Alpha-1 adrenoceptor-mediated positive inotropic effect and inositol trisphosphate increase in mammalian heart

The positive inotropic effect of the alpha-1 adrenoceptor agonist phenylephrine was accompanied by a concentration-dependent increase in inositol trisphosphate (IP3) in electrically driven left auricles isolated from rat hearts. Further analysis of the myocardial phosphoinositide pathway revealed an additional increase in inositol phosphate and inositol bisphosphate with a concomitant decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The decrease in phosphatidylinositol bisphosphate and increase in IP3 preceded the increase in force of contraction. All effects were antagonized by the alpha-1 adrenoceptor antagonist prazosin. For comparison the effects of the beta adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine but all phosphoinositide products remained unaffected. The influence of lithium and calcium ions were studied systematically. The stimulatory effect of phenylephrine on inositol phosphates was lithium-dependent. Without lithium phenylephrine did not detectably affect the phosphoinositide turnover. Phenylephrine caused a maximal increase in inositol phosphate, inositol bisphospate and IP3 at 10 mmol/l of lithium. Lithium itself had a concentration-dependent positive inotropic effect. However, lithium did not enhance the positive inotropic effect of phenylephrine. Variation of the extracellular concentration of calcium did not influence the stimulatory effect of phenylephrine on inositol phosphates indicating that inositol phosphate turnover does not require the presence of extracellular calcium. It is concluded that the stimulation of myocardial phosphoinositide breakdown generating an increased IP3 turnover may be involved in the mechanism(s) whereby alpha-1 adrenoceptor stimulation exerts an increase in myocardial force of contraction.     

Inotropic, electrophysiological and biochemical responses to histamine in rabbit papillary muscles: evidence for coexistence of H1- and H2-receptors

This study was performed to determine the subtypes of histamine receptors that are involved in the electrophysiological, inotropic and biochemical responses to histamine in isolated rabbit papillary muscles. Histamine increased force of contraction and shortened action potential duration (APD) in a concentration-dependent manner. The former was antagonized by chlorpheniramine, a H1-antagonist, whereas the latter was blocked by cimetidine, a H2-antagonist. However, even when H1-receptors were blocked entirely by chlorpheniramine, histamine still produced a positive inotropic effect, an effect which was antagonized by cimetidine. On the other hand, when H2-receptors were eliminated by cimetidine, histamine caused a H1-receptor mediated APD prolongation. Carbachol attenuated the decrease in APD but not the increase in force of contraction caused by histamine. Cyclic AMP and cyclic GMP levels both were elevated significantly by histamine. The increase in cyclic AMP level induced by histamine was abolished by cimetidine, but not altered by chlorpheniramine, whereas the converse was true for the increase in cyclic GMP level. Additionally, histamine produced a significant stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation, although its extent was far less than that produced by carbachol. The phosphoinositide response to histamine was blocked by chlorpheniramine. These data suggest that H1- and H2-receptors coexist in rabbit ventricles. Stimulation of H1- and H2-receptors with histamine independently sets off the biochemical responses linked specifically to the respective subtypes of histamine receptors. On the other hand, the inotropic and electrophysiological responses to histamine are governed predominantly by H1- and H2-receptors, respectively, and this results in an apparent restriction of the expression of the responses mediated by another subtype.     

Adenosine selectively attenuates H2- and beta-mediated cardiac responses to histamine and norepinephrine: an unmasking of H1- and alpha-mediated responses

