Regional differences in the responses of guinea-pig cardiac tissue to carbachol and their potentiation by hypothermia

The responses of guinea-pig isolated cardiac tissues to carbachol were examined. Tension responses of paced left atria and right ventricular papillary muscles, rate responses of spontaneously beating right atria and working hearts and contractility (+dP/dt) of paced and unpaced working hearts were obtained at 38 degrees C. Carbachol induced negative inotropic and chronotropic responses of atria, abolishing tension and rate at the maxima. The spontaneously beating heart also exhibited negative chronotropy. The papillary muscles displayed partial inhibition of tension but, in tissues from reserpine-pretreated animals, negative inotropy was absent. Similarly, no reduction of contractility of paced working hearts was obtained. It was concluded that muscarinic receptors mediating a direct inhibition of ventricular muscle are virtually absent and that the small response obtained in untreated tissue may be due either to inhibition of endogenous catecholamine release via presynaptic receptors or to antagonism of released norepinephrine. Lowering the temperature to 30 or 25 degrees C affected resting tension, rate and contractility and the magnitude of carbachol responses. The concentration-response curves, when plotted as a percentage of the maximum, were displaced to the left by cooling of the atria and papillary muscles. The papillary muscles now exhibited a response after reserpine pretreatment. In working hearts, the concentration-response curves for the fall in spontaneous rate were also shifted to the left, but this was not significant, probably because the temperature could be reduced to only 30 degrees C, below which contractions ceased. Cooling of guinea-pig isolated cardiac preparations therefore induced supersensitivity to the muscarinic effects of carbachol.     

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)     

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.     

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.     

LY83583: an agent that lowers intracellular levels of cyclic guanosine 3',5'-monophosphate

A novel compound, LY83583 (6-anilino-5,8-quinolinedione), was found to lower basal levels of cyclic GMP (cGMP) in fragments of guinea-pig lung incubated in vitro. The lowering of cGMP was dose-related reaching a maximum of 72% at 5 X 10(-5) M. Basal levels of cyclic AMP (cAMP) were not lowered by LY83583. cGMP concentrations were also reduced in guinea-pig heart and cerebellum after incubation with LY83583. However, the drug did not alter the levels of this cyclic nucleotide in spleen. Exposure of lung fragments from sensitized guinea pigs to ovalbumin resulted in a marked increase in cGMP and cAMP. LY83583 prevented completely the accumulation of cGMP and attenuated the rise in cAMP. Similar results were obtained in rat cerebellum stimulated with kainic acid. The compound also blocked ovalbumin-induced release of slow reacting substance of anaphylaxis (leukotrienes) from guinea-pig lung. Subcutaneous administration of LY83583 to guinea pigs did not affect cGMP concentrations in vivo in lung, but the total amount of cGMP in spleen was reduced dramatically. This was accompanied by a marked splenomegaly. LY83585 did not inhibit lung guanylate cyclase. In fact, activity was increased in a cell-free preparation from guinea-pig lung. The mechanism by which LY83583 reduced concentrations of cGMP is presently unknown. Nevertheless, our studies suggest that LY83583 will be a valuable pharmacological tool to help elucidate the role of cGMP in biological events.     

Whether phenylephrine exerts inotropic effects through α- or β-adrenoceptors depends upon the relative receptor populations

