results highlight the need for extensive pharmacological characterization of ion channels in stem cell-derived cardiomyocytes because these complex proteins contain multiple sites of drug action.     

Positive inotropic effect of carbachol and inositol phosphate levels in mammalian atria after pretreatment with pertussis toxin

The m-cholinoceptor agonist carbachol elicits a negative inotropic effect in mammalian atria. Pretreatment with pertussis toxin converts the negative to a positive inotropic effect. In this study we investigated the time course of the effects of carbachol on force of contraction and phosphoinositide products in electrically driven left auricles from guinea pig hearts after pretreatment with pertussis toxin (180 micrograms/kg i.v.; 24 hr). Inositol phosphates and phosphatidylinositols were labeled with [3H]inositol and separated with high-performance liquid chromatography and thin-layer chromatography, respectively. All experiments were performed in the presence of LiCl (10 mmol/l). The positive inotropic effect of carbachol (10 mumol/l) began within 2 min and was maximal within 15 min. Inositol 1,4,5-trisphosphate rose within 1 min followed by an increase in inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4-bisphosphate and inositol 1-phosphate beginning within 2 min. It is concluded that the carbachol-induced positive inotropic effect is associated with an increase in the presumed second messengers inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Since the increase in inositol 1,4,5-trisphosphate precedes the increase in force of contraction, it may initiate the positive inotropic effect. The increase in inositol 1,3,4,5-tetrakisphosphate may be involved in maintaining the positive inotropic effect of carbachol.     

ICI 147,798: a slowly dissociable beta adrenoceptor antagonist that causes insurmountable beta-1 and surmountable beta-2 adrenoceptor antagonism in isolated tissues

ICI 147,798 has been shown to exhibit both diuretic and beta-antagonist properties in vivo. The present study investigated the nature and selectivity of the beta-antagonism in a variety of isolated tissues. ICI 147,798 produced a concentration-dependent suppression of the maximum chronotropic response of norepinephrine in guinea pig right atria (beta-1 adrenoceptor). ICI 147,798 caused a concentration-dependent shift to the right of the salbutamol concentration-response curve in the guinea pig trachea (beta-2 adrenoceptor), and Schild analysis suggested competitive inhibition. Propranolol produced parallel shifts to the right of the norepinephrine concentration-response curve in guinea pig right atria, except at relatively high concentrations. The inhibitory effects of propranolol in guinea pig right atria were reversed by greater than 95%, whereas the effects of ICI 147,798 were only slightly reversed after a 6-hr washout period. Preincubation of propranolol with ICI 147,798 in guinea pig right atria prevented completely the suppression of the norepinephrine maximum chronotropic response. Postincubation of propranolol with ICI 147,798 partially reversed the suppression of the maximum chronotropic response. ICI 147,798 had no effect on the maximum chronotropic responses of either histamine (H2-receptor) or forskolin (adenylate cyclase activation) in guinea pig right atria and had no effect on agonist responses in a variety of other receptor systems. The insurmountable beta-1 adrenoceptor antagonism was evaluated based on the assumptions of irreversible competitive antagonism, mixed competitive and noncompetitive antagonism and slowly dissociating competitive antagonism ("hemi-equilibrium" conditions). Concentration-dependent changes in norepinephrine KA values suggested the first three possibilities were unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Bay k 8644 and nifedipine on isolated dog cerebral, coronary and mesenteric arteries

Vasoconstrictor effects of Bay k 8644, a dihydropyridine Ca++ agonist, and vasorelaxant effects of nifedipine were investigated in helical strips of dog cerebral (basilar, posterior cerebral and middle cerebral) and peripheral (coronary and mesenteric) arteries. The addition of Bay k 8644 produced a dose-dependent contraction in the absence of any contractile agent in the basilar artery with a pD2 value of 8.53. Similar sensitivity to Bay k 8644 was observed in the posterior cerebral, middle cerebral or coronary artery. Bay k 8644 was much less effective in producing a contraction in the mesenteric artery. An elevation of the concentration of extracellular K+ eliminated the difference between the responses to Bay k 8644 in the basilar and mesenteric artery. Contractile responses of the basilar artery to Bay k 8644 were antagonized competitively by nifedipine (pA2 = 8.17), but non-competitively by diltiazem. The pA2 values for nifedipine antagonism of Bay k 8644 responses with the elevated K+ were the same between the basilar and mesenteric arteries. Increased sensitivity to exogenously added K+ also was observed in cerebral and coronary arteries when compared with the mesenteric artery. The addition of nifedipine to an unstimulated strip produced a dose-dependent relaxation in cerebral and coronary arteries, but not in the mesenteric artery. When the cerebral and peripheral arteries were contracted with K+ to the same magnitude, nifedipine produced similar relaxations among these arteries. Nifedipine was less efficacious in antagonizing the contractile response to Bay k 8644 compared with the contractile response to K+ in cerebral arteries. These results suggest that 1) the voltage-dependent Ca++ channels in the cerebral and coronary arteries are in different states of activation from those in the mesenteric artery, 2) Bay k 8644 contracts the cerebral and coronary arteries by acting primarily on the same site, presumably dihydropyridine receptors of the voltage-dependent Ca++ channels at which nifedipine acts, 3) the dihydropyridine receptors were the same between the basilar and mesenteric arteries and 4) there may be a difference in the state of the Ca++ channel in the arteries between the stimulation with Bay k 8644 and K+-depolarization.