Curve For Isoproterenol Research Articles

Functional Characterization of the β-Adrenergic Receptor Subtypes Expressed by CA1 Pyramidal Cells in the Rat Hippocampus

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

Modulation of pacemaker activity in sheep cardiac Purkinje fibers by stimulation of beta-adrenoceptor subtypes.

The electrophysiological effects mediated by beta 1- and beta 2-adrenoceptors in spontaneously active sheep cardiac Purkinje fibers were investigated using the non-selective agonist (-)-isoproterenol (IPN) and the selective agonists (-)-noradrenaline (beta 1) and procaterol (beta 2) in the absence and presence of the selective antagonists bisoprolol (beta 1) and ICI 118,551 (beta 2). IPN (0.01 mumol/l) increased the spontaneous rate by 54% and the slope of diastolic depolarization by 68% of the respective control values. Further, IPN increased the action potential duration at -20 mV (APD -20 mV) from 96 to 154 ms, reduced the APD-70 mV by 17% and the duration of the diastole by 39% and slightly hyperpolarized the maximum diastolic potential. These effects were partially inhibited by ICI 118,551 (0.03 mumol/l), diminished by bisoprolol (0.1 mumol/l) and almost completely blocked by the combination of both antagonists. Concentration response curves of IPN were influenced by the selective antagonists as follows: ICI 118,551 (0.03 mumol/l) shifted the curves to the right by 0.2-0.4 log units and increased the slope factor. Bisoprolol (0.1 mumol/l) induced a greater shift to the right by 1.1-1.5 log units. Combination of bisoprolol with ICI 118,551 shifted the curves to the right by 1.5-1.7 log units. Noradrenaline (0.3 mumol/l) elicited similar actions as IPN. Bisoprolol (0.1 mumol/l) shifted the concentration response curves of noradrenaline to the right by 1.1-1.9 log units. Actions of procaterol (0.1 mumol/l) were weak, attained only 15-35% of the maximal effects of IPN and could be blocked by ICI 118,551 (0.03 mumol/l). These results show that the increase of pacemaker activity induced by catecholamines in sheep cardiac Purkinje fibers is predominantly mediated by stimulation of beta 1-receptors. However, contribution of beta 2-receptor mediated effects could be demonstrated.

ICI 147,798 causes insurmountable antagonism against T-0509, a selective beta 1-adrenoceptor agonist, but surmountable antagonism against isoproterenol.

In canine right ventricular muscle, the influence of ICI 147,798, a slowly dissociable beta 1-adrenoceptor antagonist, on the positive inotropic effect and cyclic AMP production of T-0509, a selective beta 1-agonist, and isoproterenol, a nonselective beta-agonist, were examined to elucidate the properties of the high- and low-affinity states or subtypes of beta 1-adrenoceptor. ICI 147,798 (1 x 10(-8) - 1 x 10(-5) M) produced biphasic concentration-response curves for the positive inotropic effect of T-0509 that were due to its insurmountable antagonism against the lower concentrations of T-0509. The maximum of the biphasic curves were almost the same as Camax. Analysis of hemiequilibrium antagonism by ICI 147,798 against T-0509 showed the dissociation constant (pKB) of ICI 147,798 to be approximately 8. On the other hand, ICI 147,798 produced a parallel rightward shift of the curves of isoproterenol. Schild analysis showed pA2 to be 7.98 +/- 0.04, suggesting competitive antagonism. The concentration-response curve for the increase in cyclic AMP elicited by T-0509 in the presence of 1 x 10(-6) M ICI 147,798 was also biphasic, but slightly depressed as compared with that of control, whereas that elicited by isoproterenol was shifted to the right. The positive inotropy and increase in cyclic AMP produced by T-0509 and isoproterenol in the presence of ICI 147,798 may be mediated by the low-affinity state of beta 1-adrenoceptor, which mechanism may be different from that producing the positive inotropy through the high-affinity state of beta 1-adrenoceptor.