Phenylephrine produced concentration-related positive inotropic responses in isolated left atria and papillary muscles of guinea-pigs and rats. In rat tissues, these responses were unaffected by propranolol but antagonized by prazosin and therefore mediated via alpha 1-adrenoceptors. The alpha 1-adrenoceptor agonist methoxamine also exerted positive inotropic effects in these rat tissues. The maximum alpha-adrenoceptor-mediated effect of methoxamine (relative to the isoprenaline maximum) was greater than that of phenylephrine in left atria (in the presence of propranolol), whereas in papillary muscles phenylephrine exerted the greater maximum. In guinea-pig papillary muscles, the response to phenylephrine was unaffected by prazosin but was antagonized by propranolol and therefore caused by stimulation of beta-adrenoceptors. Methoxamine had no effect in guinea-pig papillary muscles. Guinea-pig left atria produced biphasic concentration-response curves for phenylephrine, the lower portion being antagonized by phentolamine and was therefore alpha-adrenoceptor-mediated, while the upper portion was antagonized by propranolol and therefore beta-adrenoceptor-mediated. Methoxamine exerted a small inotropic response, the maximum of which was similar to that of the first component of the phenylephrine response. Phenylephrine was a partial agonist for the cardiac beta-adrenoceptor. The density of rat ventricular alpha-adrenoceptors was 4 times greater than beta-adrenoceptor density, as measured by [3H]-prazosin and [3H]-dihydroalprenolol binding. This explains why the responses of rat papillary muscles were alpha-adrenoceptor-mediated. In contrast, the density of beta-adrenoceptor binding sites in guinea-pig ventricles was 6 times greater than the alpha-adrenoceptor density. This explains why the phenylephrine responses were beta-adrenoceptor-mediated in guinea-pig papillary muscles. In the left atria of guinea-pigs, which displayed both alpha- and beta-adrenoceptor-mediated responses, the densities of alpha- and beta-adrenoceptor binding sites were similar. Thus, phenylephrine exerts positive inotropic effects through alpha- or beta-adrenoceptors depending upon their relative densities.     

Effects of cytochalasin-B and phloretin on digitalis inotropy

Phloretin and cytochalasin-B are known to inhibit sugar transport across the cell membrane of many tissues. Both of these agents at concentrations of 100 and 20 microM, respectively, blocked the inotropic effects of ouabain and acetylstrophanthidin (AS) in isolated rabbit atria and papillary muscle preparations. Neither of these agents had any effect of its own on contractile force. Addition of phloretin or cytochalasin-B after the inotropic response to ouabain was fully established did not reverse the effect. The potency of cytochalasin-B was greater in atria than in papillary muscles, 1 microM of cytochalasin caused significant inhibition of the inotropic effect of ouabain in atria without significant effect in papillary muscles. Phloretin but not cytochalasin-B decreased the binding of [3H]ouabain to a semipurified sarcolemmal preparation isolated from canine left ventricular muscle. Neither ouabain nor AS had a substantial positive inotropic effect in atria suspended in substrate-free medium. Substitution of pyruvate (5 mM) for glucose did not fully support their inotropic effect in atria. Papillary muscles behaved differently, in that substrate-free as well as pyruvate media almost fully supported the inotropic effects of ouabain and of low concentrations of AS. Higher concentrations (greater than 250 ng/ml) of As produced a negative inotropic response in substrate-free medium. The possibility that an "active" sugar transport system is required for digitalis inotropy is ruled out by the observation that 2-deoxyglucose also prevents the inotropic effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Cardiac responsiveness to alpha and beta adrenergic amines: effects of carbachol and hypothyroidism

Previous reports of cardiac beta to alpha adrenoceptor interconversion secondary to hypothyroidism left open the alternative possibility of a functional influence by hypothyroidism on the inotropic and chronotropic effects of adrenergic amines through a different mechanism. To test this possibility, the effects of hypothyroidism (thyroidectomy) were compared with those of acute carbachol pretreatment on the responses of isolated rat atria to the selective beta and alpha adrenoceptor agonists isoproterenol and methoxamine. Both hypothyroidism and acute carbachol pretreatment (3 X 10(-7) -10(-6) M): 1) reduced basal right atrial rates and left atrial tensions; 2) caused an apparent decrease in the inotropic and chronotropic potencies of isoproterenol; 3) reduced the degree of antagonism by propranolol of the responses to isoproterenol; 4) increased the maximum inotropic response of left atria to methoxamine; and 5) converted a lack of response to a positive chronotropic response of right atria to methoxamine. Equivalent reductions of basal rates by hypothermia, or of basal tensions by lowered calcium ion concentrations, did not affect the responses to isoproterenol or methoxamine. The results suggest that both carbachol pretreatment and hypothyroidism functionally antagonize the responses to isoproterenol and enhance the responses to methoxamine by means other than adrenoceptor interconversion.     

The relative efficiency of beta adrenoceptor coupling to myocardial inotropy and diastolic relaxation: organ-selective treatment for diastolic dysfunction.

