Heterogeneity and underlying mechanism for inotropic action of endothelin-1 in rat ventricular myocytes

1. To clarify the mechanisms underlying the positive inotropic action of endothelin-1 (ET-1), we investigated the effect of ET-1 on twitch cell shortening and the Ca2+ transient in rat isolated ventricular myocytes loaded with a fluorescent Ca2+ indicator indo-1. 2. There was a cell-to-cell heterogeneity in response to ET-1. ET-1 (100 nM) increased twitch cell shortening in only 6 of 14 cells (44%) and the increase in twitch cell shortening was always accompanied by an increase in the amplitude of the Ca2+ transient. 3. The ET(A)- and ET(B)-receptors antagonist TAK-044 (100 nM) almost reversed both the ET-1-induced increases in twitch cell shortening and in the Ca2+ transient. In the ET-1 non-responding cells, the amplitude of the Ca2+ transient never increased. 4. Intracellular pH slightly increased (approximately 0.08 unit) after 30 min perfusion of ET-1 in rat ventricular myocytes. However, ET-1 did not change the myofilament responsiveness to Ca2+, which was assessed by (1) the relationship between the Ca2+ transient amplitude and twitch cell shortening, and by (2) the Ca2+ transient-cell shortening phase plane diagram during negative staircase. 5. We concluded that there was a cell-to-cell heterogeneity in the positive inotropic effect of ET-1, and that the ET-receptor-mediated positive inotropic effect was mainly due to an increase in the Ca2+ transient amplitude rather than to an increase in myofilament responsiveness to Ca2+.     

Involvement of alpha1B-adrenoceptors in the positive inotropic effect of endogenous noradrenaline in rabbit myocardium

We studied the alpha1-adrenoceptor subtypes mediating the positive inotropic effects of phenylephrine and noradrenaline as well as endogenous noradrenaline released by tyramine in rabbit papillary muscle. In the presence of propranolol, both phenylephrine and tyramine produced a positive inotropic effect in a concentration-dependent manner. WB4101 (N-[2-(2,6-dimethoxyphenoxy]ethyl]-2,3-dihydro-1,4-benzodioxin+ ++-2-methanamine) and chlorethylclonidine each antagonized the positive inotropic effect of phenylephrine. On the other hand, only chlorethylclonidine significantly blocked the positive inotropic effect of tyramine. However, the presence of both antagonists was needed to block the positive inotropic effect elicited by the exogenous addition of the low concentration of noradrenaline. These data suggest that after extensive blockade of beta-adrenoceptors the positive inotropic effects of phenylephrine and exogenous noradrenaline result from stimulation of the alpha1A- and alpha1B-adrenoceptor subtypes, whereas that of endogenous noradrenaline is mediated via the alpha1B-adrenoceptor subtype. This could be explained by assuming that the alpha1B-adrenoceptor subtype population may be located on a space confronting more closely to the sympathetic nerve endings than the alpha1A-adrenoceptor subtype population.     

Alterations in alpha 1-adrenergic receptor-mediated phosphatidylinositol turnover in hypoxic cardiac myocytes

The purpose of these studies was to examine the effects of hypoxia on alpha 1-adrenergic receptor (alpha 1AR) mediated phosphatidylinositol (PI) turnover in cultured neonatal rat cardiac myocytes. Cells were pre-labeled with [3H]-inositol and incubated for 1 h in either normoxia or hypoxia. Phenylephrine, an alpha 1AR agonist, was added at various time intervals (0-60 min) before termination of the incubation. There was a time-dependent release of radioactivity from the lipid fraction to the aqueous fraction with alpha 1AR stimulation. alpha 1AR-mediated PI turnover was biphasic in normoxic cells and monophasic in hypoxic cells. Using ion-exchange chromatography, radioactivity in the inositol trisphosphate (IP3) peak was increased with acute phenylephrine stimulation (5 min) in the normoxic cells, while inositol phosphate (IP) and inositol bisphosphate (IP2) were increased with chronic stimulation (60 min). After 5 min of alpha 1AR stimulation, hypoxia did not alter total aqueous radioactivity when compared to normoxia, but there was a significant increase in IP2. However, there was decreased PI turnover in chronically stimulated (30-60 min) hypoxic cells when compared to normoxic cells. Hypoxia had no effect on radioactivity in the IP3 fraction with either 0, 5, or 60 min of alpha 1AR stimulation, but there was a significant increase in [1,4,5]-IP3 in hypoxic cells with 30 s alpha 1AR stimulation. With hypoxia, there was no difference in radioactivity in the phosphatidylinositols with either 0 or 5 min stimulation when compared to normoxia. However, after 60 min of alpha 1AR stimulation, hypoxia resulted in increased PI and PIP, when compared to normoxic cells, but PIP2 radioactivity was unchanged. There was no effect of pertussis toxin on either the acute or chronic phase of PI turnover, negating involvement of Gi or G(o). These data suggest that alpha 1AR stimulation in neonatal rat cardiac myocytes is biphasic, and that hypoxia produces a slower monophasic response during extended alpha 1-agonist exposure as would be found with ischemia.     

