Analysis of the direct and indirect effects of adenosine on atrial and ventricular cardiac muscle

The effects of adenosine were examined upon the tension developed by isolated paced left atria, left ventricular papillary muscles, and right ventricular strips, and upon the spontaneous rate of contraction of right atria of guinea pigs. Three aspects of the direct and indirect actions were examined: the direct effects upon resting developed tension and rate, the indirect activity when added to tissues prestimulated by isoprenaline, and the indirect activity upon isoprenaline concentration--response curves when added prior to exposure to isoprenaline. The direct effects on the atria were decreases in left atrial tension and right atrial rate. These responses were antagonized by 8-phenyltheophylline (8-PT) and therefore were due to stimulation of cell surface P1 purinoceptors. This antagonism was greater in the left atria than the right. The direct ventricular effects were, in contrast, increases in force of contraction, which were not antagonized by 8-PT. This positive inotropy was also unaffected by reserpine pretreatment of the guinea pigs and therefore not due to release of endogenous catecholamines. A possible intracellular effect of adenosine is discussed. Adenosine reduced the isoprenaline-prestimulated tension or rate in both atrial and ventricular tissues, and this indirect effect was susceptible to antagonism by 8-PT. In the presence of adenosine, concentration-response curves of the left and right atria for isoprenaline were displaced to the right, and the maxima were reduced. In contrast, there was no antagonism of the papillary muscle curves to isoprenaline, but the maximum developed tension was elevated. The minimal inhibitory effects of adenosine in ventricular muscles and the high concentrations required are discussed in the context of a physiological role for endogenous adenosine in attenuating cardiac overstimulation by the sympathetic nervous system or endogenously released catecholamines.     

Effects of dihydropyridine calcium channel modulators in the heart: pharmacological and radioligand binding correlations

Bay k 8644 produced a concentration-dependent positive inotropic effect followed by a negative inotropic effect in isolated and intact cardiac preparations. Nimodipine in low concentrations produced slight positive inotropy and in higher concentrations, the usual negative inotropic action. Radioligand binding experiments revealed equilibrium dissociation constants that, taken together with the pharmacological data, suggest that dihydropyridines bind to receptor subtypes and have varying intrinsic activities.     

Interactions between peripheral-type benzodiazepine receptor ligands and an activator of voltage-operated calcium channels.

The effects of the peripheral-type benzodiapine receptor (PBR) ligands Ro 5-4864 and PK 11195 were studied in the spontaneously beating guinea pig atrium and in a model for myocardial ischemia in the rat. In the former, Bay K 8644 produced positive chronotropic and inotropic responses; intracarotid administration of this agonist (5 or 10 micrograms kg-1) to anesthetized rats elicited a transient increase in mean arterial blood pressure accompanied by alterations in the ECG pattern. Ro 5-4864 and PK 11195 (10 microM) completely blocked the positive chronotropic effect of Bay K 8644 in the atrium, PK 11209, a structural analog of PK 11195 with a low affinity for PBR, was inactive, and the central benzodiazepine receptor ligand clonazepam had a marginal effect. Ro 5-4864 potentiated whereas PK 11195 inhibited the myocardial ischemia produced by Bay K 8644 in the rat. Furthermore, PK 11195 blocked the combined response to Bay K 8644 and Ro 5-4864. Addition of Ro 5-4864 (10 microM) to the organ bath potentiated the inotropic effect of Bay K 8644 in the atria; PK 11195 at the same concentration inhibited this effect. Clonazepam and PK 11209 were both inactive in this regard. Nifedipine, a potent calcium channel antagonist, completely blocked the inotropic and chronotropic responses to Bay K 8644. PK 11195 and Ro 5-4864 did not affect this action. These findings strongly suggest that there is a functional association between PBR and voltage-operated calcium channels in the guinea pig atrium and rat cardiovascular system.     