Adenosine is known to attenuate the positive inotropic and chronotropic effects of norepinephrine and histamine by reducing cyclic AMP accumulation. We assessed whether adenosine, while inhibiting the cardiac responses mediated by beta and H2 receptors, leaves unmodified the responses mediated by alpha and H1 receptors. In isolated cardiac preparations from the guinea pig, adenosine antagonized the positive inotropic effect of histamine more than that of norepinephrine. This most likely occurred because, by attenuating H2 and beta responses, adenosine unmasked the H1-negative and alpha-1-positive components of the inotropic effects of histamine and norepinephrine. Consistent with this hypothesis, the pure H2 agonist impromidine appeared to be antagonized by adenosine less than histamine, and norepinephrine less than isoproterenol. In addition, adenosine antagonized the positive inotropic effect of norepinephrine in the presence of the alpha-1 blocker prazosin, whereas it did not affect the inotropic effect of phenylephrine. In the papillary muscle depolarized by 22 mM K+, adenosine antagonized the restoration of contractile responses induced by histamine or norepinephrine. This action of adenosine was reversed by the phosphodiesterase inhibitor papaverine and by the adenylate cyclase activator forskolin, suggesting that adenosine attenuates beta and H2 responses by suppressing the cyclic AMP-dependent facilitation of Ca++ influx promoted by the two amines. Our data indicate that adenosine selectively attenuates H2 and beta but not alpha and H1 responses. Thus, when catecholamines, histamine and adenosine are released together, as in myocardial ischemia, in addition to their individual effects, negative inotropism, decreased impulse conduction velocity and coronary constriction (i.e., H1- and alpha-mediated responses) may result from the adenosine-histamine-norepinephrine interaction.     

Histamine receptors on human isolated pulmonary arterial muscle preparations: effects of endothelial cell removal and nitric oxide inhibitors.

Human isolated intact pulmonary arterial muscle ring preparations which were precontracted with serotonin (10 microM) relaxed when stimulated with low concentrations of histamine, 2-[2-thiazolyl]ethylamine or 2-[pyridyl]ethylamine (pD2 values: 8.66 +/- 0.22, 7.10 +/- 0.06 and 6.20 +/- 0.26, respectively) or contracted at higher concentrations of these agonists. This relaxant response was obliterated in endothelial denuded tissues. Chlorpheniramine (H1-antagonist; 0.25 and 2.5 microM) induced a small contractile response in the tissues at resting tone (0.08 +/- 0.03 g and 0.10 +/- 0.10 g, respectively). Chlorpheniramine also shifted the histamine relaxation curves to the right (pD2 values: control, 8.85 +/- 0.31; 0.25 microM, 6.90 +/- 0.41; and 2.5 microM, 5.58 +/- 0.30; N = 6). Dimaprit (H2-agonist) induced a small relaxation (20%) in both intact and denuded tissues. Treatment of the tissues with cimetidine (H2-antagonist; 50 microM), burimamide (H2/H3-antagonist; 10 microM) and impromidine (H2-agonist/H3-antagonist; 1 microM) did not alter histamine-induced relaxation or contraction. Indomethacin (1.7 microM) caused a small contraction in these tissues and significantly reduced the histamine relaxation. The nitric oxide inhibitors (L-NG-monoethyl-L-arginine, 30 and 300 microM; or L-NG-nitroarginine, 30 and 300 microM) induced a slight and variable contraction in the preparations. However, these inhibitors, only in the presence of indomethacin, inhibited the relaxant effects of histamine and potentiated the contractions induced by this amine. These data suggest that a dual endogenous vasodilatory mechanism is present in human isolated pulmonary arterial muscle preparations and that products of the cyclooxygenase and endothelium-derived relaxing factor-nitric oxide pathway may interact to regulate histamine stimulation of H1-receptors.     

Concentration- and time-dependence of phosphoinositide hydrolysis induced by endothelin-1 in relation to the positive inotropic effect in the rabbit ventricular myocardium.

