Philip Posner

Receptor-mediated effects of angiotensin II on neurons.

Aside from its well-known and numerous actions at peripheral tissues, the octapeptide angiotensin II (ANG II) elicits specific receptor-mediated effects within the central nervous system. In this review we focus on the receptor-mediated actions of ANG II on neurons. The distribution of ANG II receptors in the brain and physiological, electrophysiological, and cellular effects mediated by these receptors are discussed. This is extended to a review of the characteristics of ANG II receptor subtypes on cultured neurons and the cellular and genomic actions mediated by these receptors. Finally, we develop this information into speculative models for the cellular effects mediated by each ANG II receptor subtype in neurons.

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors.

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I(no)) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. Ino is the sum of all inward and outward membrane currents (minus Na+, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I(no) in cultured neurons. AII caused a reversible and concentration (0.1 nM-10 microM)-dependent increase in I(no). This effect is inhibited by the AT2 receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT1-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT2 receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM-10 microM)-dependent decrease in I(no) that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I(no) is mediated by AT1 receptors. Additionally, some neurons displayed both AT1- and AT2 receptor-mediated effects on I(no). Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.

Angiotensin II Type 2 Receptor–Mediated Regulation of Rat Neuronal K+ Channels

We have previously shown that angiotensin II (Ang II), via AT2 receptors, increases whole-cell K+ current in cultured rat hypothalamus and brain stern neurons. We have now investigated the AT2 receptor-mediated effects of Ang II on the activity of single delayed rectifier K+ channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K+; pipette, 140 mmol/L K+), two voltage-dependent channels were recorded with conductances of 34 +/- 4 and 56 +/- 6 pS, respectively (n = 6). When patches were excised, the channels reversed near a membrane potential expected for a K+ channel. In cell-attached patches (-40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K+ channel from 0.03 +/- 0.01 to 0.21 +/- 0.05 (n = 3). The selective AT2 receptor antagonist PD 123319 (1 mumol/L) but not the AT1 receptor antagonist losartan (1 mumol/L) blocked the actions of Ang II (n = 3). The selective AT2 receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, tau 1 = 0.9 +/- 0.1 milliseconds, tau 2 = 2.3 +/- 0.3 milliseconds; Ang II, tau 1 = 3.1 +/- 0.4 milliseconds, tau 2 = 12.1 +/- 2.4 milliseconds), and PD 123319 blocked this effect (n = 3). The closed-time histogram was not affected by Ang II PD 123319, or losartan. These results suggest that activation of AT2 receptors modulates rat hypothalamus and brain stern neuronal whole-cell K+ current by increasing the open probability of a 56-pS K+ channel.

Angiotensin II regulation of intracellular calcium in astroglia cultured from rat hypothalamus and brainstem.

Abstract: This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca2+]i) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1–100 nM) increased [Ca2+]i in both polygonal and stellate astroglia when measured using fura‐2 imaging fluorescence microscopy. Ang II increased [Ca2+]i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrodes solution (containing 2 mM CaCl2), the Ang II‐stimulated increase in [Ca2+]i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady‐state plateau of free Ca2+. In both cell types, the selective Ang II type 1 receptor subtype (AT1) antagonist losartan (1 µM) inhibited the Ang II‐stimulated increase in [Ca2+]i. The selective AT2 antagonist PD 123319 (1 µM) did not inhibit the Ang II‐stimulated increase in [Ca2+]i in either cell type. To define the sources of Ca2+ that participate in the Ang II‐stimulated increase in [Ca2+]i in astroglia, experiments were performed in a nominally Ca2+‐free Tyrodes solution. In either cell type, this resulted in only an initial transient increase of Ca2+ and no sustained plateau of Ca2+ when challenged with Ang II. Thapsigargin (5 µM), cyclopiazonic acid (10 µM), and ryanodine (10 µM), but not caffeine (1–10 mM), inhibited the initial rise in [Ca2+]i. The plateau increase of [Ca2+]i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 µM) and nifedipine (10 µM); in contrast, gadolinium (100 µM) had no effect on the plateau increase of [Ca2+]i. These results indicate that Ang II, in physiological concentrations, can activate AT1 receptors to stimulate both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space to increase [Ca2+]i of polygonal and stellate astroglia.

The effect of verapamil on potassium fluxes in canine cardiac purkinje fibres

Electrophysiological studies of verapamil on isolated cardiac tissue show its effects to occur during Phase 2 and Phase 4 of the cardiac action potential and to be a result of blocking the slow inward Ca2+ current. It is important to know if verapamil effects the transmembrane movement of ions other than Ca2+. By studying uptake and efflux of 42K by canine cardiac Purkinje fibres in the presence and absence of verapamil (1.1 x 10-6 M-4.4 X 10-6 M), it was found that the drug inhibits both 42K uptake (15%) and efflux (30%).

