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.     

Inhibition of the cardiac inotropic effect of phenylephrine by a tetramine disulfide with irreversible alpha-adrenoceptor blocking activity.

The newly synthesized alpha-adrenoceptor blocking drug BHC (N,N'-bis[6-(10-methoxybenzyl-amino)-a-hexyl]cystamine) was found to block irreversibly the positive inotropic effect of the sympathomimetic drug phenylephrine on the isolated rat left atrium. BHC was used to test the adrenoceptor interconversion hypothesis which claims that low temperature converts inotropic beta-adrenoceptors in rat atrium and frog ventricle to alpha-adrenoceptors. There was no evidence of adrenoceptor 'interconversion.' In the rat atrium low temperature did not increase the BHC antagonism of phenylephrine and did not cause BHC to inhibit the inotropic effect of noradrenaline or isoprenaline. In the perfused frog heart low temperature did not lead BHC to inhibit the inotropic effect of phenylephrine, adrenaline, or isoprenaline.     

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3 X 10(-9)-3 X 10(-7) M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10(-5) M) and adenosine (10(-4) M) reduced the positive inotropic effect of hCGRP at concentrations of 3 X 10(-9) and 3 X 10(-8) M, but not at that of 3 X 10(-7) M. Ouabain (5 X 10(-5) M) inhibited the effect of hCGRP in concentrations of 3 X 10(-7) and 3 X 10(-8) M, but not in that of 3 X 10(-9) M. Simultaneous pretreatment with verapamil (10(-5) M) and ouabain (5 X 10(-5) M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10(-6) M) potentiated only the positive inotropic effect of 3 X 10(-7) M hCGRP. Metoprolol (10(-7) M) and theophilline (10(-3) M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by beta-adrenoceptors but by two distinct mechanisms of action, which was inhibited by verapamil but not by ouabain (facilitation of Ca++ influx in lower concentrations of hCGRP) and which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na+/Ca++ exchange mechanism at higher concentrations of hCGRP).     

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.     

Effects of chronic streptozotocin-induced diabetes on the cardiac responses to milrinone

We have studied the effects of milrinone on various cardiac preparations obtained from 6-week streptozotocin diabetic rats. The basal rate of spontaneously beating right atrium from diabetics was significantly lower as compared with controls. Milrinone (5 X 10(-5) to 8 X 10(-4) M) produced a dose-dependent positive inotropic and positive chronotropic effect in left atrium and right atrium, respectively. The positive chronotropic response to milrinone was slightly increased in right atria from diabetic animals. In papillary muscle neither the maximum response nor the pD2 value of milrinone was altered significantly in diabetic animals. The pD2 values of milrinone in right atrium and left atrium were found to be significantly higher in diabetic preparations compared with controls. The data indicate that the responses to milrinone are either unchanged or enhanced in hearts from diabetic animals.     

The topological characteristics of rat myocardial reactivity to noradrenaline, acetylcholine and a change in the electrostimulation frequency]

Dose-dependent effects of noradrenaline (10-7-10-6M), acetylcholine (10-8-3x10-6M) and stimulation rate (0.2-2.0 Hz) were obtained in experiments on myocardium preparations of the right and left atria and ventricles in rat. Three types of topological differences of the rat myocardium reactivity were observed: between the atria and ventricles (A/V), between the right and left atria and ventricles (R/L), between the right atrium (RA) and other cardiac chambers. A/V differences were most pronounced in the reactivity to acetylcholine (the atria were more reactive), the highest R/L differences were observed in the reactivity to noradrenaline (the myocardium of the right chambers was more reactive). RA reactivity greatly exceeded reactivity of other myocardial preparations to all three test influences. Topological peculiarities of chrono-inotropism permit supposing, that inotropic effects of rate changes in vivo are able to compensate, to some extent, the regional nonuniformity of cholin- and adrenergic regulatory inotropic effects.     

Pharmacological evidence for catecholamine-independent contractile effects of X-537A on the isolated working rat heart preparation.