Scattered light intensity fluctuation in the canine ventricular myocardium: correlation with inotropic drug effect

Scattered light intensity fluctuation (SLIF) of coherent light by a strip of ventricular muscle during diastole is believed to be due to asynchronous cellular motion within the myocyte as a result of spontaneous release of Ca from the sacoplamic reticulum. Previous studies have shown a correlation between inotropic agents, such as ouabain and elevated extracellular Ca or decreased extracellular Na, and SLIF. The purpose of this study was to see if this correlation could be extended to other inotropic agents. The digitalis genin, ouabagenin, produces inotropy by increasing intracellular free Ca. In toxic concentrations the drug produces abnormal aftercontractions by spontaneous Ca release from the sarcoplasmic reticulum. On the other hand, the Ca channel agonist BAY k 8644 is also positively inotropic, but its effect is associated with a decrease in Ca release from the sarcoplasmic reticulum, manifested by conversion of "rest potentiation" to "rest depression." The effects of these inotropic agents on the power spectra of SLIF were dissimilar. Both frequency and amplitude of SLIF were increased after ouabagenin (1 microM), but these changes were most marked after the onset of toxicity, at which time contractility was decreased, rather than during the positive inotropic response. In contrast, BAY k 8644 (1 microM) decreased SLIF at all levels of inotropic response. The beta-adrenoceptor stimulant drug, dobutamine, and the adenylate cyclase activator, forskolin, produced minimal increase in SLIF at inotropic concentrations but caused a large increase in SLIF only after the onset of toxicity. These results suggest that SLIF is a better indicator of intracellular Ca overload and toxic oscillatory contractions in the presence of an inotrope and not of increased inotropy, per se.     

Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts.

1. Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The EC50 values for the negative inotropic effect of acetylcholine were 3.3 x 10(-7) M in rat and guinea pig atria and 4.1 x 10(-6) M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atrial acetylcholinesterase with soman reduced the EC50 of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.     

The inotropic effect of endothelin-1 on rat atria involves hydrolysis of phosphatidylinositol

Endothelin-1 induces a positive inotropic response in isolated left atria of the rat with an IC50 value of 20 nM. The contractile effect of endothelin is larger than that of other inotropic hormones such as phenylephrine and epinephrine and smaller than that of Bay K8644. In the spontaneously active right atria, endothelin induces a positive inotropic effect with no chronotropic effect. Endothelin does not modify intracellular levels of cAMP under basal conditions or after stimulation with isoproterenol but stimulates the formation of inositol phosphates. Mobilization of inositol phospholipids is observed in the same range of concentrations as for the contractile action of endothelin. The contractile action of endothelin is not mediated by protein kinase C. It is antagonized by blockers of L-type Ca2+ channels, low external Ca2+ concentrations and drugs such as caffeine and ryanodine that interfere with Ca2+ release by the sarcoplasmic reticulum.     

Direct actions of prostaglandins E1, A1, and F2α on myocardial contraction

The inotropic and chronotropic actions of prostaglandin (PG) types PGE₁, PGA₁, and PGF_2α were studied in isolated guinea pig right and left atria, and papillary muscles; rabbit atria; and toad ventricular strips in order to more completely define the cardiac contractile properties of PG. All three prostaglandins, in muscle bath concentrations of 10μg/ml, exerted positive inotropic and chronotropic actions on guinea pig atrium. These contractile effects were persistent after removal of PG from the muscle bath and appeared to limit the relative response to a subsequent dose of PG. The inotropic action of PGE₁ was present over a wide range of bath calcium concentrations (1.1 to 4.4 mM/L). Beta adrenergic receptor blockade, histamine blockade, and pretreatment with reserpine failed to significantly affect the inotropic actions of PG. Norepinephrine and histamine produced more potent inotropic and chronotropic effects on guinea pig atria than did PG and these contractile effects did not exhibit persistence or tachyphylaxis. The prostaglandins did not significantly affect dose response curves for norepinephrine inotropic and chronotropic actions. The prostaglandins had no effect on the force or frequency of contraction in rabbit atria. PGE₁ exerted a positive inotropic effect on toad ventricular myocardium whereas PGA₁ had no effect and PGF_2α had a negative inotropic action.     