Negative inotropic effect of adrenomedullin in isolated adult rabbit cardiac ventricular myocytes

BACKGROUND: Adrenomedullin (AM) is a potent vasodilator peptide. AM-induced vasodilatation is mediated by an increase of NO as well as cAMP. Both AM and binding sites for this peptide have been found in cardiac tissue, indicating the possible existence of an autocrine or paracrine system of AM in the heart. METHODS AND RESULTS: Myocytes were isolated by use of retrograde coronary perfusion with physiological solution containing collagenase and hyaluronidase from adult rabbit ventricles. Contraction of cardiac myocytes was traced with a video motion detector, and [Ca2+]i was measured with indo 1 at 37 degrees C. The Ica was measured with a whole-cell patch clamp at 23 degrees C. AM and calcitonin gene-related peptide (CGRP), another member of the same peptide family, showed a concentration-dependent negative inotropic effect (10(-7) mol/L AM: contraction amplitude, 64 +/- 7% of control; [Ca2+]i, 52 +/- 5% of control; n = 10; 10(-6) mol/L CGRP: contraction amplitude, 64 +/- 25%; [Ca2+]i, 70 +/- 3%; n = 5; mean +/- SD). Ica was decreased to 60 +/- 39% by superfusion with AM after the cessation of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor. Pretreatment with L-NMMA (10 mumol/L) abolished the negative inotropic effect of AM, whereas switching from AM+L-NMMA to AM+L-arginine (1 mmol/L) restored it. Superfusion with 8-bromo-cGMP also showed a negative inotropic effect. AM significantly increased the intracellular content of cGMP, a second messenger of NO, but not that of cAMP. AM (10 nmol/L) blunted the effect of 1 mumol/L forskolin. CONCLUSIONS: AM has a negative inotropic effect and decreased both [Ca2+]i and Ica, with these effects being at least party mediated via the L-arginine-NO pathway in adult rabbit ventricular myocytes.     

Role of alpha1-adrenoceptor subtypes in production of the positive inotropic effects in mammalian myocardium: implications for the alpha1-adrenoceptor subtype distribution

Both alpha1A- and alpha1B-adrenoceptor subtypes are present in mammalian myocardium. Alpha1-adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are proposed: an increase in myofilament Ca2+ sensitivity and an increase in transsarcolemmal Ca2+ influx during action potential prolongation that accompanies the transient outward current inhibition. We suggest that the former is mediated by the alpha1B-adrenoceptor subtype and the latter is by the alpha1A-adrenoceptor subtype. The alpha1B-adrenoceptor subtype may be located on a space more proximal to the sympathetic nerve endings than the alpha1A-adrenoceptor subtype, because the positive inotropic effect of endogenous norepinephrine was mediated entirely by the alpha1B-adrenoceptor subtype. In some species, the sustained positive inotropic effect develops following the transient negative inotropic effect, which is mediated by the alpha1A-adrenoceptor subtype.     