The effects of endothelin-1 on phosphoinositide hydrolysis in relation to the positive inotropic effect of the compound were studied in the rabbit ventricular myocardium. Endothelin-1 elicited a concentration- and time-dependent accumulation of [3H]inositol monophosphate (IP1) in ventricular muscle slices prelabeled with myo-[3H]inositol in concentrations similar to those that caused a positive inotropic effect. The EC50 value of endothelin-1, both for the induction of [3H]IP1 accumulation and for the positive inotropic effect was 6 x 10(-9) M. The endothelin-induced positive inotropic effect was linearly related to [3H]IP1 accumulation. Endothelin-1 induced a small transient negative inotropic effect before the onset of a gradually developing and sustained positive inotropic effect which reached a steady level at 30 min. After administration of endothelin-1, [3H]IP3 accumulated rapidly and transiently before the development of the positive inotropic effect. [3H]IP2 accumulated slowly and peaked at 20 min. [3H]IP1 accumulation occurred more slowly and of the inositol phosphates its time course coincided best with that of the positive inotropic effect. Phorbol 12,13-dibutyrate inhibited both the [3H] IP1 accumulation and positive inotropic effect induced by endothelin-1. The present results indicate that an acceleration of phosphoinositide hydrolysis induced by activation of endothelin-1 receptors may be responsible for induction of the positive inotropic effect of endothelin-1 on rabbit ventricular myocardium.     

High efficiency coupling of alpha-1 adrenergic receptors to inositol phospholipid metabolism revealed by denervation of rat vas deferens

The effect of surgical denervation on alpha-1 adrenergic receptor-stimulated inositol phosphate (IP) formation was examined in rat vas deferens. Rings of tissue from acutely reserpinized animals were incubated with [3H]inositol in the presence of lithium to block IP degradation and desmethylimipramine to block neuronal uptake of norepinephrine. Eighteen days after denervation the potency of norepinephrine in stimulating [3H]IP accumulation was increased 10-fold. The potency of epinephrine was increased only 3.5-fold, and the potency of phenylephrine was not altered significantly. The potency of norepinephrine in control tissues incubated with 0.1 microM desmethylimipramine was unaffected by addition of cocaine to further block neuronal uptake; however, addition of pyrogallol and pargyline to block metabolic degradation increased the potency of norepinephrine in these tissues by 6-fold. Two days after denervation there was a similar 5-fold increase in the potency of norepinephrine. In denervated tissues, the potency of norepinephrine in stimulating [3H]IP accumulation was decreased about 40-fold after receptor inactivation with 1 microM phenoxybenzamine. These results suggest that there is a substantial alpha-1 adrenergic receptor reserve for stimulating [3H]IP accumulation in rat vas deferens which is normally obscured by rapid inactivation of norepinephrine. The increase in the potency of norepinephrine after denervation appears to be due to removal of these inactivation mechanisms.     

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.     

Serotonergic control of phenylephrine-induced cyclic 3,5'-adenosine monophosphate and inositol phosphates formation in rat hippocampus

Chemical lesion of serotonergic afferents to the hippocampus resulted in a potentiation of phenylephrine (PHE) stimulation of cyclic AMP (cAMP) formation in rat hippocampal slices. The concentration-response curve of the alpha-1-agonist in lesioned rats was shifted markedly to the left with the peak increase occurring at a concentration 200 times lower (500 nM) than that required to raise cAMP to the same extent in slices of the sham-operated controls. Adenylate cyclase activity measured on crude homogenate preparations was increased by the lesion, too. The alpha-1 antagonist WB-4101 (dimethyloxyphenoxy-ethylamino-methyl benzo-1,4-dioxan) was the most potent inhibitor of PHE in lesioned rats with an IC50 of 125 nM, 110 times lower than that of prazosin (14 microM); yohimbine and propranolol, respectively, alpha-2 and beta-antagonists caused inhibition only at very high concentrations. Serotonin (10 microM) induced a marked rightward shift in the concentration response of PHE in lesioned rats, without any effect of its own on the cAMP level. PHE stimulated inositol phosphates accumulation in hippocampal slices in a concentration-dependent manner with an EC50 of 20 microM. Chemical lesion of the serotonergic median raphe nuclei caused a significant reduction of PHEs maximal effect on inositol phosphate accumulation by 50% with no change detectable in the EC50. The results imply that both second messenger responses to PHE stimulation are modulated by serotonin. Whether cAMP and phosphatidylinositol-4,5-bisphosphate generated second messengers act in a cooperative manner, or independently, is discussed.     