Effects of Tertatolol on the Responsiveness of Isolated Femoral, Mesenteric, and Renal Resistance Arteries to Adrenergic Stimuli

We evaluated effects of the beta-adrenoceptor antagonist tertatolol on responses of resistance arteries to isoproterenol and phenylephrine and compared them with those of propranolol. The experiments were performed in femoral, mesenteric, and renal resistance arteries that had been isolated from adult Wistar-Kyoto rats, chemically sympathectomized and mounted for recording of contractile reactivity in vitro. In renal resistance arteries that had been pretreated with phenoxybenzamine, isoproterenol did not affect contractile responses to potassium. Under these conditions, isoproterenol induced relaxation in mesenteric resistance arteries. Low concentrations of both SR-tertatolol and SR-propranolol shifted concentration-response curves for isoproterenol to the right. Affinity for competitive beta-adrenergic antagonism did not differ between both agents. Unlike SR-propranolol, SR-tertatolol reduced maximal relaxing responses to isoproterenol. As compared with R-tertatolol, S-tertatolol was 100 times less potent as a competitive beta-adrenergic antagonist and failed to affect maximal relaxing responses to isoproterenol. In femoral, mesenteric, and renal resistance arteries preconstricted by phenylephrine, concentrations of SR-tertatolol greater than 1 microM induced relaxation. This relaxing effect did not differ between SR-tertatolol and SR-propranolol or between S-tertatolol and R-tertatolol. These data indicate that in resistance arteries tertatolol is a stereoselective beta-adrenoceptor antagonist that exhibits both a competitive and a noncompetitive mechanism of action. In addition, high concentrations of tertatolol relax resistance arterial smooth muscle. This effect was neither stereoselective nor selective for the renal vascular bed and was also observed with propranolol.

An Explanation of Bell-Shaped, Dose-Positive Inotropic Effect Curves for Denopamine in Canine Right Ventricular Muscle

We investigated mechanisms by which denopamine produces bell-shaped, cumulative concentration-response curves for positive inotropic effect in canine right ventricular muscle. As in our previous study, the curves ascended at 10(-8) to 10(-6) M, reached a maximum at 3 X 10(-6) to 10(-5) M, and descended at higher concentrations. The ascending limb gave 6.58 +/- 0.21 for the pD2 value. Denopamine, when administered cumulatively up to 3 X 10(-5) to 3 X 10(-4) M, caused rightward shifts of concentration-response curves for the positive inotropic effect of isoproterenol. Application of a two-component fitting model to the concentration-response curves for isoproterenol in the presence of denopamine yielded two pA2 values of denopamine; 6.59 +/- 0.19 for the high- and 5.05 +/- 0.21 for the low-affinity state of beta 1 receptors. The bell-shaped curves for denopamine were successfully computer-simulated with the following assumptions. (a) Denopamine produces a positive inotropic effect by binding to the high-affinity state of beta 1 receptors with an apparent dissociation constant of 10(-6.59) M. (b) The fraction of the high-affinity state decreases as denopamine binds to the low-affinity state with an apparent dissociation constant of 10(-5.05) M. Cyclic AMP levels were changed by denopamine, isoproterenol, and a combination of both, roughly in a parallel way to changes in positive inotropic effect.

Evidence for Dual Adrenergic Receptor Regulation of Ovine Pineal Function

Both alpha 1- and beta-adrenergic receptors are present on ovine pineals. In the rat these two receptors interact so that activation of the alpha 1-receptor potentiates the beta-receptor-mediated changes in cyclic AMP and the correlated changes in pineal N-acetyltransferase (EC 2.3.1.87). Here we investigate possible interactions between alpha 1- and beta-receptors through changes in cyclic AMP and assess the importance of each receptor to the melatonin response in ovine pineal punches/slices in vitro. The adrenergic agonists isoproterenol (ISO), noradrenaline (NA), and phenylephrine (PHE) stimulated dose-dependent changes in cyclic AMP with the order of potency of ISO greater than NA greater than PHE, consistent with their relative binding affinities for the beta-receptor. The beta-receptor antagonist, propranolol, showed dose-dependent inhibition of the ISO effect, whereas the alpha 1-selective antagonist, prazosin, had no effect. The S-shape of the stimulation and inhibition curves for ISO reflects cyclic AMP changes mediated by the beta-receptor only without interaction through the alpha 1-receptor. Each of the adrenergic agonists stimulated indistinguishable dose-dependent increases in melatonin release. The ability of PHE to stimulate changes in melatonin release in the absence of concomitant changes in cyclic AMP indicates an important role for the alpha 1-receptor. Prazosin inhibits this response, substantiating this conclusion. However, as propranolol is also inhibitory, it seems that the alpha 1-receptor response is absolutely dependent upon a small level of beta-receptor stimulation, thus providing evidence for adrenergic receptor interaction at a step other than cyclic AMP.