The ionophore X-537A increased heart rate and contractility of the isolated, working rat heart preparation. The increased heart rate appeared to be caused solely by release of catecholamines as the response was completely eliminated by reserpine pretreatment or addition of propranolol to the perfusate. The inotropic response, however, had an apparent catecholamine-independent component as neither propranolol, nor propranol in combination with phentolamine, completely eliminated the inotropic response to X-537A. On the other hand, reserpine pretreatment did abolish the inotropic effect of the ionophore but this action appeared to be a nonspecific one as the responses to norepinephrine and to CaCl2 were substantially diminished.     

Cardiac adrenoceptors at low temperature: what is the experimental evidence for the adrenoceptor interconversion hypothesis?

Isolated heart preparations of frog and rat were used to test the validity of the adrenoceptor interconversion hypothesis. This hypothesis claims that low temperature converts the inotropic beta-adrenoceptors in isolated frog and rat heart to alpha-adrenoceptors. The present results do not support the adrenoceptor interconversion hypothesis. In the isolated frog ventricle, lowering the temperature from 24 C to 14 C did not significantly alter the inotropic potency of the sympathomimetic drugs isoprenaline, epinephrine, and phenylephrine and did not reduce the potency of the beta-adrenoceptor blocking drug propranolol as an epinephrine antagonist. In the isolated rat left atrium, lowering the temperature from 31 C to 17-19 C did not significantly change the inotropic potency of isoprenaline, norepinephrine and phenylephrine, did not diminish the potency of propranolol, and did not increase the potency of the alpha-adrenoceptor blocking drug phentolamine.--Benfey, B. G. Cardiac adrenoceptors at low temperature; what is the experimental evidence for the adrenoceptor interconversion hypothesis?     

The effects of adenyl compounds on the heart of the axolotl (Ambystoma mexicanum)

In the spontaneously beating axolotl atrium, adenosine 5'-triphosphate (ATP) produced initial excitation followed by inhibition and then by a secondary excitation. This third phase of the ATP response was only seen in electrically driven preparations in the presence of 8-phenyltheophylline (8-PT), an adenosine receptor antagonist. alpha,beta-Methylene ATP (APCPP), a stable analogue of ATP, produced only excitatory effects, while adenosine and beta,gamma-methylene ATP (APPCP), a slowly degradable ATP analogue, produced inhibition or inhibition followed by excitation. 2-Chloroadenosine produced inhibition. The excitatory effects were not antagonized by 8-PT, indomethacin or propranolol and phentolamine. The negative inotropic responses of these compounds were antagonized by 8-PT and, with the exception of 2-chloroadenosine, potentiated by dipyridamole, an adenosine uptake blocker. In the ventricle, ATP, APCPP and APPCP produced positive inotropic effects, which were not affected by 8-PT, indomethacin or propranolol and phentolamine. Adenosine produced a negative inotropic effect which was not antagonized by 8-PT nor atropine nor potentiated by dipyridamole. The effects of adenyl compounds on the axolotl (urodele) heart suggest that, like the frog (anuran) heart, both P1- and P2-purinoceptors are present in the axolotl atrium and that only P2-purinoceptors are present in the axolotl ventricle, although adenosine does produce an inhibitory effect on the ventricle which is probably mediated via the release of a neurotransmitter other than acetylcholine.     

Mechanism of action of ethanol on enkephalinase A activity in the rat brain

The influence of ethanol, its metabolites and some opiates on enkephalinase A activity was studied in rat experiments in vitro after acute and chronic administration of ethanol. It was demonstrated that addition of ethanol to the reaction mixture activated enkephalinase A of the midbrain and hypothalamus of intact rats, the maximal effect being attained at an ethanol concentration of 10(-3) M. Multiple washings with buffer of the ethanol-preincubated membranous fraction of these brain structures in the control rats did not lead to a significant reduction in the activating effect of ethanol on enkephalinase A. No activation was recorded upon the use of an enzymatic preparation of the brain from chronically alcoholized animals. Morphine, naltrexon, beta-carbolines, salsolinol (10(-4) M) and acetaldehyde (10(-8)-10(-2) M) did not activate the enzyme. It is suggested that enkephalinase A activation in rats given ethanol is determined by a direct action of ethanol on the enzyme.     