Effect of forskolin, isoprenaline and propranolol on spontaneous contractions of the isolated atrium of guinea pigs

Forskolin (0.375 mumol 1(-1)) produced positive inotropic and chronotropic effects on the isolated, spontaneously beating atria of the guinea pig. The same effects were also observed or even increased in the presence of various concentrations of isoprenaline (0.024 and 0.12 mumol 1(-1)) in the organ bath. The effects of forskolin on the isometric contraction and the atrial rate of the isolated, spontaneously beating atria of the guinea-pig were significantly inhibited by propranolol (9 mumol 1(-1)). These results indicate that the action of forskolin on the spontaneously beating atria of the guinea-pig is mediated by stimulation of the adenylate cyclase system, but in some steps of this action, a direct stimulation of beta-adrenoceptors might also be implicated.     

Altered inotropic reactivity in diabetic rabbit right ventricular myocardium

Alloxan monohydrate was used to induce diabetes in rabbits, which were maintained for a 3-month period with or without daily insulin replacement along with age-matched controls. Isolated right ventricular myocardial strips were used to generate dose-response curves to isoproterenol, forskolin, and Bay K 8644. Basal developed force was significantly elevated in diabetic ventricular strips. While isoproterenol acted as a full inotropic agonist, diabetic preparations revealed a consistent but insignificant decrease in the maximum developed force. While both sensitivity to isoproterenol and beta-adrenoceptor density were decreased in preparations from diabetic rabbits, there was no associated increase in circulating plasma catecholamines. In contrast, forskolin and Bay K 8644 were partial agonists in control preparations but full inotropic agonists in diabetic preparations, demonstrating significant increases in maximum developed force. This hyperresponsiveness was not associated with altered calcium channel density. Finally, insulin replacement reduced or prevented all diabetic-related changes. These data indicate that the hyperresponsiveness to forskolin and Bay K 8644 represents an altered utilization of intracellular calcium in the diabetic rabbit, converting them into full agonists similar to isoproterenol. The decrease in sensitivity to isoproterenol correlated with a decrease in beta-adrenoceptor density but not elevated circulating catecholamines as previously observed in diabetic rats.     

Species differences in the negative inotropic effect of acetylcholine and soman in rat,guinea pig,and rabbit hearts

1. Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit.2. The EC₅₀ values for the negative inotropic effect of acetylcholine were 3.3 × 10⁻⁷ M in rat and guinea pig atria and 4.1 × 10⁻⁶ M in rabbit atria.3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors.4. Inhibition of atrial acetylcholinesterase with soman reduced the ec₅₀of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine.5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.     

Possible reasons for the variability of the inotropic insulin effect in papillary muscles of ground squirrel myocardium

The effects of insulin (0.1–50 nM) on isometric twitch force (0.1 to 1.0 Hz; 30 ± 1°C; 1.8 mM Ca²⁺) were studied in right ventricular papillary muscles from active ground squirrels of different seasons (summer, n = 14; autumn, n = 16 and winter interbout, n = 16) in control conditions and after one-hour pretreatment of PM with 2 μM nifedipine (an L-type Ca²⁺-channel inhibitor) and 1.0 mM orthovanadate (a tyrosine phosphatase inhibitor). In active animals of different seasonal periods insulin causes both positive and negative inotropic effects. At low frequencies (0.1–0.5 Hz), insulin of low concentrations (0.1–1.0 nM) induces a transient (within the first 20 min after application) positive effect (about 15–25%). Application of high hormone concentration (10 nM) in a low range of stimulation frequencies causes a biphasic effect (a small initial positive inotropic effect followed by a marked negative one). At frequencies above 0.5-Hz stimulation, insulin of 10 nM concentration causes presumably a negative inotropic effect. It was proposed that ICaL is possibly involved in the insulin-induced negative inotropy in ground squirrels hearts. Alteration of protein phosphorylation in tyrosine residues is known to be a major link in the mechanism of insulin action. We performed a study on sodium orthovanadate action (a known inhibitor of tyrosine phosphatase) on the inotropic insulin effect. In the group of summer animals the pretreatment of papillary muscles with sodium orthovanadate (100 μM) does not change the negative inotropic effect of insulin in a low range of stimulation frequencies but almost completely removes this effect at stimulation frequencies above 0.3 Hz (n = 4). Nifedipine (1–1.5 h pretreatment), a blocker of L-type calcium channel, reduces the inhibitory effect of insulin in autumn and winter animals, and on the contrary intensifies it in summer animals. This fact indicates that different mechanisms must be involved in insulin actions in animals of summer and winter periods. The main findings of the present study are that insulin induces positive, negative or no inotropic effects in papillary muscles of ground squirrels myocardium. The character of the effects of insulin depends on the physiological state of animals; time and concentrations of the hormone applied; affected by conditions that alter cellular Ca²⁺ loading and the ratio of protein-tyrosine kinases/phosphatases activity.     