The relative effects of drugs which elevate cytosolic cyclic AMP on inotropy and diastolic relaxation (lusitropy) of guinea pig atria were quantified in vitro. There was a temporal difference between these responses in that inotropy reached peak response considerably faster than lusitropy. Also, although the relaxation response was sustained to an elevated steady state, the inotropic responses to beta adrenoceptor agonists were transient and returned to base line over 90 min. However, the inotropic responses to forskolin and dibutyryl cyclic AMP (cAMP) were sustained. For all of the drugs tested, the lusitropic response was at least 4 times more sensitive than the inotropic response (i.e., the concentration response curve for relaxation was shifted to the left of the curve for inotropy). In the case of beta adrenoceptor agonists, these differences were greater, presumably because of the fading inotropic response over 90 min. It was found that although high efficacy beta adrenoceptor agonists such as isoproterenol (and the direct activator of adenylate cyclase forskolin) produced both inotropy and lusitropy, lower efficacy agonists produced predominant lusitropy. The low efficacy agonist prenalterol produced insignificant inotropy but 60% maximal lusitropy. These data were modeled mathematically by a "differential coupling model" which assumed that a uniform cytosolic level of elevated cAMP activated two biochemical processes of differing sensitivity. Thus, the lusitropic response (phosphorylation of phospholamban) was coupled more efficiently to the cAMP response than the inotropic response (phosphorylation of calcium channels). A second model ("differential messenger concentration model") which calculated the effects of a compartmentalization of cAMP concentration within the cardiac cell by restricted diffusion and/or selective degradation by phosphodiesterases also was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Non-cyclic AMP-dependent, positive inotropic cyclodepsipeptides with negative chronotropy.

The effects of natural cyclodepsipeptides (CDPs) on isolated rat cardiac tissue preparations were examined in vitro. Destruxin A, destruxin B (DB), roseotoxin B (RB), and roseocardin (RC), a novel CDP, each caused a concentration-dependent increase in the contraction force of the right atrium and the papillary and trabecular muscles of the right ventricle at 0.6 to 600 microM. RB, destruxin A, and DB did not affect the half-decay time of relaxation of the papillary muscles, but RC slightly prolonged it, although to a much lesser extent than BA 41899, a calcium sensitizer. This inotropic effect is accompanied by a prolongation of the automatic atrial contraction intervals. The RB-induced increase in the contraction force of papillary muscle was not affected by phentolamine, propranolol, pyrilamine, or cimetidine. RB- and RC-induced increases in the contraction force of papillary muscles were not affected by 3-isobutyl-1-methylxanthine or carbachol. Neither peptide changed the cyclic AMP levels in trabecular muscles. Neither RB nor RC affected the activity of Na(+),K(+)-ATPase from rat kidney. Neither RB, RC, nor DB affected the resting membrane potential or the apparent input resistance of papillary muscles. These results suggest that these CDPs produce both non-cyclic AMP-dependent positive inotropic and negative chronotropic effects.     

Partial agonist activity of oxotremorine at muscarinic acetylcholine receptors in the embryonic chick heart

Previous work has demonstrated that prolonged treatment of chick embryos in vivo with muscarinic agonists leads to a decrease in the number of muscarinic acetylcholine binding sites in the heart and a decrease in the sensitivity of isolated atria to the negative chronotropic response to muscarinic stimulation. We show here that treatment with the agonist carbachol leads to greater decreases in receptor number than does treatment with oxotremorine. Simultaneous treatment with oxotremorine and carbachol resulted in partial blockage in the maximal decrease in receptor number seen after treatment with carbachol alone. After treatment of embryos in vivo with carbachol to decrease the reserve of spare receptors in the atria, oxotremorine could no longer elicit a significant negative chronotropic response under conditions in which carbachol was still effective. These results demonstrate that oxotremorine is a partial agonist at the muscarinic acetylcholine receptor in the embryonic chick heart.     

