Henry R. Besch

Sympathetic nerve activity: role in regulation of blood pressure in the spontaenously hypertensive rat.

Sympathetic nerve activity (SNA) and high pressure baroreceptor regulation of SNA were studied in the Okamoto strain of spontaneously hypertensive rat (SHR). Mean arterial pressure (MAP) and SNA were not significantly affected by anesthesia with low doses of pentobarbital (20–25 mg/kg). Thus, most of these studies were performed in anesthetized rats. SNA in visceral sympathetic nerves increased rapidly with age up to 24 weeks and slowly thereafter. MAP increased with SNA, following the same time course. Both SNA and MAP in SHR were significantly greater than that found in normotensive Wistar control rats of comparable ages. Abolition of ganglionic transmission with hexamethonium in both SHR and normotensive controls reduced postganglionic SNA and MAP to comparable levels. In SHR less than 16 weeks old, increased baroreceptor stimulation effectively inhibited SNA with the same sensitivity as found in Wistar control rats. However, older SHR appeared to lose their ability to completely inhibit SNA during induced hypertension, whereas in Wistar control rats as old as 52 weeks, elevation of blood pressure to 165.3 ± 2.3 mm Hg completely suppressed SNA. These results suggest that SNA may play an important role in the development and maintenance of hypertension in SHR, and that central sympathetic centers, uninhibited by baroreceptor afferents, become active during the development of hypertension in the SHR.

Cyclic Adenosine Monophosphate Modulation of Slow Calcium Influx Channels in Guinea Pig Hearts

The relationship between cellular levels of cyclic adenosine monophosphate (AMP) and slow inward calcium ion (Ca2+) current was studied in isolated perfused guinea pig hearts in which fast sodium ion (Na+) channels had been inactivated by depolarization with potassium ions (K+) or blockade with tetrodotoxin. Perfusion with 22 mM K+ depolarized cardiac tissue to approximately −40 mv and rendered hearts inexcitable. Tetrodotoxin (3 × 10−5M) blocked excitability without altering resting membrane potential. Excitability and contractions could be restored to these hearts with a variety of inotropic agents that also raised the measured tissue levels of cyclic AMP or with high concentrations of Ca2+. The magnitude of steady-state tension developed by restored hearts was directly related to the external Ca2+ concentration as well as to the concentration of the restoring agent used. The tension of restored hearts was markedly reduced by Ca2+-channel antagonists. Elevation of cyclic AMP levels preceded restoration of excitability to inactivated hearts. A highly significant positive correlation was found between the magnitude of Ca2+-dependent tension developed by restored hearts and the level of cyclic AMP in those hearts. Glucagon and ouabain, inotropic drugs that do not elevate myocardial levels of cyclic AMP, failed to restore depolarized or tetrodotoxin-blocked hearts. Therefore, cyclic AMP appears to modulate slow Ca2+ influx channels in myocardial cells.

Characterization of [3H](+/-)carazolol binding to beta-adrenergic receptors. Application to study of beta-adrenergic receptor subtypes in canine ventricular myocardium and lung.

[3H](±)Carazolol, a newly available β-adrenergic receptor antagonist, has been used to characterize β-adrenergic receptor subtypes present in membrane vesicles derived from canine ventricular myocardium and canine lung. [3H](±)Carazolol binding is saturable, of high affinity, and in displaceable by β-adrenergic agents in accordance with their known pharmacological potencies. The interaction of carazolol with β-adrenergic receptors is stereospeclflc; the (−) stereoisomer demonstrates greater potency than the (+) stereoisomer. Kinetic analysis of [JH}(±)carazolol interaction with β-adrenergic receptors suggests the presence of two phases of interaction, consistent with initial rapidly reversible “low” affinity association of ligand with receptor, followed by Isomerization to form a high affinity, slowly reversible complex. Through use of a [3H](±)carazolol binding assay based on the high affinity complex, pharmacological specificities of £-adrenergic receptor populations of canine myocardium and lung were quantified. Analysis using computer-assisted techniques suggests a β1/β2 receptor ratio of approximately 85%/15% for canine myocardium and 5%/95% for canine lung. In the absence of added guanine nucleotides, comparison of potencies of β-adrenergic agonists in the two membrane systems suggests significant β, selectivity of l-isoproterenol and l-eplnephrine, and nonselectivity of norepinephrine. In the presence of saturating levels of guanine nucleotides, comparison of agonist potencies confirms the non-selectivity of l-isoproterenol and l-epinephrine, and β1 selectivity of norepinephrine. These results demonstrate that the state of guanine nucleotide regulation of receptors should be defined when examining agonist selectivities for β-adrenergic receptor subtypes in vitro.

Isolation and characterization of adult rat heart cells

Abstract Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca 2+ at concentrations greater than 10 −5 m .

Thyroxine and propylthiouracil effects on alpha- and beta-adrenergic receptor number, ATPase activities, and sialic acid content of rat cardiac membrane vesicles

Membrane vesicle preparations from the hearts of euthyroid, hyperthyroid, and hypothyroid rats were analyzed in an attempt to identify biochemical changes which might correlate with known functional changes occurring in these thyroid states. Several sarcolemmal constituents which have been shown to influence myocardial contractility were measured in membrane vesicle preparations from the hearts of animals in the three thyroid states. These constituents included the apparent number of alpha- and beta-adrenergic receptors (judged from specific binding of radiolabeled adrenergic antagonists) and Na-, K--ATPase activity. As a control for the recovery of sarcolemma in the preparations, the sialic acid content was measured in all preparations. The activity of K-, Ca2--ATPase, a sarcoplasmic reticulum enzyme which regulates intracellular ionized Ca2- concentration, was also measured. Membrane vesicles of the thyroxine-treated hyperthyroid rats showed a decrease (41%, p < 0.01) in the total apparent number of alpha-adrenergic receptors relative to euthyroid controls and an increase in the total apparent number of beta-adrenergic receptors (43%, p < 0.001). Membrane vesicles prepared from propylthiouracil-treated hypothyroid rats showed a decrease relative to euthyroid controls in the total apparent number of both alpha-adrenergic receptors (29%, p < 0.01) and beta-adrenergic receptors (23%, p < 0.05). The ratio of beta- to alpha-receptors approximately doubled in the hyperthyroid animals but remained unchanged compared to controls in the membranes isolated from hypothyroid animals. In the same membrane vesicle preparations, total sialic acid content was similar regardless of thyroid state. In addition.