Beta-2 adrenergic activation of L-type Ca++ current in cardiac myocytes

The whole-cell patch-clamp and intracellular perfusion techniques were used for studying the effects of a beta-2 adrenergic receptor activation on the L-type Ca current (ICa) in frog ventricular myocytes. The beta-2 adrenergic agonist zinterol increased ICa in a concentration-dependent manner with an EC50 (i.e., the concentration of zinterol at which the response was 50% of the maximum) of 2.2 nM. The effect of zinterol was essentially independent of the membrane potential. The stimulatory effect of zinterol was competitively antagonized by ICI 118,551, a beta-2 adrenergic antagonist. The maximal stimulatory effect of zinterol was comparable in amplitude to the effect of a saturating concentration (1 or 10 microM) of isoprenaline, a nonselective beta adrenergic agonist. Moreover, 3-isobutyl-1-methylxanthine (100 microM), a nonselective phosphodiesterase inhibitor, or forskolin (10 microM), a direct activator of adenylyl cyclase, had no additive effects in the presence of 0.1 microM zinterol. Zinterol had a long lasting action on frog ICa because after washout of the drug, ICa returned to basal level with a time constant of 17 min. An application of acetylcholine (1 microM) during this recovery phase promptly reduced ICa back to its basal level suggesting a persistent activation of adenylyl cyclase due to a slow dissociation rate constant of zinterol from its receptor. Zinterol also increased ICa in rat ventricular and human atrial myocytes, and the maximal effect was obtained at 10 and 1 microM, respectively. In all three preparations, intracellular perfusion with 20 microM PKI(15-22), a highly selective peptide inhibitor of cAMP-dependent protein kinase, completely antagonized the stimulatory effect of zinterol on ICa. We conclude that beta-2 adrenergic receptor activation produces a strong increase in ICa in frog, rat and human cardiac myocytes which is due to stimulation of adenylyl cyclase and activation of cAMP-dependent phosphorylation.     

Possible mechanism of the negative inotropic effect of alpha1-adrenoceptor agonists in rat isolated left atria after exposure to free radicals

1. This study was designed to investigate the mechanism(s) of the negative inotropic effects of alpha1-adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals. 2. Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine-induced phospholipase C and Na+/K+ ATPase activities were measured. 3. Methoxamine (300 microM) increased contractile force by 1.6 +/- 0.2 mN in control atria but decreased contractile force in electrolysis-treated atria by 2.0 +/- 0.1 mN (P < 0.05), as determined 10 min after methoxamine addition. In contrast, the positive inotropic effects of endothelin-1 (30 nM) and isoprenaline (10 microM) were reduced from 2.6 +/- 0.3 to 1.3 +/- 0.1 mN and from 2.6 +/- 0.3 to 1.7 +/- 0.2 mN, respectively, by electrolysis treatment (P < 0.05), but not converted into a negative inotropic action. 4. In an inositol phosphate assay we observed that the stimulation of phospholipase C by methoxamine was attenuated by electrolysis when the (electrolyzed) medium from the organ bath was used, but the phospholipase C responses were restored by the use of fresh medium. However, fresh medium did not counteract the negative inotropic effect of methoxamine. Accordingly, the negative inotropic effect of methoxamine is not directly related to the impaired phospholipase C responses seen in atria subjected to electrolysis. 5. Ouabain (10 microM) and the protein kinase C inhibitor calphostin C (50 nM), completely prevented the negative inotropic effect of 300 microM methoxamine in electrolysis-treated atria. 6. Measurement of the Na+/K+ ATPase activity, revealed that in control atria, alpha1-adrenoceptor stimulation with 300 microM methoxamine, decreased the Na+/K+ ATPase activity by 14.4 +/- 7.7%. In contrast, methoxamine increased the Na+/K+ ATPase activity by 48.8 +/- 8.9% (P < 0.05) in electrolysis-treated atria. Interestingly, this increase in Na+/K+ ATPase activity was completely counteracted by calphostin C (1.4 +/- 0.1% over basal). 7. These results indicate that the negative inotropic effects of alpha1-adrenoceptor agonists, observed in rat isolated left atria exposed to free radicals, are likely to be caused by protein kinase C-mediated phosphorylation and subsequent activation of the Na+/K+ ATPase.     

Alpha1-adrenergic stimulation of sarcolemmal Na+-H+ exchanger activity in rat ventricular myocytes: evidence for selective mediation by the alpha1A-adrenoceptor subtype

Alpha1-adrenoceptor (alpha1-AR) stimulation increases sarcolemmal Na+-H+ exchanger (NHE) activity. The present study was designed to determine the role(s) of alpha1-AR subtype(s) in mediating this response. As an index of NHE activity, acid efflux rates (JHs) were determined in single rat ventricular myocytes loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after 2 consecutive intracellular acid pulses in bicarbonate-free medium. JH at pHi 6.90 did not change significantly during the second pulse relative to the first in control cells but increased in a dose-dependent manner when the second pulse occurred in the presence of phenylephrine (nonselective alpha1-AR agonist) or A61603 (alpha1A-AR-selective agonist), with EC50 values of 1.24 micromol/L and 3.6 nmol/L, respectively (both agonists given together with 1 micromol/L atenolol). Stimulation of NHE activity by 10 micromol/L phenylephrine was inhibited in a dose-dependent manner by the competitive antagonists prazosin, WB4101, and 5-methylurapidil, with IC50 values of 12, 32, and 149 nmol/L, respectively. Analyses of the relative EC50 and IC50 values obtained (and Ki values estimated from the antagonist IC50s) in relation to the relative potencies of these agents at native rat alpha1-AR subtypes and their relative affinities for recombinant rat alpha1-ARs suggest that alpha1-adrenergic stimulation of sarcolemmal NHE activity is likely to be mediated selectively by the alpha1A-AR.     