Endothelin and Ca++ agonist Bay K 8644: different vasoconstrictive properties

The mechanism of vasoconstriction induced by endothelin was investigated in rat isolated aorta in comparison with the Ca++ agonist, Bay K 8644. Endothelin (EC50 = 4 nM) induced a slow and sustained contraction in control medium whereas the one elicited by Bay K 8644 (EC50 = 14 nM) necessitating a partly K+ depolarized medium was fast with superimposed rhythmic contraction. By opposition with Bay K 8644, endothelin contraction was not inhibited by the calcium antagonists (1 microM), nifedipine, diltiazem and D 600, and substantially persisted in Ca++ free medium or after depletion of intracellular Ca++ by phenylephrine (1 microM). These data show that endothelin does not act as an activator of potential dependent Ca++ channels but probably through specific receptor(s) as suggested by its mode of vasoconstriction.     

Changes in sensitivity to histamine of guinea pig cardiac muscles during postnatal development

Histamine stimulates the heart by interacting with cardiac histamine receptors. We investigated the postnatal changes in histamine sensitivity with spontaneously beating right atria and electrically driven left atria and right ventricular papillary muscles from 0-, 5-, and 10-day-old and adult guinea pigs. The positive chronotropic response to histamine in right atria was antagonized by cimetidine but not by chlorpheniramine at any age. Chlorpheniramine did not antagonize the positive inotropic effect of histamine and 2-(2-pyridyl)ethylamine in the immature left atria but it blocked the positive inotropic effect in the adult; cimetidine had no effect. The positive inotropic effect of histamine in right ventricular muscles was not affected by chlorpheniramine in immature right ventricular muscles but was antagonized in the adult. These results suggest that, in immature left atria and right ventricular muscles, there is no H1-receptor system mediating the positive inotropic effect of histamine and that, as age advances, this system begins to mediate the positive inotropic effect. In immature left atria, non-H1 and non-H2 receptors exist and mediate the positive inotropic effect of histamine.     

Roles of PKA, PI3K, and cPLA2 in the NO-mediated negative inotropic effect of beta2-adrenoceptor agonists in guinea pig right papillary muscles

Although beta(2)-adrenoceptors represent 15-25% of beta-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of beta(2)-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+36% at 6 microM) due to activation of beta(1)-adrenoceptors. In the presence of 4 microM atenolol, the concentration-dependent NIE (-12% at 6 microM) was biphasic, best described by a double logistic equation with respective EC(50) values of 3 and approximately 420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/cytosolic phospholipase A(2) (cPLA(2)) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by cPLA(2). The possibility that these effects are due to an equilibrium between different affinity states of the receptor (G(s)/G(i) coupled and G(i) independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that beta(2)-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state.     