Effect of adaptation to physical loading on the adrenoreactivity of the isolated rat atrium

The experiments on the isolated atria of rats, previously adapted to swimming exercises, have established a significant decrease in the heart rate and an increase in positive inotropic effect of noradrenaline (4 X 10(-7) and 6 X 10(-7) M). After beta-adrenoceptor blockade with propranolol (2 X 10(-6) M) the inotropic effect of an alpha-adrenoceptor agonist mezaton on the isolated atrium of the adapted rats was greater than in nonadapted controls.     

Age-related changes in inotropic effects of noradrenaline and acetylcholine on the myocardium of guinea pigs]

The inotropic effects of noradrenaline (10(-7)-10(-5) M) and acetylcholine (10(-8)-10(-6) M) were studied in experiments carried out on preparations of the right atria and on papillary muscles of the right ventricle in adult (4-5 months) and old (18-24 months) guinea pigs. An age-related decrease in inotropic noradrenaline effects and the displacement of dose-effect relationships to the right was revealed. Similar changes of the dose-related effects of acetylcholine superfused against the background of noradrenaline action were observed. The direct inotropic action of the acetylcholine did not change with ageing. A lack of the essential atrial-ventricular differences in age-related changes in myocardial reactivity is apparently very significant for support of effective functional coupling of cardiac chambers in ageing.     

Noradrenergic-mediated potentiation of acetylcholine release from the phrenic nerve: evidence for presynaptic alpha 1-adrenoceptor involvement

This study, conducted in the rat phrenic nerve-diaphragm preparation, was designed to establish more direct evidence that norepinephrine enhances acetylcholine (ACh) release from motor neurons and characterize the alpha-adrenoceptor type mediating this action. Norepinephrine (50 microM, alpha 1 + alpha 2 agonist) increased nerve-stimulated release by 183%, as determined by radioenzymatic assay. This effect was completely abolished by pretreatment with the alpha-adrenoceptor antagonists phentolamine (alpha 1 + alpha 2) and by WB 4101 (alpha 1) but only modestly reduced by yohimbine (alpha 2). Clonidine (alpha 2 agonist) did not enhance ACh release or nerve-stimulated muscle contractions, while phenylephrine (alpha 1 agonist) and norepinephrine increased muscle contractions up to 19.5-22.4%. These results support the hypothesis that norepinephrine increases ACh release from somatic motor nerves via a presynaptic alpha 1 interaction.     

Effect of methionine-enkephalin on the spontaneous electrical and mechanical activity of the smooth muscle of the rat portal vein

In the longitudinal smooth muscle of the isolated rat portal vein, methionine-enkephalin (Met-enkephalin) increased the spontaneous contraction with a concentration as low as 10(-8)M. When the membrane activity was recorded using a microelectrode, Met-enkephalin enhanced the spike burst activity but without any effect on the resting membrane potential. Naloxone, phentolamine, atropine and reserpine pre-treatment did not inhibit the excitatory effect of Met-enkephalin on the spontaneous contraction. These results suggest that the excitatory effect of Met-enkephalin on the mechanisms involved in the automaticity may be a direct action on smooth muscle or relate to presynaptic action on a non-adrenergic non-cholinergic system.     