Does cyclic AMP mediate rat urinary bladder relaxation by isoproterenol?

Cyclic AMP is the prototypical second messenger of beta-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon beta-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying beta-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 microM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 microM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 microM), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 30 microM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 microM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 microM), tetraethylammonium (3 microM), apamin (300 nM), and glibenclamide (10 microM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in beta-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.     

Alpha-1 adrenergic stimulation of 1,4,5-inositol trisphosphate formation in ventricular myocytes

We demonstrated previously that alpha-1 adrenergic catecholamines modulate cardiac automaticity in a manner that is dependent upon the function of a pertussis toxin sensitive guanine nucleotide binding protein (G protein). Furthermore, we demonstrated that alpha-1 adrenergic receptor stimulation promotes the accumulation of inositol monophosphate (IP1). In the present study we used high-pressure liquid chromatography to resolve individual inositol phosphate isomers formed in norepinephrine-stimulated cultured rat ventricular myocytes. Norepinephrine stimulated a rapid, transient increase in 1,4,5-inositol trisphosphate (1,4,5-IP3) which was followed by slower, sustained increases in 1,3,4-IP3, inositol bisphosphate (IP2) and IP1. IP1 was composed of two major isomers with retention times characteristic of 1-IP1 and 4-IP1. 4-IP1 was the predominant IP1 isomer formed during stimulation with norepinephrine suggesting that the polyphosphoinositides rather than phosphatidylinositol are the principal targets of norepinephrine-stimulated phospholipase C activity in the heart. This was confirmed in studies performed on myocyte membranes which demonstrated proportionately greater IP2 and IP3 (relative to IP1) accumulation in response to norepinephrine. G protein regulation of alpha-1 adrenergic-dependent inositol phospholipid hydrolysis also was examined. In myocyte membranes, guanosine-5'-0-(3-thiotriphosphate) induced the accumulation of IP2 and IP3 and was required for the stimulatory effect of norepinephrine. This response was not impaired after pretreatment with pertussis toxin. These results indicate that the myocyte alpha-1 adrenergic receptor is coupled to a polyphosphoinositide-specific phospholipase C by a pertussis toxin insensitive G protein and suggest that under certain conditions IP3 may serve an important role in alpha-1 adrenergic modulation of cardiac function.     

Effects of histamine-trifluoromethyl-toluidide derivative (HTMT) on intracellular calcium in human lymphocytes

We have studied the effects of histamine trifluoromethyl-toluidide derivatives on calcium mobilization in human peripheral blood lymphocytes using spectrofluorometric analysis. HTMT (compound 1) induced two phases of increase in intracellular calcium concentration--a rapid intracellular calcium concentration peak (10-60 sec), partial recovery (1-3 min) and a sustained moderate elevation that persisted for more than 5 min. The EC50 value was 1.9 X 10(-5) M. Pretreatment of lymphocytes with the agonist resulted in receptor desensitization that recovered after 15 min when the cells were drug free. The presence of extracellular ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid did not abrogate the early phase of the calcium rise, suggesting that the calcium appearing in the cytosol during the early phase was derived from intracellular stores. The increase in intracellular calcium concentration by this compound was competitively antagonized by high concentrations of histamine but not by classic histamine receptor antagonists (H1, H2 or H3). Other cyclic AMP elevating agents, with the exception of prostaglandin E2, did not affect the increase in calcium levels induced by compound 1. Compound 1 caused phosphatidylinositol metabolism resulting in inositol phosphate production, suggesting that inositol triphosphate may be the second messenger for the mobilization of intracellular Ca2+ by compound 1. The data imply a specific binding site for histamine trifluoromethyl toluidide derivative on lymphocytes that is different than the classic H1, H2 or H3 receptors.     