Forskolin‐ and diacylglycerol‐like potentiation of isoproterenol‐induced positive inotropic effect in guinea pig papillary muscles by the musk component, musclide

AbstractMusclide (ML) is a glycerol‐rich mixed component extracted from musk, a dried secretion from the preputial follicle of musk deer, Moschus moschiferus Linn. ML (50 μg/mL) that caused no direct inotropic effect potentiated isoproterenol (Isp)‐induced positive inotropic effect in isolated guinea pig papillary muscles. This cardiotonic action has been attributed to β‐adrenergic potentiation. The mechanisms were further investigated using pertussis toxin (IAP), cholera toxin (CTX), forskolin (FK) and dioctanoylglycerol (DOG). IAP pretreatment (50 μg/kg, i.p., the 3rd day before tissue isolation) and/or DOG (200 μM, preliminarily applied for 5 min) increased the maximal response of the concentration‐response curve for isoproterenol. CTX pretreatment (0.1 μM, for 3 h) increased the affinity. ML (50 μg/mL) or FK (82.9 nM) preliminarily applied for 5 min caused increases in both the maximal response and affinity. The extent of potentiation produced by ML or DOG was not changed in normal muscles or IAP‐pretreated muscles. The extent of potentiation by ML was greater in CTX‐pretreated muscles than in normal ones. ML or DOG, when combined with FK, caused only the increase in the maximal response. These results suggest that the potentiation mechanisms of ML are partially FK (adenylate cyclase activator)‐like and partially DOG (protein kinase C activator)‐like.

Changes in Sensitivity of the Rat Stomach Fundus to Various Drugs in Streptozotocin-Induced Diabetic Rats

The changes in sensitivity of the rat stomach fundus to acetylcholine (ACh), norepinephrine (NE), isoproterenol (ISO) and to vasoactive intestinal peptide (VIP) were examined in control rats and in streptozotocin (STZ)-induced diabetic rats. The dose-response curves for drugs were constructed 8 weeks after treatment with STZ. The dose-response curve for ACh in diabetic rats was shifted to the left as compared to the control curve, whereas the dose-response curves for NE, ISO and VIP were shifted to the right. These results suggest that functional changes in the autonomic nervous systems of the rat stomach fundus may occur in STZ-induced diabetic rats.

Changes in sensitivity of the rat stomach fundus to various drugs in streptozotocin-induced diabetic rats.

The changes in sensitivity of the rat stomach fundus to acetylcholine (ACh), norepinephrine (NE), isoproterenol (ISO) and to vasoactive intestinal peptide (VIP) were examined in control rats and in streptozotocin (STZ)-induced diabetic rats. The dose-response curves for drugs were constructed 8 weeks after treatment with STZ. The dose-response curve for ACh in diabetic rats was shifted to the left as compared to the control curve, whereas the dose-response curves for NE, ISO and VIP were shifted to the right. These results suggest that functional changes in the autonomic nervous systems of the rat stomach fundus may occur in STZ-induced diabetic rats.

Antitachycardiac Effect of OPC-8212, a Novel Cardiotonic Agent, on Tachycardiac Responses of Guinea Pig Isolated Right Atria to Isoproterenol and Histamine

In isolated right atria of guinea pigs, OPC-8212 produced a concentration-dependent decrease in spontaneous beating rate that had previously been elevated by isoproterenol or histamine in the presence of nadolol. OPC-8212 scarcely affected the baseline rate, however, OPC-8212 depressed the maximum increase in right atrial rate induced by isoproterenol or histamine in the presence of nadolol. These effects of OPC-8212 were discernible from the effects of its solvent sulfolane. We propose the term "antitachycardiac" to describe such effects of OPC-8212 to distinguish it from those of bradycardic agents. When the concentration-response curves of isoproterenol or histamine for positive chronotropy were normalized, these curves were shifted in a parallel way to the left with OPC-8212, indicating that OPC-8212 retains activity as a cyclic AMP phosphodiesterase inhibitor. The virtual absence of the chronotropic effect of OPC-8212 on the baseline rate and the antitachycardiac effect are discussed in terms of the opposing effects in increasing the inward calcium current on one hand and in decreasing the outward potassium and the inward current activated by hyperpolarization on the other.