Action-potential duration and contractility in canine cardiac tissues: action of inotropic drugs

The goal of this study was to determine whether neuronally derived nitric oxide mediates responses of cerebral blood flow (CBF) to N-methyl-D-aspartate (NMDA). In anesthetized Sprague-Dawley rats, regional CBF of the parietal cortex was monitored by laser-Doppler flowmetry. Topical application of either NMDA or acetylcholine produced concentration-related increases in CBF. Responses of CBF to NMDA (10(-5) M) but not to acetylcholine were inhibited (0+/-3% vs 21+/-5%, p < 0.05) by 7-nitroindazole (50 mg/kg, i.p.). MK-801 (0.5 mg/kg, i.v.) and tetrodotoxin (10(-6) M, topical application) also inhibited NMDA-induced responses. These results suggest that nitric oxide of neuronal origin mediates NMDA-induced increases in CBF.     

Prostaglandin F2alpha as a mediator of pulmonary changes during platelet aggregation

Increases in pulmonary arterial pressure, tracheal insufflation pressure, and blood levels of the prostaglandin F2alpha metabolite, 15-keto-13, 14-dihydro F2alpha, were observed after protamine chloride or thrombin-induced platelet aggregation and release reaction in dogs. These effects were largely eliminated after administration of acetylsalicylic acid, an inhibitor of prostaglandin synthesis. The platelet aggregation was not noticeably affected. It is suggested that release of prostaglandin F2alpha from platelets is an important factor for the pulmonary changes during induced platelet aggregation. The necessity of measuring metabolite levels, rather than prostaglandin F2alpha levels, in blood during in vivo conditions is demonstrated.     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth.     

Doxorubicin cardiotoxicity: diastolic cardiac myocyte dysfunction as a result of impaired calcium handling in isolated cardiac myocytes

We examined intracellular calcium transients of isolated single cardiac myocytes from rats with doxorubicin (DOX)-induced cardiomyopathy with simultaneous measurement of cell motion. DOX was administered i.p. to Sprague-Dawley rats at 2.5 mg/kg once a week for 10 weeks. Field-stimulated calcium transients and simultaneous cell motion in single myocytes were measured in the presence or absence of isoproterenol using fura-2/AM. Histopathologic examination revealed slight changes. The time courses of both calcium transients and cell motion were significantly prolonged by DOX. There was a slight but not significant reduction in parameters of contractility in both calcium transients and cell motion. The beta-adrenoceptor responsiveness of both calcium transients and cell motion was not significantly impaired compared with the controls. Our data indicated that, despite the slight histologic changes in the heart in DOX-induced cardiomyopathy, impaired sequestration of intracellular free calcium ions in individual myocytes may be one factor leading to diastolic dysfunction. Monitoring of diastolic function is important to detect early cardiotoxicity caused by DOX.     

Effects of WB4101 and chloroethylclonidine on the positive and negative inotropic actions of phenylephrine in rat cardiac muscle

This study was designed to determine if the positive and negative inotropic actions of alpha-1-adrenergic agonists in rat atrial and ventricular myocardium are mediated via different alpha-1-adrenergic receptor (AR) subtypes. Inotropic effects of phenylephrine were examined in isolated left atrial and papillary muscle before and after treatment with prazosin, WB4101 (N-[2-(2,6-dimethoxyphenoxy)ethyl]-2,3-dihydro-1,4-benzodioxin+ ++-2-methanamine), chloroethylclonidine (CEC) and WB4101 plus CEC. Phenylephrine (10 microM) elicited a monophasic positive inotropic response in left atrial muscle and a triphasic inotropic action in papillary muscle (transient positive, then negative inotropic components preceding a sustained positive inotropic response). CEC, WB4101 and prazosin each antagonized the monophasic response in isolated left atria and the sustained positive inotropic response in papillary muscle. CEC and prazosin each antagonized the transient negative inotropic component in papillary muscle. The transient positive inotropic response was not affected by CEC, WB4101 or CEC plus WB4101, but was antagonized by higher concentrations of prazosin. These data suggest that the sustained positive inotropic effect of alpha-1-adrenergic agonists in rat atrial and ventricular myocardium results from stimulation of alpha-1A and alpha-1B ARs, whereas the transient negative inotropic component of the triphasic response in ventricular preparations is mediated via alpha-1B ARs. However, present data do not exclude the possibility that the CEC-sensitive inotropic responses elicited by phenylephrine may be mediated in part by other recently described alpha-1 subtypes. The receptors involved in the transient positive inotropic action cannot be identified by current results.     