Pharmacological evidence for distinct muscarinic receptor subtypes coupled to the inhibition of adenylate cyclase and to the increased generation of inositol phosphates in the guinea pig myometrium

In the guinea pig myometrium, muscarinic receptor activation leads to contraction and elicits two biochemical responses viz. an increased formation of inositol phosphates (via a guanine nucleotide regulatory protein, distinct from the stimulatory and inhibitory G proteins of the adenylate cyclase system and a decreased synthesis of cyclic AMP involving inhibitory G protein activation. We now describe two major differences in the effects of muscarinic agonists. First, the greater potency of carbachol in inhibiting cyclic AMP formation (EC50 = 8 nM) than in stimulating the accumulation of inositol phosphates and tension (EC50 = 15 and 2 microM, respectively). Second, carbachol, oxotremorine and pilocarpine were equally effective in eliciting cyclic AMP inhibition but the order of potency for inositol phosphate formation was carbachol greater than oxotremorine and pilocarpine was without effect. The partial agonists, pilocarpine and oxotremorine, inhibited carbachol-mediated inositol phosphate formation. Pirenzepine, selective for muscarinic M1 receptor subtype, displayed a low affinity for antagonizing cyclic AMP inhibition, inositol phosphate generation and tension due to carbachol (Ki = 286, 92 and 110 nM, respectively). AF-DX116 (11-[( 2-[(diethylamino)methyl]-1- piperidinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine- 6-one), selective for cardiac M2 receptors blocked cyclic AMP inhibition with high affinity (Ki = 1.14 nM) while it antagonized inositol phosphate formation with low affinity (Ki = 346 nM). Both high (Ki = 1 nM) and low (Ki = 100 nM) affinities were displayed by AF-DX116 in antagonizing contractions due to carbachol (24 and 76% inhibition, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular reactivity of isolated thoracic aorta of the C57BL/6J mouse

We characterized the thoracic aorta from the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, in response to vasoactive substances. Strips of aorta were mounted in tissue baths for measurement of isometric contractile force. Cumulative concentration-response curves to agonists were generated to observe contraction, or relaxation in tissues contracted with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). In endothelium-denuded strips, the order of agonist contractile potency (-log EC(50) [M]) was norepinephrine > phenylephrine = 5-hydroxytryptamine > dopamine > PGF(2alpha) > isoproterenol > KCl. Angiotensin II and endothelin-1 were weakly efficacious (15% of maximum phenylephrine contraction), as were UK14,304, clonidine, histamine, and adenosine. In endothelium-intact strips, agonists still caused contraction and both angiotensin II and endothelin-1 remained ineffective. In experiments focusing on angiotensin II, angiotensin II-induced contraction was abolished by the AT(1) receptor antagonist losartan (1 microM) but was not enhanced in the presence of the AT(2) receptor antagonist PD123319 (0.1 microM), tyrosine phosphatase inhibitor orthovanadate (1 microM) or when angiotensin II was given noncumulatively. Prazosin abolished isoproterenol-induced contraction and did not unmask isoproterenol-induced relaxation. Angiotensin II and endothelin-1 did not cause endothelium-dependent or -independent relaxation in phenylephrine- or PGF(2alpha)-contracted tissues. Acetylcholine but not histamine, dopamine, or adenosine caused an endothelium-dependent vascular relaxation. These experiments provide information as to the vascular reactivity of the normal mouse thoracic aorta and demonstrate that the mouse aorta differs substantially from rat aorta in response to isoproterenol, angiotensin II, endothelin-1, histamine, and adenosine.     

