Angiotensin II receptor activation neurons in vitro CHARLES Neurosciences Yang, Charles
R. YANG,
M. IAN PHILLIPS,
Unit, Ottawa
R., M. Ian Phillips,
Civic Hospital,
and Leo P. Renaud.
NERVOUS SYSTEM is now perceived as a site for the synthesis of angiotensin (32), and a substantial body of evidence supports the notion that angiotensin II (ANG II) may have a neurotransmitter role in select neural circuits that mediate body fluid homeostasis and cardiovascular regulation. For instance, angiotensin-like immunoreactivity is present within neurons and pathways connecting principal autonomic and cardiovascular regulatory regions of the brain stem, hypothalamus, and forebrain (16), with a high density of angiotensin binding sites distributed along the lamina terminalis (7, 18, 25), an important site for regulation of body fluid homeostasis (13). Within this area neurons are influenced by systemic, central, and local administration of angiotensin to elicit behavioral (e.g., drinking), autonomic (e.g., elevation in arterial pressure), and neuroendocrine (e.g., release of neurohypophysial hormones) responses (23). At the level of the magnocellular neurosecretory neurons, extracellular electrophysiological data obtained during exogenous application of ANG II suggest that they possess functional ANG II receptors whose activation results in an increase in firing rate, an action that is reversibly blocked by the angiotensin peptide antagonist saralasin (12, 20, 21). To date, however, there are scant intracellular data as to the actual transmembrane events that accompany the actions of angiotensin on these mammalian magnocellular neurosecretory cells. The recent development of nonpeptide ANG
THE CENTRAL
$2.00
Copyright
rat supraoptic
AND LEO P. RENAUD Ottawa,
Angiotensin II receptor activation depolarizes rat supraoptic neurons in vitro. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1333-R1338, 1992.-Functional studies indicate that hypothalamic magnocellular neurosecretory neurons are a target for angiotensin. The present investigation used intracellular recordings to characterize the nature and type of angiotensin II receptors on rat supraoptic nucleus neurons maintained in superfused hypothalamic explants. Of 68 cells transiently exposed to either Va15- or Ile5-angiotensin II (maximum peak concentration 1-25 PM), 34 responded with a gradual membrane depolarization (l-15 mV) that peaked in 2.2 t 0.4 (SD) min and was accompanied by a 17.6 t 4.8% reduction of input resistance. Responses persisted (and were actually enhanced) in media containing tetrodotoxin (0.5-1.0 PM) and/or nominally zero calcium, indicating a direct postsynaptic action. In 19 responsive cells, the mean reversal potential for the angiotensin-induced response was -26.4 t 2 mV. Bath application of the nonpeptide type- 1 angiotensin receptor antagonist DuP753 (5-20 PM) reversibly blocked the angiotensin-induced depolarization in all of 11 cells tested. By contrast, equimolar applications of the type-2 antagonist PD123177 were ineffective in all seven angiotensin-responsive cells tested. These observations provide novel evidence for the existence of functional type-l receptors on rat supraoptic nucleus neurons. The reversal potential for the angiotensin-induced response suggests mediation through a nonselective cationic conductance. angiotensin; receptors; electrophysiology; supraoptic nucleus
0363-6119/92
depolarizes
0 1992
Ontario
Kl Y 4E9, Canada
II antagonists permits differentiation between two types of angiotensin receptors in the adrenal gland (3). In the rat brain, at least two types of binding sites for angiotensin are demonstrated (7, 28, 33), and the hypothalamic magnocellular neurons have one of these subtypes (11, 33). However, no intracellular electrophysiological correlate of angiotensin on magnocellular neurosecretory neurons has been reported. In the present study, we used intracellular recordings obtained from neurons of rat supraoptic nucleus (SON) maintained in superfused hypothalamic explants to evaluate transmembrane events and receptor types involved in responses to exogenously applied angiotensin II. The data indicate that a portion of the vasopressinand oxytocin-secreting neurons in the rat supraoptic nucleus appear to contain type-l angiotensin receptors whose activation induces membrane depolarization and an increase in membrane conductance, possibly mediated through nonselective cationic channels. A preliminary report has been presented (24). METHODS Experiments used acutely prepared hypothalamic explants of adult (200-250 g) male Long-Evans rats (35). Within 4 min of decapitation, the brain was removed and a horizontal explant of basal forebrain containing the supraoptic