Defibrotide, an Antithrombotic Substance That Preserves Postsynaptic α- and β-Adrenergic Function in Post Acute Infarcted Rabbit Hearts

Defibrotide, a simple strand polydeoxyribonucleotide of mammalian origin with a molecular weight of 20,000 daltons, given intravenously to the rabbit before and after production of left ventricular infarction, prevents the alteration of the contractile response to postsynaptic adrenergic stimulation tested in isolated perfused heart preparations 3 days after coronary artery occlusion. According to the dose-response curves for isoproterenol and tyramine, left ventricular dP/dtmax was significantly depressed in infarcted hearts, whereas the dose-response curve for the inotropic effect of phenylephrine was markedly enhanced. These alterations were prevented by pretreatment of the rabbits with defibrotide (32 mg/kg/h i.v. for 6 h). In fact the potency ratios, calculated from the dose-response curves related to dP/dtmax of isoproterenol, tyramine, and phenylephrine in infarcted and control hearts excised from defibrotide treated and shamoperated rabbits, are nearly 1. The observed alterations in myocardial contractility in infarcted hearts seem to be specific for postsynaptic alpha and beta-adrenoceptors since the dose-response curve of left ventricular dP/dtmax for histamine is not different from control. The results obtained with defibrotide reflect the ability of this substance to protect the myocardial tissue from ischemic damage: this is also supported by the capacity of defibrotide (8 mg/kg/h i.v. for 6.5 h) to prevent the reduction of CPK-activity in the infarcted ventricle. Finally, we suggest that the observed beneficial effect of defibrotide in rabbit heart may also be explained by the antithrombotic effect of this substance, which is based on its profibrinolytic activity and PGI2-release.

Effects of magnesium on the action of vasodilatory agents.

Studies have shown that alterations of the magnesium (Mg2+) concentration can alter the response of certain vasoactive compounds. Responses of rabbit thoracic aorta to verapamil, diltiazem, nitroglycerin and isoproterenol were examined in zero Mg2+ (0.0 M), N Mg2+ (1.2 mM), 2 N Mg2+ (2.4 mM) and 4 N Mg2+ (4.8 mM). Preconstriction was induced with norepinephrine and cumulative dose-response curves were obtained for the vasodilators. The dose-response curve for isoproterenol was shifted to the right in zero Mg2+ while there was no effect on the other vasodilators. Elevation of the Mg2+ concentration to 4.8 mM produced a shift to the left in the dose-response curves for diltiazem, nitroglycerin and isoproterenol with no effect on verapamil. Therefore, Mg2+ deficiency does not appear to affect the vasodilatory actions of the agents tested except for the beta-receptor agonist, isoproterenol. Elevated Mg2+, however, potentiated the actions of isoproterenol, nitroglycerin and diltiazem, but not verapamil.

Age-dependent changes in electrophysiologic characteristics of fast and slow action potentials in rat papillary muscle.

Isolated papillary muscles from juvenile (about 2 months old, average weight of 250 g) and young adult rats (about 4 months old, average weight 485 g) were studied for age-dependent differences in the characteristics of fast and slow action potentials (APs). The fast and slow APs were recorded in 5.4 mM and 25 mM K+-Tyrode solutions, respectively (stimulation rate of 1 Hz). For the slow APs, the dose-response curves for isoproterenol versus Vmax (the maximum rate of rise of the APs), overshoot, and AP amplitude were linear between 10(-9) M and 10(-6) M (10(-5 M in some cases) in the juvenile and young adult rats. Isoproterenol pretreatment (1 mg/kg s.c., 1 h prior) decreased the slope of the dose-response curve, and saturation was achieved at a lower concentration. The Vmax, overshoot, and amplitude of both the fast and slow APs were somewhat smaller in the young adult rats than in the juvenile rats; there were no differences in the resting potential, AP duration, or threshold voltage. These results suggest that activation of a greater fraction of the beta-adrenergic receptors is coupled directly or indirectly to activation of a greater fraction of the slow channels. The pretreatment data suggest that down-regulation of the beta-adrenergic receptor may occur. The conductance per channel for the fast Na+ channels and slow channels, and (or) the number of both types of functional channels, may decrease with age.