Stimulation of glycogenolysis by beta adrenergic agonists in skeletal muscle of mice with the phosphorylase kinase deficiency mutation (I strain)

The mechanism by which beta adrenergic agonist stimulate glycogenolysis in intact skeletal muscle was investigated in mice with the phosphorylase kinase deficiency mutation (I strain). Although extracts of I strain diaphragm muscle had only 3.7% of the phosphorylase kinase activity found in extracts of the control strain (C57BL), incubation of I strain hemidiaphragms in Krebs-Ringer bicarbonate buffer with either isoproterenol or epinephrine resulted in a stimulation of the rate of glycogenolysis. In C57BL diaphragms, the EC50 values for isoproterenol and epinephrine were 2 and 14 nM, respectively. With I strain diaphragms, dl-isoproterenol or l-epinephrine stimulated glycogenolysis as a linear function of the log of the drug concentration with no apparent plateau of response up to concentrations of 30 to 40 mugM. For each 10-fold increase in drug concentration, isoproterenol and epinephrine stimulated glycogenolysis in I strain muscles an additional 0.37 to 0.42 mg/g/hr, a slope in the concentration-response relationship of 0.17 and 0.37, respectively, of that measured in C57BL diaphragms at concentrations around the EC50. The highest glycogenolytic response measured in I strain hemidiaphragms (at 40 mugM isoproterenol) was 80% of the maximal catecholamine-stimulated glycogenolysis in C57BL diaphragms. Both 4 nM and 4 mugM isoproterenol, in a concentration-dependent manner, stimulated phosphorylase b to a conversion in I and C57BL diaphragms and increased cyclic adenosine 3':5'-monophosphate (cyclic AMP) concentrations. The glycogenolytic response to 10.1 nM dl-isoproterenol in both I and C57BL diaphragms was blocked by 34 nM l-propranolol but not by 34 nM d-propranolol. The response to 4 mugM isoproterenol was enhanced by the cyclic nucleotide phosphodiesterase inhibitors papaverine (27 mugM) or dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 3 mugM). From the results of these studies, we conclude: 1) Catecholamines stimulate glycogenolysis in skeletal muscle of I mice, as in C57BL mice, by interacting with the beta adrenergic receptor, thereby increasing tissue cyclic AMP concentrations and stimulating phosphorylase b to a conversion. 2) alternative hypotheses for the mechanism of the catecholamine-stimulated decrease in glycogen concentration in I skeletal muscle-inhibition of glycogen synthesis, hyposia and 5'-AMP stimulation of phosphorylase b activity-have been ruled out. 3) the activity of the mutant phosphorylase kinase, although it is only 3.7% of that in extracts of C57BL muscle, is sufficient to produce phosphorylase b to a conversion and thereby account for the glycogenolytic response of I strain muscle to catecholamines.     

Effects of vesnarinone, a novel orally active inotropic agent with an immunosuppressive action, on experimental cardiac transplantation in rats

