Thomas E. Tenner

Alterations in the myocardial β-adrenoceptor system of streptozotocin-diabetic rats

Abstract Previous investigations in our laboratory revealed subsensitivity of right ventricular tissue, isolated from one month STZ-diabetic rats, to the inotropic effects of isoproterenol. The present study was concerned with the characterization of this subsensitivity phenomenon. Observations of supersensitivity to methoxamine accompanied by decreased responsiveness to glucagon without a change in responsiveness to forskolin suggested a specific effect of diabetes on pathways involving receptor-mediated activation of adenylate cyclase. Radioligand binding analysis further revealed a specific decrease in the population of the high affinity state of the β-adrenoceptor. Since the high affinity receptor state is a necessary intermediate for adenylate cyclase activation and enhanced myocardial contractility, it is proposed that the specific decrease in the high affinity population of the β-adrenoceptor contributes to myocardial subsensitivity to isoproterenol observed in the diabetic animals. It is further proposed that the decrease in receptor population is related to increases in circulating epinephrine levels which were evident in the diabetic animals.

Reserpine-induced supersensitivity and the proliferation of cardiac β-adrenoceptors

Chronic reserpine pretreatment resulted in an increase in the basal developed force generated by papillary muscles isolated from the right ventricle of the rabbit. In addition, supersensitivity to the inotropic effects of isoproterenol and a concomitant increase in beta-adrenoceptor number were demonstrated. These results indicate that reserpine-induced supersensitivity is not only associated with alterations at sites beyond the level of receptor activation but also at the level of specific postjuctional membrane receptors.

Effect of insulin replacement on streptozotocin-induced effects in the rat heart

Streptozotocin (65 mg/kg) was used to induce diabetes in male Sprague-Dawley rats. Isolated cardiac tissue exhibited a systematic depression in atrial pacemaker function and an enhancement in ventricular function accompanied by a supersensitivity to calcium relative to control animals. beta-Adrenoceptor density was found to be significantly lowered in the treated animals. However, no change in responsiveness of the tissues to isoproterenol was observed. The systematic changes in atria and ventricle were found to be completely and partially reversed respectively, by daily administration of 4-5 units of Ultralente (U-100) insulin, whereas the decrease in beta-adrenoceptor number and supersensitivity to calcium were completely reversed. These results suggest that STZ by itself might not have toxic effects in the heart and that its effects may be overcome by chronic insulin-replacement.

Characterization of H2-receptor mediated relaxation in rabbit aortic strips

Rabbit aortic strips contracted with either KC1 (60 mM), phenylephrine (10−5 M) or angiotensin-II (10−7 M) were exposed to cumulative concentrations of the H2-receptor agonists impromidine, histamine, 4-methyl histamine and dimaprit in the presence of the H1-receptor antagonist, pyrilamine (10−6 M). All of the H2-receptor agonists produced a similar degree of relaxation in strips contracted with phenylephrine. The order of potency was impromidine>histamine>4-methyl histamine⩾dimaprit. All of the H2-receptor agonists except 4-methyl histamine were able to produce significant degrees of relaxation in aortic strips contracted with angiotensin-II. In contrast, none of the H2-receptor agonists, in doses which relaxed phenylephrine and angiotensin-II contracted muscles, we able to relax aortic strips contracted by KCl. When the relaxant ability of the H2-receptor agonist, impromidine, was compared to that of D-600, isoproterenol and adenosine, it was found to produce relaxation similar to that characterized by isoproterenol. Impromidine, like isoproterenol, more readily relaxed aortic strips contracted by either phenylephrine or angiotensin-II.

The role of calcium in supersensitivity to the inotropic effects of norepinephrine

Experiments using electrically stimulated rabbit left atria have demonstrated that supersensitivity to the inotropic effects of norepinephrine can be induced by either chronic reserpine pretreatment or hypothermia (lowering the temperature of the bathing medium). These two experimental conditions for inducing supersensitivity were not additive implying that they shared a common mechanism of action. Norepinephrine had no significant effect on the amplitude of a potentiated contraction of the rabbit atrium when the temperature was reduced from 37 to 30 degrees C or following pretreatment with reserpine (30 or 37 degrees C). Under these same conditions the ED50 of norepinephrine on the normal contraction was reduced. It is concluded that both reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of norepinephrine by enhancing the cellular store of activator calcium while not affecting the ability of norepinephrine to release activator calcium.