Cardiac effects of adenosine and adenosine analogs in guinea-pig atrial and ventricular preparations: evidence against a role of cyclic AMP and cyclic GMP

The effects of adenosine, the Ri site adenosine receptor agonist (-)-N6-phenylisopropyladenosine (PIA), the Ra site agonist 5'-N-ethylcarboxamideadenosine (NECA) and the P site agonist 2',5'-dideoxyadenosine (DIDA) on force of contraction, cyclic AMP (cAMP) and cyclic GMP (cGMP) content and on transmembrane action potential were studied in isolated electrically driven left auricles and papillary muscles from guinea pigs. Furthermore, the effects on adenylate cyclase activity in a particulate membrane preparation were investigated. In the auricles, adenosine, PIA and NECA had negative inotropic effects which were accompanied by a shortening of the action potential. Theophylline antagonized these effects which are likely mediated by R site adenosine receptors. DIDA was ineffective. Except for a small positive inotropic effect of adenosine the analogs were ineffective in the papillary muscles. None of the mechanical effects was accompanied by a change in cAMP and cGMP content in the intact preparations. In the broken cell preparation PIA and NECA had no effect on adenylate cyclase activity. Adenosine and DIDA inhibited the enzyme. The latter effects can be classified as P site-mediated effects. In conclusion, distinct mechanical, i.e., negative inotropic effects of adenosine and its analogs in the heart are observed in auricular preparations only. These effects are unlikely to be related to the cAMP and/or cGMP system. Instead, they are probably due to a direct shortening of the action potential which, in turn, is conceivably due to an increase in K+ outward current and a secondary decrease in Ca++ inward current. This effect is apparently mediated by cardiac R site adenosine receptors which are not detectably coupled to adenylate cyclase.     

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.     

Activation of muscarinic cholinergic receptors in mouse neuroblastoma x rat glioma hybrid cells: rapid induction of enhanced capacity of prostaglandin E1 receptors to stimulate cyclic AMP accumulation

Many cells develop an adaptive increase in the capacity of adenylate cyclase to synthesize cyclic AMP (cAMP) after prolonged (hours or days) exposure to drugs which initially inhibit enzyme activity. Recent evidence suggests that adaptive increases in cAMP responses can be induced within minutes by inhibitory drugs. We have investigated the kinetics for induction and decay of this phenomenon in mouse neuroblastoma x rat glioma hybrid cells. The muscarinic cholinergic agonist carbachol induced an increase in prostaglandin E1-stimulated cAMP accumulation within 2 min of pretreatment with carbachol; the increase was 70 to 100% above control values after exposure to carbachol for 30 min. Enhanced cAMP responsiveness decayed with a half-life of about 8 min after removal of carbachol. Pretreatment with carbachol for 30 hr led to an enhanced cAMP response which decayed in two components, a rapid component and an additional, more stable component which persisted for at least 2 hr after withdrawal of carbachol. Pertussis toxin prevented these effects of carbachol. Prevention of carbachol-induced inhibition of cAMP accumulation below basal concentrations with a phosphodiesterase inhibitor did not prevent the ability of carbachol to acutely induce augmented prostaglandin E1-stimulated cAMP accumulation. Mouse neuroblastoma x rat glioma hybrid cells exhibit an enhanced cAMP response after both acute and chronic exposure to a muscarinic cholinergic agonist although these processes decay with different time courses. The signal for this acutely induced adaptation does not appear to be the decrease in cellular cAMP concentration resulting from inhibition of adenylate cyclase but does require a pertussis toxin-sensitive substrate.     

Effects of K+-channel blockers on A1-adenosine receptor-mediated negative inotropy and chronotropy of guinea-pig isolated left and right atria

Adenosine has previously been shown to stimulate K(+)-efflux and to block L-type calcium channels in atrial myocytes. The aim of the present study was to evaluate the contribution of K(+)-channels in the development of the negative inotropic and chronotropic responses to adenosine agonists in guinea-pig left and right atria, respectively. Tetraethylammonium (TEA) potentiated the negative inotropic and chronotropic responses to R-(-)-N6-(2-phenyl-isopropyl)-adenosine (R-PIA), seen as leftward shifts of the concentration-response curves. Glibenclamide had no effect on the negative inotropic response to R-PIA but increased the rate of onset of the negative chronotropic response in right atria. 4-Aminopyridine (4-AP, 10 mM), potentiated the left atrial inotropic responses to R-PIA, seen as a leftward shift of the concentration-response curve, but slowed the speed of onset of the response to a single concentration (10(-6) M) of R-PIA. This reduction in speed of onset of the response can explain the differences in effects of 4-AP on concentration-response curves reported here and previously. In the right atria, 4-AP (10 mM) inhibited the negative chronotropic responses to R-PIA, seen as a rightward shift of the concentration-response curve and reduction of the maximum response. 4-AP also slowed the onset of the right atrial rate response to R-PIA. These results support the theory that K(+)-efflux plays only a minor role in the negative inotropic responses of guinea-pig left atria to R-PIA. This apparently controls the speed of onset of the response. The negative chronotropic response of guinea-pig right atria to R-PIA appears to be much more dependent upon K(+)-efflux than for the negative inotropic response of the left atria.     