BACKGROUND: Vesnarinone (VES) has been used for treatment of patients with congestive heart failure. In addition to inotropic effects, it seems to have immunosuppressive action. We tested the hypothesis that VES suppresses graft rejection, inotropic dysfunction caused by early rejection, and chronic coronary obstruction in a heterotopic rat cardiac transplantation model. METHODS: (1) To study acute rejection, hearts from Lewis-Brown Norway (LBN) rats were transplanted into Lewis rats, which were treated with or without VES (50 or 100 mg/kg/day orally). (2) In a functional study, LBN hearts with or without VES (100 mg/kg/ day) were isolated and perfused on day 3 after transplantation to assess inotropic response to isoproterenol (3 x 10(-8) M). (3) To study chronic rejection, Lewis hearts were transplanted into Fisher 344 rats, which were treated with or without VES (50 mg/kg/day) for 90 days. Coronary obstructive disease was assessed by morphometric analysis. There were five to six animals in each group. RESULTS: (1) VES (100 mg/kg/day) prolonged LBN heart survival (11.7 +/- 0.7 vs. 9.6 +/- 0.7 days in control; P < 0.05). (2) Left ventricular developed pressure was depressed in transplanted hearts regardless of VES treatment (84 +/- 12, 90 +/- 8 vs. 144 +/- 16 mmHg in untransplanted hearts; P < 0.01). The developed pressure after administration of isoproterenol in VES-treated hearts (184 +/- 20 mmHg) was higher than transplanted hearts without VES (118 +/- 16 mmHg; P < 0.05), and similar to untransplanted hearts (203 +/- 27 mmHg; P = NS). (3) Transplanted hearts treated with or without VES showed similar grades of rejection (2.0 +/- 0.3 vs. 2.6 +/- 0.2; P = NS), intimal area (6,996 +/- 3,186 vs. 13,441 +/- 5,165 microns2; NS), and coronary luminal obstruction (45 +/- 16% vs. 67 +/- 14%; NS). CONCLUSIONS: VES produces mild prolongation in survival of rat heart grafts, but has no significant effect on chronic graft atherosclerosis. VES preserves the positive inotropic effects of isoproterenol that are otherwise deteriorated by early acute rejection.     

The involvement of sodium ions in the positive inotropic effect of naloxone

1. The opiate antagonist naloxone induces a positive inotropic effect in isolated cardiac muscles. 2. The response to naloxone is dependent on the presence of Na+ in the bathing solution, is proportional to the rate of electrical stimulation, and increased in the presence of veratridine. 3. Lowering [K+]o to 50% augments the response, while complete removal of K+ from the extracellular solution attenuates the response to naloxone. 4. Maximal concentration of naloxone decreases the inotropic effect of the cardiac glycoside ouabain. 5. The results indicate the involvement of intracellular sodium accumulation in the positive inotropic effect of naloxone, probably through the inhibition of the sarcolemmal Na(+)-K+ pump.     

Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

1. When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 microM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5'-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity. 2. ATP can be metabolized by ecto-nucleotidases to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine. 3. We now show that both AMP and ATP (50 microM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5'Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml(-1)), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 microM), AMP deaminase prevented the depressant effect of AMP on discharge rate. 4. AMP deaminase, at a concentration of 0.2 u ml(-1) which annulled the effect of AMP (50 microM), prevented the inhibitory activity of ATP (50 microM). A higher concentration of ATP (200 microM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase. 5. Superfusion of the slices with 5'-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges. 6. The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the effect of ATP. A corollary to this is that, when examining the pharmacology of ATP, care must be taken to inactivate AMP with AMP deaminase, as well as adenosine with adenosine deaminase, before a direct action of ATP on P1 receptors can be postulated. Failure to do so may have led to erroneous conclusions in some previous studies of nucleotide activity on nucleotide receptors.     

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.     

Comparison of the action potential prolonging and positive inotropic activity of DPI 201-106 and BDF 9148 in human ventricular myocardium

The aim of our study was to compare the effects on contractile function and action potential duration of the new Na+ channel modulator BDF 9148 with the parent compound DPI 201-106 in human ventricular myocardium. Right ventricular papillary muscles were obtained from explanted hearts of heart transplant recipients or from non-failing hearts not suitable for transplantation. BDF 9148 induced an increase in force of contraction that was accompanied by prolongation of action potential duration. The action potential duration prolonging effect of BDF 9148 was not significantly different to that of DPI 201-106. The effects of BDF 9148 were similar in muscles obtained from non-failing and failing hearts. Using Na(+)-sensitive electrodes, we have demonstrated that the positive inotropic effect of BDF 9148 is accompanied by an increase in intracellular Na+ activity. Our results indicate: (i) that BDF 9148 is as effective as DPI 201-106 in increasing force of contraction and prolonging action potential duration in human ventricular myocardium: (ii) that BDF 9148 is effective in enhancing force of contraction, in spite of heart failure; (iii) that the positive inotropic effect is related to an increased Na+ load; and (iv) due to action potential duration prolongation, changes in Q-T interval of the electrocardiogram could be possible during in vivo use of BDF 9148.