nucleus was dissected and pinned to the Sylgard base of a temperature-regulated (33°C) chamber. Each explant was superfused with oxygenated (95% 02-5% CO,) artificial cerebral spinal fluid (aCSF) delivered by gravity at a rate of 0.9-1.3 ml/min. The standard aCSF (pH 7.4) contained (in mM) 126 NaCl, 3 KCl, 1.3 MgC12, 25.9 NaHCO,, 2.4 CaCl,, and 10 glucose and had a freezing point osmolality of 290-295 mosmol/kg. Modifications for different experiments are specified in the text. Levels of aCSF were adjusted with cotton wicks to minimize electrode capacitance. Recordings commenced after 3 h of equilibration. Both [Ile”] ANG II and [Val”]ANG II (Peninsula, Belmont, CA) were shown to be effective in exciting rat neurons in previous electrophysiological studies (2, 9, 20, 21) and thus they were used as ANG II agonists in the present study. Nonpeptide antagonists were DuP753 (Losartan, 2-n-butyl-4-chloro-5hydroxymethyl-l-[2’-(l~-tetrazole-5-yl)biphenyl-4-y methyl] imidazole, potassium salt; Refs. 3, 15, 19, 29) and PD123177 (hydrochloride salt of EXP655, l-(4-amino-3-methylphenyl) methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-lH-imidazole[4,5-Clpyridine-6-carboxylic acid; Refs. 3, 15, 33; both are gifts from Du Pont). Both the agonists and the antagonists were prepared as concentrated stock in aCSF and stored in siliconecoated (Sigmacote, Sigma) vials at -25°C until use, when appropriate dilutions were achieved. All syringes used to deliver ANG II were also silicone-coated. Drugs were either bath applied or injected as a lo- to 40-~1 bolus over 2-4 s into the superfusion line proximal to the explant by a timer-controlled pump. As described elsewhere (35), the final concentration of the drug reaching the supraoptic nucleus was an estimate based on the volume and concentration of the injected drug and the volume of aCSF overlying the nucleus. The values quoted in the The
American
Physiological
Society
R1333
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R1334
ANGIOTENSIN
DEPOLARIZES
text refer to the transient peak concentration; effective concentrations reaching a cell are likely to be much lower because of diffusion and metabolism within the tissue. Intracellular recordings were obtained through micropipettes containing a mixture of potassium acetate (3-4 M) and potassium chloride (0.05 M) at pH 7.0. Signals were processed with an Axoclamp-2A amplifier. A Ag-AgCl electrode connected to the bath solution via a KCl-agar bridge served as reference. Bridge balance was maintained throughout the experiments by eliminating the time-independent component of the voltage response occurring at the onset and offset of rectangular current pulses applied through the recording electrode. Resting membrane potentials were recorded as the difference between the potential inside the cell and that in the extracellular space. Input resistance was determined from the slope of voltage-current (V-I) plots obtained from membrane voltage deflections after delivery of a series of 100-ms depolarizing and hyperpolarizing current pulses. Amplified signals were digitized by a Neuro-Data (DR-384) pulse-code modulator, sampled on-line using pCLAMP software (Axon Instruments), and recorded on video tapes for analysis off-line. In experiments using nominally calcium-free media, the effectiveness of calcium removal was assessed by the disappearance of a [Ca”+]-dependent postburst afterhyperpolarization (AHP) evoked by a l-s depolarizing current pulse (26). Group data are expressed as means t SD and all pairwise comparisons of the group data were made using the Student’s t test. RESULTS
The data base was derived from stable intracellular recordings in 68 supraoptic neurons whose resting membrane potentials ranged between -55 and -63 mV and whose action potentials exceeded 70 mV and input resistances ranged between 75 and 300 MO. All cells demonstrated two features typical of recordings from supraoptic neurons, i.e., activity-dependent action potential broadening and the presence of an “A’‘-notch, signifying a transient outward K+-current when depolarized from holding potentials more negative than -70 mV (26) (Fig. 1). Of the 68 cells tested, 34 cells (50%) demonstrated membrane depolarization (range l-15 mV) after bolus infusions of either Ile” or VaPANG II at transient maximum concentrations estimated between 10 and 25 PM. A typical response, illustrated in Fig. 1, was a gradual membrane depolarization that peaked in 2.2 t 0.4 min and subsided after 5-7 min. Successive responses to ANG II could be reproduced provided that consecutive applications were at least 10 min apart in the case of small (53 mV) depolarizations or at least 30 min apart in the case of larger depolarizations.