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)     

Alpha adrenoceptor activation and postreceptor contractile mechanisms in the human digital artery

In order to compare postreceptor mechanisms of different alpha adrenoceptors, polyphosphoinositide hydrolysis and extracellular calcium entry stimulated by alpha-1 and alpha-2 adrenoceptor activation has been evaluated in the human digital artery, a tissue which contains both receptors. [3H]Inositol-I-PO4 accumulation during a 60-min exposure to an alpha-1 or alpha-2 agonist in the presence of 5 mM LiCl was used as an index of phosphatidylinositol-4,5-bisphosphate hydrolysis. Norepinephrine (1-30 microM) produced a concentration-related increase in [3H]inositol phosphate formation with an EC50 of 2.3 microM. Equieffective contractile concentrations of TL-99 (1 microM) and methoxamine (100 microM) produced similar increases in [3H]inositol-I-PO4 formation (1.41- and 1.70-fold increases over control, respectively). Norepinephrine EC50 values of 0.54, 1.7 and 1.0 microM were obtained for contractile responses in 1.25 mM Ca++, 0 mM Ca++ and 0 mM Ca++ + 0.1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, respectively. Calcium omission (no ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) produced similar inhibition of alpha-2 and alpha-1 adrenoceptor-mediated responses, assessed as inhibition of the area under the concentration-effect curve. Calcium omission generally produced a slightly greater inhibition of TL-99- or methoxamine-induced contractile responses than did nifedipine (0.1 or 1 microM), but the combination of calcium-omission and nifedipine practically abolished the contractile responses. However, the combination of a calcium omission and nifedipine did not prevent the accumulation of inositol monophosphate stimulated by TL-99 or methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses mediated via beta-1 adrenoceptors but not beta-2 adrenoceptors exhibit supersensitivity after chronic reserpine pretreatment

The purpose of this study was to examine whether reserpine pretreatment induces supersensitivity of both beta-1 and beta-2 adrenoceptor-mediated responses. Guinea pigs received reserpine (0.5 mg kg-1 s.c. or i.p.) daily for 7 days. Isolated tissues were set up in the presence of phentolamine (5 microM) and metanephrine (10 microM) and the sensitivity to isoproterenol and, where possible, a partial agonist (ritodrine, salbutamol or prenalterol) was determined. The beta adrenoceptor-mediated responses were recorded as the increase in rate and tension of right and left atria, inhibition of carbachol-induced contractions of ileum, relaxation of aortic spirals contracted with histamine, inhibition of transmurally stimulated vas deferens and relaxation of tracheal spirals and lung strips with intrinsic tone. The atria exhibited supersensitivity after reserpine pretreatment (s.c. and i.p.) as a leftwards shift of the isoproterenol concentration-response curve and elevation of the prenalterol maximum response. The ileum was also supersensitive, but only when tissues from animals receiving i.p. reserpine were compared with shams, which themselves were subsensitive or when reserpine was administered s.c. The trachea was also supersensitive, but not the aorta, lung and vas deferens, the responses of which are mediated via beta-2 adrenoceptors. In contrast, beta-1 adrenoceptors are involved in the atrial, ileal and tracheal responses. Therefore, only responses mediated via beta-1 adrenoceptors exhibited reserpine-induced supersensitivity which supports the hypothesis that beta-1 but not beta-2 adrenoceptors receive a sympathetic innervation.     

Beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart: relationship to extra- and intracellular calcium

We have investigated the contribution of extra- and intracellular Ca++ and calmodulin to beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart perfused with Krebs-Henseleit buffer. Administration of isoproterenol (100 ng) increased the output of immunoreactive 6-keto-prostaglandin F1 alpha and prostaglandin E2 as well as heart rate and developed tension; the coronary perfusion pressure was reduced. Isoproterenol-induced output of prostaglandins was positively correlated with the extracellular Ca++ concentration (0-5 mM). Infusion of the Ca++ channel blockers diltiazem (22 microM) or nifedipine (0.27 microM) inhibited isoproterenol-stimulated output of prostaglandins and the positive inotropic but not the positive chronotropic effect of the amine. Administration of the intracellular Ca++ antagonists 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (23 microM) or ryanodine (1.6 microM) reduced the outflow of prostaglandins and the positive chronotropic and inotropic effect elicited by isoproterenol. The calmodulin inhibitors trifluoperazine (50 microM) or calmidazolium (1 microM) failed to alter isoproterenol-induced output of prostaglandins; trifluoperazine but not calmidazolium reduced the developed tension and coronary perfusion pressure without altering heart rate. The prostaglandin synthesis elicited by arachidonic acid (3 micrograms) was inhibited by indomethacin but not by alterations in extracellular Ca++, Ca++ channel blockers, intracellular Ca++ antagonists or calmodulin inhibitors. These data suggest that activation of beta adrenergic receptors promotes cardiac prostaglandin synthesis and myocardial contractility by increasing the trans-sarcolemmal flux of Ca++, which releases intracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does the positive inotropic action of a novel cardiotonic agent, MCI-154, involve mechanisms other than cyclic AMP?