Mechanism for amine modulation of the neurogenic Limulus heart: evidence for involvement of cAMP

The role of cyclic nucleotides as intracellular second messengers mediating the excitatory chronotropic and inotropic actions of octopamine (OCT) and dopamine (DA) on the neurogenic Limulus heart was investigated. Tissue levels of cAMP, but not cGMP, were significantly increased in isolated cardiac ganglia and cardiac muscle following 10 min exposure to 10(-5) M OCT or 10(-5) M DA. In both tissues, OCT elicited larger increases in cAMP than did DA. Amine-induced cAMP accumulation in the cardiac ganglion and in the cardiac muscle was prevented by the alpha-adrenergic blocker phentolamine. The adenylate cyclase activator forskolin and the phosphodiesterase inhibitor IBMX produced amine-like chronotropic and inotropic effects when applied to the isolated heart preparation. However, the kinetics of the responses differed for the two agents. Additional pharmacological agents (RO-20-1724, papaverine, SQ 20,009, and 8-parachloro-phenylthio cAMP) also had amine-like effects but to a lesser extent. The chronotropic, but not inotropic, effects of OCT and DA were potentiated in the presence of IBMX. These data suggest that a cAMP-dependent mechanism underlies the excitatory effects of the neuromodulators OCT and DA on the Limulus heart.     

CGRP antagonist activity of short C-terminal fragments of human alpha CGRP, CGRP(23-37) and CGRP(19-37).

The activity of short C-terminal fragments of human alpha calcitonin gene-related peptide (CGRP), CGRP(19-37), and CGRP(23-37), and of the N-terminally acetylated (Ac) derivative, AcCGRP(19-37), has been investigated in the guinea pig isolated left atria (electrically driven) for their ability to antagonize the positive inotropic effect of human alpha CGRP. All the peptides tested produced a rightward displacement of the curve to the agonist without depressing the maximal response: apparent pA2 values were 5.39 and 4.81 for CGRP(19-37) and CGRP(23-37), respectively, as compared to 6.81 for CGRP(8-37). AcCGRP(19-37) was a more potent antagonist than the parent peptide, with an apparent pA2 value of 6.03.     

Absence of blocking effects on cardiac slow calcium channels by the intracellular calcium antagonist 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene

Extensive pharmacological evidence supports the contention that 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene hydrochloride (pr-MDI) is a calcium antagonist with a predominantly intracellular site of action. On the other hand, electro-physiological evidence points to a possible membrane slow inward calcium channel blocking property of this agent. To gain further insight as to the site of action of pr-MDI, the interactions between the negative inotropic action of this agent and the positive inotropic actions of excess extracellular calcium (which directly penetrates the myocardial cells through the slow calcium channels), isoproterenol (which indirectly augments calcium influx through the slow calcium channels), and ouabain (which enhances calcium influx through membrane calcium entry routes distinct from the slow calcium channels) were investigated in the isolated, electrically drive guinea pig left atrium. Although excess extracellular calcium, isoproterenol, and ouabain reversed the negative inotropic effect of pr-MDI, an analysis of the concentration-response relationships to all three positive inotropic agents in the presence and the absence of pr-MDI demonstrated that this agent did not significantly inhibit the contractile effects of calcium, isoproterenol, or ouabain, at pr-MDI concentrations which exhibit intrinsic negative inotropic effects. It is concluded that pr-MDI does not block the membrane slow inward calcium channel nor other presumptive membrane routes of calcium entry into myocardial cells at concentrations of 10(-4) M or less. At very high concentrations (3 X 10(-4) M) some inhibition of slow channel calcium influx may occur.     

Temporal dynamics of inotropic, chronotropic, and metabolic responses during beta1- and beta2-AR stimulation in the isolated, perfused rat heart