Effect of insulin on the myocardium of the active, hibernating and wakening ground squirrel Citellus undulatus

The effect of insulin (0.1-100 nM) on isometric force of contraction in isolated ground squirrel papillary muscle was investigated. In summer, autumn and winter active animals, insulin had a negative inotropic effect on papillary muscles, decreasing the amplitude of contraction by about 30% of the control value. In some cases, predominantly in the summer group of animals, insulin produced different effects on contractility: low doses (0.1-0.5 nM) caused a transient activation of isometric contraction by about 10-15% of control, whereas high doses produced a negative inotropic effect by about 30% of the control level. During deep hibernation (at 5-6 degrees C of heart temperature) and during arousal from hibernation (from 3 to 20 degrees C), insulin had no significant effect on contractility. Opposite inotropic effects of insulin at concentrations of 0.1-50 nM were found during arousal: from 26 to 31 degrees C of heart temperature--a positive inotropic effect by about 20-25% of control, and from 32 to 36 degrees C--a negative one by about 30-40% of the control value.     

Negative inotropic response to cerium in ventricular papillary muscle is mediated by reactive oxygen species

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl₃ and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl₃ was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations ≤5μM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations ≤5 μM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce³⁺, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.     

Characterization of the effects of AHN 086, an irreversible ligand of "peripheral" benzodiazepine receptors, on contraction in guinea-pig atrial and ileal longitudinal smooth muscle

The effects of AHN 086 and its reversibly acting structural analogue Ro 5-4864 were studied in the spontaneously beating guinea-pig atria and field-stimulated guinea-pig ileal longitudinal smooth muscle in the presence and absence of dihydropyridine calcium channel modulators. The treatment of guinea-pig atria with AHN 086 followed by extensive washing did not alter contraction. However, AHN 086 (0.5 microM) potentiated (88%) the positive inotropic responses by BAY K 8644, an effect that was not reversed by extensive washing of the tissue. Higher concentrations of AHN 086 (greater than 2 microM) irreversibly inhibited the intropic, but not the chronotropic responses to BAY K 8644, nifedipine, and isoproterenol. Ro 5-4864 (10 microM) produced a reversible enhancement of the inotropic responses and block of the chronotropic responses to BAY K 8644. In guinea-pig ileal longitudinal smooth muscle, both AHN 086 and Ro 5-4864 reversibly inhibited field-stimulated contractions. Neither Ro 5-4864 nor AHN 086 affected the ability of nifedipine to inhibit field-stimulated contractions of ileal longitudinal smooth muscle. Treatment of intact atrial with 5 microM AHN 086 followed by extensive washing resulted in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors and of [3H]nitrendipine binding to voltage-operated calcium channels, but did not affect [3H]dihydroalprenolol binding to beta-adrenergic receptors on atrial membranes. The same treatment applied to intact ileal longitudinal smooth muscle affected neither [3H] (-)-quinuclidinyl benzilate binding to muscarine receptors nor [3H]nitrendipine binding, but did result in a significant inhibition (30-50%) of [3H]Ro 5-4864 binding to ileal longitudinal smooth muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

垂体后叶素和加压素对离体心肌的直接作用

本实验采用大鼠离体右心房和右心室肌条模型,观察了垂体后叶素和加压素对右心房和右心室肌的直接作用。结果表明:垂体后叶素对右心房的自主性收缩频率和幅度及右心室肌的收缩幅度均有剂量依赖性抑制作用;加压素对右心房和右心室肌收缩幅度也有剂量依赖性抑制作用,但对右心房自主节律无影响;催产素对右心房的收缩频率和幅度则均无影响。加压素V_1、V_2受体拮抗剂d(CH_2)_5Tyr(Me)AVP和d(CH_2)_5(D-Ile~2,Ile~4,Ala(NH_2)~9)AVP对垂体后叶素的负性变力作用具有不同程度的阻断作用,但对垂体后叶素的负性变时作用无阻断作用。以上结果提示,垂体后叶素的负性变力作用主要是由加压素产生的,加压素对心肌有直接的负性变力作用;垂体后叶素的负性变时作用可能是非加压素和催产素成分的作用结果。     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.