MCI-154 is a new positive inotropic agent with vasodilating property. Experiments were carried out in the canine isolated right ventricular muscle in order to elucidate whether or not cyclic AMP is involved in the positive inotropic effect (PIE) of MCI-154. MCI-154 (10(-7) to 10(-4) M) produced a concentration-dependent PIE amounting to 75% of the maximal effect of isoproterenol. MCI-154 did not affect the time to peak tension and had a tendency to shorten the relaxation time and total duration of contraction. Pindolol, reserpine-pretreatment or tetrodotoxin did not modify the PIE of MCI-154. MCI-154 increased the cyclic AMP levels only at 3 X 10(-4) M, whereas CI-914, of which chemical structure is similar to that of MCI-154, elevated definitely the cyclic AMP at the lower concentrations (10(-5) to 10(-4) M). Carbachol at a concentration known to decrease markedly the PIE of amrinone, milrinone and papaverine, did not affect the PIE of MCI-154. MCI-154 inhibited the activity of a crude phosphodiesterase (PDE) from the canine ventricular muscle and it enhanced the PIE of isoproterenol, which implied the involvement of cyclic AMP. However, the maximal inhibition of PDE by MCI-154 remained less than 18%. Amrinone, milrinone and papaverine inhibited more potently the PDE activity than MCI-154. These results suggest that the elevation of cyclic AMP levels is only partially involved in the PIE of MCI-154 in the canine right ventricular muscle, and that MCI-154 may have novel mechanisms of action different from those of amrinone, milrinone and CI-914 that are largely cyclic AMP-dependent.     

Regulation of alpha-1 adrenergic receptor density and functional responsiveness in rat brain

The effects of changes in norepinephrine availability on alpha-1 adrenergic receptor density and responsiveness were determined in several regions of rat brain. Receptor density was determined from specific 125I-BE 2254 binding, and responsiveness was determined by norepinephrine-stimulated increases in inositol phosphate accumulation and in cyclic AMP accumulation in the absence or presence of adenosine in slices where beta adrenergic receptors had been inactivated. Adrenergic input was reduced by destroying noradrenergic neurons with DSP-4, depleting amines with chronic reserpine treatment or blocking alpha-1 adrenergic receptors with chronic prazosin treatment. All three treatments caused similar increases in alpha-1 adrenergic receptor density in cerebral cortex but not in striatum or hippocampus. DSP-4 treatment increased the maximal cyclic AMP response to both norepinephrine and phenylephrine but did not alter the maximal inositol phosphate response or the maximal potentiation of the cyclic AMP response to adenosine. DSP-4 treatment also increased the potency of norepinephrine in activating all three responses in cerebral cortex. Adrenergic input was increased by chronic treatment with desmethylimipramine to block norepinephrine reuptake, chronic i.c.v. infusion of norepinephrine or chronic yohimbine treatment to block presynaptic autoreceptors. None of these treatments caused significant changes in alpha-1 adrenergic receptor density or functional responsiveness in any region studied. The results suggest that alpha-1 adrenergic receptor density and responsiveness in rat brain can be increased but not easily decreased. They also suggest that different brain regions are affected differently by alterations of adrenergic input and that the different responses are not coregulated.