Acetylcholine antagonism of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers.

The purpose of these experiments was to determine whether or not acetylcholine modulated the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers. Conventional microelectrode techniques were used. Predictably, isoproterenol produced shortening of action potential duration; acetylcholine significantly blunted this effect of isoproterenol. Isoproterenol restored excitability to fibers exposed to 22 mM potassium solutions, and acetylcholine abolished this isoproterenol-restored excitability. Both of these antagonistic effects of acetylcholine were blocked by atropine. Acetylcholine alone did not affect action potential duration in polarized fibers or excitability in potassium-depolarized fibers. Furthermore, acetylcholine had no effect on the decrease in action potential duration induced by premature electrical stimulation or by acetylstrophanthidin administration, or on excitability of fibers exposed to a zero sodium, high calcium superfusant-These data demonstrate a direct cellular basis for cholinergic antagonism of the electrophysiological effects of β-adrenergic stimulation of canine cardiac specialized intraventricular conducting tissue. cire Res 44: 378-383, 1979

Effect of l-dopa on sympathetic nerve activity and blood pressure in the spontaneously hypertensive rat.

We examined the effect of L-dopa, after peripheral L-amino acid decarboxylase inhibition, on sympathetic nerve activity (SNA) and blood pressure in spontaneously hypertensive rats (SHR) and in normotensive control rats. L-Dopa reduced SNA in both groups of animals. The SHRs were significantly more sensitive to the depressor effect of L-dopa than were the control animals, the threshold dose for reduction of SNA being 3 mg/kg in SHR and 15 mg/kg in control rats. Similarly, the magnitude of inhibition of SNA was substantially greater in the SHR than in normotensive rats. The reduction in SNA in the SHR was accompanied by a parallel fall in blood pressure. In contrast, blood pressure in control rats did not change significantly, even though SNA was diminished. Studies of the penetration of L-dopa into the cerebral parenchyma revealed that equivalent amounts of the amino acid entered the brains of the two groups of rats. These results suggest that the SHRs are more sensitive to the SNA-inhibiting effects of L-dopa than are normotensive rats. In addition, they confirm our previous suggestion that excessive SNA plays a causative role in the hypertension of the SHR.

Isolation of Canine Cardiac Sarcolemmal Vesicles

Isolation and characterization of sarcolemmal membrane fragments from the heart has been a goal of several research laboratories for some time (Wollenberger and Will, 1978). This is because of the fundamental importance of the sarcolemma in maintaining the electrophysiological and contractile properties of the heart by regulating transmembrane fluxes of the inotropically active cations Na+, K+, and Ca2+ between intracellular and extracellular compartments. Both purity and morphological form of the isolated membranes are equally important considerations in any isolation scheme for cardiac sarcolemma (Besch et al., 1976). In analogy to results first observed with cardiac sarcoplasmic reticulum membrane preparations, it seems highly advantageous to isolate sarcolemmal membrane fragments from the heart in the form of tightly sealed vesicles so that not only enzymic activity but also active and passive transport of ions can be measured in the same membrane preparation.

alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

We determined the effect of α-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of α-adrenergic receptors with either phenylephrine (10-8 M to 10-6 M) or epinephrine (10-8 M to 10-6 M) plus propranolol (10-6 M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10-6 M). Phenylephrine (10-5 M) attenuated cyclic AMP generation in response to isoproterenol (10-8 M and 10-5 M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10-6 M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by β-adrenergic receptors, whereas decreases appear to be mediated by α-adrenergic receptors.

Adrenergically Mediated Ventricular Fibrillation in Probucol‐Treated Dogs: Roles of Alpha and Beta Adrenergic Receptors

A high incidence of sudden death due to ventricular fibrillation (VF) has been observed in dogs under chronic treatment with probucol, a new hypocholesterolemic agent. The present study describes the cardiac electrophysiologic properties of probucol‐treated dogs and characterizes the electrophysiological response of these animais to manipula‐tion of the autonomic nervous system. There was no significant difference in the spontaneous sinus cycle length, the QT interval, refractory period of the atrium, ventricle or A‐V junction between normal and probucol‐treated dogs. Epinephrine produced VF with few and sometimes no preceding premature ventricular extra‐systoles. Electrical stimulation of the left stellate ganglion induced VF in 16/19 dogs whereas stimulation of the right stellate ganglion induced VF in 1/19 dogs. Phenyl‐ephrine induced VF in 0/19 dogs, isoproterenol in 5/19 dogs, but phenylephrine + isoproterenol induced VF in 9/11 dogs in which isoproterenol did not produce VF. α(phentolaminej or β (propranolol) blockade prevented initiation of VF by epi‐nephrine, phenylephrine + isoproterenol, and left stellate stimulation but a blockade did not prevent induction of VF by isoproterenoJ when isoproterenol alone produced VF. In this nonischemic model, we conclude that left stellate stimulation is a far more potent initiator of VF than right stellate stimulation and that induction of VF appears to require both α and β adrenergic receptor stimulation.