LY 83583 interferes with the release of endothelium-derived relaxing factor and inhibits soluble guanylate cyclase

LY 83583 (6-anilino-5,8-quinolinedione) has been reported to lower intracellular cyclic GMP by an unknown mechanism. The objective of the present study was to investigate the effect of LY 83583 on different types of vasorelaxation and to study its mechanism of action. Low concentrations of LY 83583 (less than or equal to 0.1 microM) inhibited endothelium-dependent relaxations of rabbit aortic strips induced by acetylcholine or by the calcium ionophore A23187. Higher concentrations (greater than or equal to 0.3 microM) were required to produce partial inhibition of relaxation to sodium nitroprusside and glyceryl trinitrate. Cyclic AMP-mediated relaxations, induced by isoprenaline or forskolin, were not affected by LY 83583 (10 microM). The site of interference of LY 83583 with endothelium-dependent relaxation was examined with endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells that were grown on microcarrier beads and stimulated by superfusion with ATP or thimerosal. EDRF in the superfusate was detected by endothelium-denuded segments of rabbit femoral artery, which responded with dilation and, simultaneously, by purified soluble guanylate cyclase (GC) in test tubes, which was activated by EDRF. When LY 83583 was added to the glutathione-containing GC-assay or to the superfusate from cultured endothelial cells, it did not affect stimulation of soluble GC by EDRF but it slowly reversed the dilator response of the arterial detector segment. Superfusion of cultured endothelial cells with LY 83583 (1 microM), rapidly and reversibly inhibited EDRF release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of endogenous opioid peptides and nitric oxide in the blunted chronotropic and inotropic responses to beta-adrenergic stimulation in cirrhotic rats

It is well known that chronotropic and inotropic responses to beta-adrenergic stimulation are impaired in cirrhosis, but the exact reason is not clear. Considering the inhibitory effect of endogenous opioid peptides and nitric oxide (NO) on beta-adrenergic pathway, we examined their roles in hyporesponsiveness of isolated atria and papillary muscles to isoproterenol stimulation in cirrhotic rats. Cirrhosis was induced by chronic bile duct ligation. Four weeks after ligation or sham operation, the responses of the isolated atria and papillary muscles to isoproterenol stimulation were evaluated in the absence and presence of naltrexone HCl (10(-6) m), N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) m), and naltrexone plus L-NAME in the organ bath. Considering the role of inducible NOS (iNOS) in hemodynamic abnormalities of cirrhotic rats, the chronotropic and inotropic responses of cirrhotic rats to isoproterenol stimulation were also assessed in the presence of aminoguanidine (a selective inhibitor of iNOS, 3 x 10(-4) m). Sham operation had no significant effect on basal atrial beating rate, contractile force, and maximal time derivatives for the development and the dissipation of papillary muscle tension. The basal atrial beating rate of cirrhotic rats did not show any significant difference compared with the sham-operated ones; however, the basal contractile parameters were significantly decreased in cirrhosis. Although the maximum effects of isoproterenol on chronotropic and inotropic responses were significantly reduced in cirrhotic rats, there was no difference in half-maximal effective concentrations of isoproterenol in these concentration-response curves. The basal abnormalities and the attenuated chronotropic and inotropic responses to isoproterenol were completely corrected by the administration of naltrexone, L-NAME and aminoguanidine. Concurrent administration of naltrexone and L-NAME also restored to normal the basal abnormalities and the blunted responses to isoproterenol in cirrhotic rats, and did not show any antagonistic effect. Based on these findings, both the endogenous opioid peptides and NO may be involved in the attenuated chronotropic and inotropic responses to beta-adrenergic stimulation in cirrhosis. It seems that the iNOS activity results in NO-induced hyporesponsiveness to beta-adrenergic stimulation in cirrhosis.