Functional characterization of β-adrenoceptor subtypes in rabbit right atria

Abstract The advent of radioligand binding studies has allowed the classification of receptor subtypes in various tissues. However, the presence of a receptor subtype in a heterogenous tissue does not insure that the receptor has a significant physiological role. β1- and β2-Adrenoceptors have been reported to coexist in the rabbit right atria. The purpose of the present investigation was to determine the physiological role of β-adrenoceptor subtypes in catecholamine-induced chronotropic responses in the rabbit right atria through comparison of data from functional and radioligand binding studies. Rank order of potency was determined using isoproterenol, epinephrine and norepinephrine for both chronotropic and inotropic responses in the rabbit right atria and right ventricular papillary muscles, respectively. These studies indicated that the β1-adrenoceptor was primarily responsible for catecholamine-induced responses. Next, the β1-selective antagonist, atenolol, was found to inhibit the chronotropic responses of the nonselective β-agonist, isoproterenol, and the β2-selective agonist, terbutaline, to the same extent. These data indicate that terbutaline produces its chronotropic effects in the rabbit right atria through stimulation of β1-, not β2-adrenoceptors. Finally, competition studies for [125I] iodocyanopindolol and the relatively selective β1- and β2-adrenoceptor antagonists (ICI 89406 and ICI 118551, respectively) indicated that the ratio of β1- to β2-adrenoceptor subtypes is 6:1. It is concluded that while both receptors may be present in the rabbit right atria, the β1-adrenoceptor is the predominant subtype both in density and physiological significance, while the β2-adrenoceptor plays little, if any role, in the chronotropic responses induced by catecholamines.

Propranolol withdrawal supersensitivity in rat cardiovascular tissue, in vitro.

The purpose of the present study was to determine if propranolol withdrawal supersensitivity could be demonstrated under in vitro conditions. Rats pretreated for 14 days with propranolol were studied 24, 48, 72 and 96 h after propranolol withdrawal. Right atria, right ventricle strips and thoracic aortic strips were isolated for study under in vitro conditions. Dose-response curves and geometric mean ED50 values revealed the presence of supersensitivity to the chronotropic and inotropic effects of isoproterenol in vitro at 72 and 96 h after withdrawal. No change in sensitivity was found 24 or 48 h after propranolol cessation. In like manner, supersensitivity to the vasorelaxant action of isoproterenol was demonstrated in aortic strips precontracted with angiotensin II at 72 and 96 h after propranolol withdrawal. In contrast, subsensitivity to the contractile actions of methoxamine was observed 48 h after withdrawal. These data indicate that chronic propranolol pretreatment and withdrawal results in the unmasking of supersensitivity to both beta 1 and beta 2 adrenoceptor stimulation. There would also appear to be a compensatory change in alpha-adrenoceptor-induced responses in rat aorta following propranolol withdrawal.

Determination of beta adrenoceptor density on rabbit mononuclear leukocytes

The purpose of the present study was to devise a technique for the isolation of a relatively homogeneous mononuclear leukocyte (MNL) preparation from rabbit whole blood and determine the density and affinity of beta-adrenoceptors on MNL. A modified method based upon that by Böyum was developed to maximize isolation of MNL from red blood cells (RBC), granulocytes, and platelets. The method involved an initial centrifugation to remove platelets and two centrifugations with a Ficoll solution to eliminate RBC and granulocytes. beta-Adrenoceptor density as determined with [125I]cyanopindolol and MNL membrane or whole cell preparations ranged between 317 and 360 sites per cell. Affinity for the binding sites was dependent upon whether membrane or whole cell preparations were studied, being 56.3 +/- 9.9 and 11.4 +/- 1.4 pM, respectively. Binding sites were found to be saturable and noncooperative. In addition, the binding sites demonstrated selectivity and stereospecificity for beta-adrenoceptor ligands. It is concluded that the modified method of harvesting MNL from rabbit whole blood provides a relatively homogeneous cell suspension that can be used to study the beta-adrenoceptor system.

Propranolol withdrawal-induced supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit

The present study was undertaken to determine if chronic administration and abrupt withdrawal of propranolol would induce supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit. Eight rabbits were pretreated with propranolol (40 mg/kg/day, i.p.) for one week. Dose-response curves were obtained one week prior to pretreatment and 24, 48, 72 and 168 hours after the last dose of propranolol. Isoproterenol (0.01 to 10 micrograms/kg) was injected via the marginal ear vein and heart rate was monitored continuously by recording the ECG. During the withdrawal period no significant rebound increase in basal heart rate was noted. Geometric mean ED50 values and their corresponding 95% confidence intervals were calculated and used as an index of potency. Twenty-four hours after withdrawal of propranolol pretreatment, the ED50 value for isoproterenol was significantly greater than control indicating continued surmountable beta-adrenoceptor blockade. In contrast, 72 hours after withdrawal, the geometric mean ED50 value was significantly less than control. The maximum chronotropic response to isoproterenol was not found to be different from control at any time during the withdrawal period indicating that a change in responsiveness was not induced. These data are interpreted as evidence of a true supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit following propranolol withdrawal.

Characterization of β-adrenoceptor subtypes in rabbit mononuclear leukocytes

Computer analysis of [125I]iodocyanopindolol competition studies using the relatively selective β1-adrenoceptor antagonist, ICI 89406, and the β2-selective antagonist, ICI 118551, on rabbit mononuclear leukocyte plasmalemmal preparations favored a two-site model indicating that both β1- and β2-adrenoceptor subtypes were present in approximately equal numbers. In contrast, similar studies performed on rabbit cardiac sarcolemma favored a one site model consistent with the presence of β1-adrenoceptors. Consequently, rabbit mononuclear leukocytes may provide a useful model for studying selective modulatory mechanisms of β1- and β2-adrenoceptors.