RAT
SUPRAOPTIC
CELLS
Tests applied at shorter intervals yielded responses of diminished magnitude. There was virtually no difference in the magnitude of the membrane depolarization or conductance change among cells responding to either VaPANG II or Ile5ANG II; therefore the data on these parameters in this group were pooled. However, sequential testing of both peptide analogues on 27 cells revealed apparent differences in responsivity: four responded to both peptides with membrane depolarization, five responded to only one of the two peptides, and 18 showed no response to either peptide. Five cells were tested by bath application of ANG II. The lowest effective concentration of ANG II to induce a depolarization of 3 mV was 1 pM. ANG II-responsive neurons included five neurons whose spontaneous activity demonstrated clear phasic discharges. This firing pattern is frequently associated with magnocellular neurosecretory neurons that are immunoreactive to vasopressin (4, 34). After ANG II applications, all cells demonstrated prolongation in the duration of each phasic discharge. In another six cells, whose spontaneous firing appeared to be of a continuous nature and might therefore be either vasopressin or oxytocin secreting (26), exposure to ANG II produced an increase in firing without a change in pattern. ANG II did not transform continuous firing neurons into phasic firing cells. Cells in both categories were among the neurons not responding to ANG II (Fig. 2). Measurements derived from the slope of V-I plots indicated that a mean decrease of 17.6 t 4.8% in membrane resistance accompanied the ANG II-induced depolarization. The mean reversal potential for the ANG II-induced response in 19 cells was -26.4 t 2 mV, as extrapolated from the intercept of the linear range (-60 to -120 mV) of the V-I plots obtained in normal aCSF at resting membrane potentials and during drug-induced membrane depolarizations (Fig. 2A). In five cells tested in media containing tetrodotoxin (TTX, 1.0 ,uM), the ANG II-induced depolarizations and reduction of input resistance persisted, with a mean reversal potential of -30 t 2 mV for the peptide-induced response that differed little from the control. However, in two of these five neurons, marked increases in the ANG II-induced membrane depolarization were observed, and they were associated with a greater reduction in their slope resistance from 12 and 15% in control media to 35 and 38% in TTX, respectively. However, their reversal
ANGII -
Fig. 1. Top trace: intracellular current clamp recording from a supraoptic neuron (resting membrane potential -62 mV) after a brief bolus infusion (bar) of Ile5ANG II into the perfusion media sufficient to attain a transient maximum concentration of 10 PM. Note gradual onset of prolonged membrane depolarization. Full spike amplitude is attenuated by chart recorder. Downward &flec-
~IIIIIMI~~
I 0.1 nA 2 min
current pulses (100 ms, -0.05 nA, bottom trace) delivered to monitor input resistance; these are reduced by 22% during the drug-induced depolarization.
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ANGIOTENSIN
DEPOLARIZES
RAT
SUPRAOPTIC
R1335
CELLS
Fig. 2. Left: extrapolations of the linear range of voltage-current (V-I) plots (-120 to -60 mV) obtained from supraoptic neurons (SON) under control conditions (open symbols) and during membrane depolarizations follow a bolus infusion of Va15ANG II (maximum peak concentration, 10 PM; closed symbols) showed a reversal potential of -27 mV from the ANG II-induced depolarization. ANG II depolarized this cell by 7 mV and induced a reduction of slope resistance of 8%. Right: V-I plots from another neuron recorded in control artificial cerebrospinal fluid (aCSF) containing tetrodotoxin (1 PM) before (open symbols) and after (closed symbols) bolus infusion of Va15ANG II (maximum concentration, 10 PM) reveals a large drug-induced membrane depolarization (25 mV) and a 38% reduction of slope resistance, with a virtually identical reversal potential (-28 mV). Suppression of sodium spikes by tetrodotoxin permitted voltage readings in the depolarized range. Regression lines through linear range are fitted by eye.