Reconstitution of a hormone-sensitive adenylate cyclase with membrane extracts from Neurospora and avian erythrocytes.

Adenylate cyclase activity associated to wild type Neurospora membranes is highly dependent on Mn2+ and insensitive to fluoride, guanyl nucleotides, and cholera toxin. These membranes are able to interact with components of detergent extracts from turkey erythrocyte ghosts. The reconstituted cyclase system is catalytically active in the presence of Mg2+ and it is activated by guanyl-5'-yl imidodiphosphate plus isoproterenol and fluoride. When detergent extracts were prepared from avian erythrocyte membranes treated with cholera toxin, the reconstituted system was stimulated by guanyl-5'-yl imidodiphosphate in the absence of isoproterenol and cyclase activities were higher than those observed with extracts from membranes not treated with the toxin. Dose-response curves for isoproterenol and fluoride in the reconstituted system were similar to those reported for avian erythrocyte and liver membranes, respectively.

Micropuncture evaluation of beta-receptors in pial arteries of cats.

The existence of β-receptors and their possible contribution to the vascular tone of pial arteries was investigated using the microapplication technique combined with the measurement of vascular diameter. Concentration response curves for (+) and (−)propranolol revealed an identical course with no vascular reaction occurring between 2.5×10−10 and 2.5×10−5 M propranolol. Since both (+) and (−)propranolol were without effect on vascular diameter over a wide concentration range it is concluded that the vascular tone is not influenced by β-receptors under these experimental conditions. The increase in vascular diameter (60%) observed at 2.5×10−4 M (+) and (−)propranolol is supposed to be due to an unspecific effect. Concentration response curves for isoproterenol revealed no vascular reaction between 2.5×10−11 and 2.5×10−8 M, dilations of 4–5% at 2.5×10−7 and 2.5×10−6 M, constrictions of about 5% at 2.5×10−4 and of 3% at 1.25×10−3 M isoproterenol. These weak vascular reactions cannot be explained by reduced biological activity of isoproterenol due to autoxidation during storage. It is concluded that β-receptors are of little or no physiological significance for regulation of the vascular tone of pial arteries.

In vitro demonstration of the existence of ?-adrenoceptors in human saphenous and canine femoral veins

Helical strips of human saphenous (normal and varicose tissues) and canine femoral veins relaxed in response to isoproterenol when there was pre-existing tone. This effect was antagonised by the β-adrenoceptor blocking drugs pronethalol and pindolol demonstrating the presence of β-adrenoceptors. Using the canine femoral vein, dose-response curves for isoproterenol were determined in the presence of phenoxybenzamine and a depolarising concentration of potassium. Under these conditions, both β-adrenoceptor blocking agents caused a parallel shift to the right of the dose-response curves. Approximately one hundred times more pronethalol than pindolol was needed to produce a similar degree of blockade.

COMPETITIVE ANTAGONISM BETWEEN ISOPROTERENOL AND A NEW BETA-RECEPTOR ADRENERGIC BLOCKING AGENT, PROPRANOLOL.

SummaryThe effects of graded doses of isoproterenol on mean systemic arterial pressure and myocardial contractile force were studied in anesthetized dogs before and after administration of graded doses of a new beta-receptor blocking agent, propranolol. It was found that, with geometrically increasing doses of propranolol, the effects of given doses of isoproterenol on systemic arterial pressure and myocardial contractile force decreased progressively. However, the larger doses of isoproterenol surmounted the blocking effect of propranolol on myocardial contractile force. Dose-response curves of isoproterenol after graded doses of propranolol indicate the existence of competitive antagonism between isoproterenol and propranolol. In addition, after administration of propranolol, the effect of isoproterenol on systemic arterial pressure was found to become biphasic, an initial increase being followed by a decrease. Hence, it is postulated that isoprotenerol possesses a vasoconstrictor (alpha-receptor stimul...