The effect of bromoacetylalprenololmentane on rat atrial tension development and β-adrenoreceptors

In isolated rat atria, 1 microM bromoacetylalprenololmentane shifted the tension development curve for isoproterenol to the right of the control curve. After a 2 h washout period, no significant recovery of the isoproterenol concentration-response curve had occurred. Pretreatment of rat atrial membranes with 1 microM bromoacetylalprenololmentane resulted in an 83% decrease in the concentration of beta-adrenoceptors with no change in the KD value for [125I]iodocyanopindolol binding to the receptors left. The results indicated that bromoacetylalprenololmentane was an irreversible beta-blocker.

The effect of the beta-adrenoreceptor antagonist Ro03-7894 on rat atrial tension development and (-)-[3H]dihydroalprenolol binding to cardiac and lung membranes.

The ability of 1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol (Ro03-7894) to irreversibly inactivate beta-adrenoreceptors was studied. In isolated rat atria Ro03-7894 (500 microM) depressed and shifted the tension development curve for isoproterenol to the right. After a 2 hour washout period the dose response curve for isoproterenol was further depressed. At a lower dose of Ro03-7894 (50 microM), the isoproterenol dose response curve was also depressed and shifted to the right although after a 2 hour washout, the sensitivity to isoproterenol was restored but the maximum response was still depressed. Ro03-7894 (50 microM) also depressed the tension development response to increasing concentrations of external calcium. The concentration of Ro03-7894 that inhibited (-)-[3H]dihydroalprenolol (DHA) binding by 50% in cardiac and lung membranes was 20 microM. Incubation of rat ventricular or lung membranes for 1 hour with 100 microM Ro03-7894 followed by washing did not change the concentration of beta-adrenoreceptors or the KD values for [3H]DHA binding. Furthermore, neither the concentration of beta-adrenoreceptors nor the KD for [3H]DHA binding was changed in cardiac and lung membranes at 4 or 24 hours after an i.p. injection of 20 mg/kg of Ro03-7894. The results suggested that Ro03-7894 was a relatively weak beta-adrenoreceptor antagonist which under the conditions used did not irreversibly inactivate the receptor but probably depressed tension development in intact atria nonspecifically.

Negative chronotropic effect of chronic ethanol ingestion in the rat

The chronic consumption of ethanol has been correlated with the development of arrhythmias. This study looked at the effect of chronic ethanol ingestion on the action potential of sino-atrial cells. The studies were carried out on hearts excised from male Long-Evans hooded rats, pair-fed on ethanol (E) or control (C) liquid diet. The ethanol diet supplied 35-39% of calories as ethanol. The studies of isolated sino-atrial tissue were carried out after 18-20 weeks, 30-32 weeks and 40-42 weeks on the diet. Sino-atrial cells from E and C rats were compared for changes in spontaneous rate, action potential amplitude, time to repolarize to -70 mV, and resting membrane potential. At 18-20, 30-32 and 40-42 weeks the spontaneous rate of firing of the sinus node was significantly lower in the E group as was the maximum response to isoproterenol. The time to repolarize to -70 mV was longer in E. The endogenous level of catecholamines was also lower in the E group.

Cardiac effects of toxin A-III from the heteronemertine worm Cerebratulus lacteus (Leidy)☆

Reversible and irreversible (cytolytic) effects of pure Cerebratulus toxin A-III, a heteronemertine polypeptide toxin, upon canine cardiac Purkinje fibers were investigated using standard microelectrode techniques and a fluorometric assay for measuring release of cell protein. Sublytic concentrations (0·25-1·0 μg/ml) of A-III reversibly depolarized the resting membrane if the duration of exposure was brief. Tetrodotoxin and saxitoxin at concentrations which block the Purkinje fiber sodium channel failed to prevent the A-III induced resting membrane depolarization. The amplitude and duration of the action potential were also reversibly reduced by low concentrations of A-III. The Ca2+-dependent slow response observed in TEA-Tyrode was also blocked without alteration of the resting potential. Increasing [Ca2+]0 reduced the effectiveness of A-III in altering transmembrane potentials. Higher concentrations (> 2 μg/ml) of A-III irreversibly damaged Purkinje cells, causing release of intracellular macromolecular constituents. Toxin A-III actions upon cardiac tissue were very similar to those reported by Ho et al. (1975) for an elapid snake cardiotoxin.

Depressed atrial inotropic response in the rat with chronic ethanol ingestion.

Consumption of ethanol for long periods of time has been correlated with cardiac dysfunction as well as changes in function of the autonomic nervous system. This study looked at the effect of chronic ethanol ingestion on atrial contractility and atrial muscarinic and beta adrenoreceptors. Male Long-Evans hooded rats were pair-fed on ethanol (E) or normal (N) liquid diet for 40 weeks. The E diet supplied 35-39% of calories as ethanol. The atria from E rats had significantly lower baseline and peak contractility. They also showed a higher incidence of failure induced by isoproterenol. There was no difference in concentration or binding characteristics of beta-adrenoreceptors or muscarinic receptors. The data suggest that the negative inotropism caused by ethanol ingestion is the result of some mechanism other than changes in autonomic receptors.