During the beta-adrenergic receptor (beta-AR)-mediated stress response in the heart, the relations between functional responses and metabolism are ill defined, with the distinction between beta1- and beta2-AR subtypes creating further complexity. Specific outstanding questions include the temporal relation between inotropic and chronotropic responses and their metabolic correlates. We sought to elucidate the relative magnitudes and temporal dynamics of the response to beta1- and beta2-AR stimulation and the energy expenditure and bioenergetic state related to these responses in the isolated perfused rat heart. Inotropic [left ventricular developed pressure (LVDP) and dP/dt], chronotropic [heart rate (HR)], and metabolic responses were measured during beta1- (n = 9; agonist: norepinephrine) and beta2- (n = 9; agonist: zinterol) AR stimulation. Myocardial oxygen consumption (MVO2) was measured using fiber-optic oximetry, and high-energy phosphate levels and intracellular pH were measured using 31P NMR spectroscopy. A multiple-dose protocol was used, with near-maximal beta-AR stimulation at the highest doses. In both beta1 and beta2 groups, there were dose-dependent increases in LVDP, dP/dt, HR, and MVO2. The inotropic response showed more rapid onset, washout, and variation during dose than did the chronotropic response and was closely correlated with MVO2. This suggests that the myocardial bioenergetic state is more closely related to the inotropic response than to the chronotropic response. In addition, beta1-AR stimulation resulted in a greater magnitude and rate of onset of inotropic and MVO2 responses than did beta2-AR stimulation during maximal stimulation. However, a similar decrease in intracellular energy charge was seen in the two groups, consistent with a greater rate of oxidative phosphorylation during beta1- than during beta2-AR stimulation.     

The right ventricular working heart preparation

An isolated working rat heart preparation was modified to study right ventricular (RV) performance. All hearts were perfused with a Krebs-Henseleit bicarbonate buffer via a Langendorff column at 90 mm Hg. Right atrial filling (preload) was varied by raising a buffer reservoir from 5 cm below to 10 cm above the right atrium while pulmonary artery outflow resistance remained fixed. RV systolic pressure and the maximum rise and decrease in pressure development (+/- dP/dt) were measured via a catheter in the RV. Cardiac output was collected with a catheter placed in the pulmonary artery. One group of hearts, monitored at a fixed preload (0 cm H2O) for 2 hr, and another group of hearts, in which two ventricular function curves were performed, demonstrated the stability and reproducibility of the preparation. Additionally, the ability of this preparation to measure changes in inotropy was studied. A negative inotropic effect was measured after verapamil (5 X 10(-8) M) treatment. Positive dP/dt showed the greatest depression (30%) and was significantly lower at every preload. A positive inotropic effect was demonstrated by reducing the buffer Ca2+ concentration to 1.9 mM for the first work curve followed by an addition of Ca2+ (2.8 mM final concentration) or ouabain (5 X 10(-5) M) for the second work curve. Again, the greatest effect was found in the dP/dt measurements (elevated by 20 and 30%, respectively). Thus, this preparation manifests qualities similar to those used in studying the left ventricle and allows investigation of various cardiac diseases which may affect RV pump function.     

Alanine and glutamine synthesis and release from skeletal muscle. IV. beta-Adrenergic inhibition of amino acid release.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Epinephrine reduced the release of alanine and glutamine in a concentration-dependent manner. Measurable inhibition was observed at 10(-9) M epinephrine, and maximal inhibition was obtained at 10(-5) M. Norepinephrine also reduced alanine and glutamine formation and release but the concentration required for maximal inhibition was approximately 100-fold greater than for epinephrine. Isoproterenol (beta agonist), but not phenylephrine (alpha agonist), reproduced the effects of epinephrine, and propranolol (beta antagonist), but not phentolamine (alpha antagonist), blocked the effect of the catecholamine. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate reproduced the effects of epinephrine and theophylline potentiated the effect of submaximal concentrations of the hormone. Glucagon and prostaglandin E2 had no observable effect on amino acid release. Insulin did not modify the inhibition of alanine and glutamine release produced by epinephrine. Alanine and glutamine formation from added precursor amino acids was unaffected by epinephrine or cyclic adenosine 3':5'-monophosphate. Epinephrine reduced alanine formation in muscles obtained from diabetic rats or animals treated with thyroxine or cortisone. These findings indicate that physiological levels of catecholamines reduce alanine and glutamine formation and release from skeletal muscle. This effect is mediated by a beta-adrenergic receptor and the adenylate cyclase system and can be accounted for by an inhibition of muscle protein degradation.