potentials remained similar to those obtained in control media (Fig. 2B). In five cells tested in control aCSF where the mean ANG II-induced membrane depolarization was small (1.5 t 0.2 mV), the same ANG II test in media containing nominally zero [Ca2+10 and 6 mM Mg2+/Mn2+ induced a significantly greater depolarization (10.6 t 1.1 mV) (Figs. 3 and 4). Reversal potentials remained at -30 mV (not illustrated). Two other patterns of response were detected in a minority of instances. Two neurons displayed a weak membrane hyperpolarization with no consistent change in membrane resistance when tested with Ile5ANG II. In three other cells, ANG II applications were associated with an increase in spontaneous inhibitory postsynaptic potentials (IPSPs) blocked in each instance by bath application of 25-50 PM bicuculline (Fig. 5). Evidently there are y-aminobutyric acid-ergic inputs to supraoptic neurons that are activated by ANG II. To address the type of receptor that might mediate these ANG II-induced responses in supraoptic neurons, cells were tested in the presence of nonpeptide angio-
A
CONTROL
ANG - II
tensin antagonists recently reported to distinguish between two types of ANG II receptors in the adrenal gland and in the brain (3, 15, 19, 29). In 11 cells responding to ANG II, bath application of the nonpeptide ANG II type-l antagonist DuP753 (5-20 PM) had no appreciable influence on membrane potential or spontaneous firing. For this reason we chose to use this concentration range of antagonist. The ANG II-induced response in all 11 cells was blocked during DuP753 application, with partial recovery of the ANG II response evident some 15-20 min after return to control media (Fig. 6). We did not seek the lowest effective antagonist concentration. Figure 7 summarizes the actions of DuP753 on their ANG II-induced depolarizations. In three neurons in which bath-applied DuP753 (5 FM) was evaluated for its specificity of action, in each instance membrane depolarizations evoked by ANG II were reversibly attenuated, whereas those evoked by bolus application of glutamate (50 PM) were unaffected (data not illustrated). Bath application of the ANG II type-2 receptor antagonist PDl23177 (5-20 PM) induced small changes in
B
I 2s
5 mV
I(2nA
ANG II -
O[Ca++]. + 6 mM Mn”
0.5 mill
Fig. 3. Removal of extracellular calcium enhances action of angiotensin. A: application of IlesANG II (solid bar; final concentration, 20 PM) induces no apparent membrane response in normal aCSF (control). B: left, in normal aCSF, a [ Ca2+],-dependent postburst after hyperpolarization (arrow; AHP) is elicited by intracellular injection of a depolarizing pulse (1 s, 0.1 nA); right, 15 minutes after application of zero [Ca2+], media containing 6 mM Mg2+/Mn2+, the same intracellular injection of depolarizing pulse no longer evoked an AHP indicating effective removal of [Ca2+10. C: in the absence of extracellular [Ca2+],,, reapplication of the ANG II onto this previously unresponsive cell induced a prolonged membrane depolarization and action potential discharge. Calibration bars in C also apply to A.
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ANGIOTENSIN
DEPOLARIZES
RAT
SUPRAOPTIC
CELLS
CONTROL
(n=5) *
ACSF
ANG II
o[ca++](-j
Fig. 4. Histogram of data from 5 neurons demonstrating < 0.001) increase of the net depolarization by ANG [Ca2+10. Values are means t SD.
a significant II in absence
(P of
membrane potential in both depolarized and hyperpolarized directions (2 mV) with no detectable change in input resistance. PD123177 did not alter the response to ANG II in any of the seven cells tested. DISCUSSION
The present study provides novel intracellular data to indicate that rat supraoptic nucleus neurosecretory neurons contain functional angiotensin AT1 receptors whose activation results in membrane depolarization. These results are consistent with previous reports of a predominantly excitatory action from iontophoretically applied ANG II on rat and cat supraoptic neurons (20,21). Moreover, if (in the majority of instances) the firing patterns of these cells are a true reflection of their ability to synthesize either vasopressin or oxytocin (4, 34), then the present data would indicate that a population of both cell types are likely to contain type-l ANG II receptors (12). As noted earlier, not all cells responded to both forms of ANG II used in this study. This may reflect further heterogeneity of the ANG II receptor subtypes. Although the ionic mechanism mediating the excitatory actions of ANG II on supraoptic neurons remains to be detailed, a linear V-I relationship and a mean reversal potential of -26 mV for the ANG II response suggests activation of a voltage-independent nonselective cationic conductance. This is similar to their response to dopamine (35) and hypertonic media (1) and bears a close resemblance to the cationic conductance that has been demonstrated in invertebrate bursting neurons and glandular secretory cells (22). ANG II also induces membrane depolarization in hippocampal (9) and sympathetic ganglia neurons (2), although the ionic basis of these responses remains to be defined. A prerequisite for the activation of nonselective cationic conductance is the mobilization of intracellular calcium (22). Stimulation of ANG II receptors on a variety of cell types will promote hydrolysis of inositol triphosphate, and a concomitant rise in intracellular calcium (14, 17, 19, 29). While such mobilization of cytosolic [Ca2+] may itself activate a calcium-dependent nonselective cation conductance, the details surrounding the intracellular messengers induced by ANG II in central neurons remain controversial (31). The depolarizing response to ANG II suggests a direct activation of ANG II receptors on SON neurons, since this response persists after synaptic blockade through removal of extracellular TCa2+l or addition of TTX. How-
ANG II + BMI
4J
10 mV 0.5 min
Fig. 5. Top: 5 traces of control baseline voltage recordings from a supraoptic neuron (3 M KAc-filled micropipette) held at -60 mV with current injection (-0.01 nA) display transient membrane fluctuations, some of which are presumably inhibitory postsynaptic potentials. A&& dZe: traces obtained during application of Va15ANG II (10 PM) reveal a prominent increase in magnitude and frequency of inhibitory postsynaptic potentials (IPSPs). IPSPs did not summate so there was no apparent membrane hyperpolarization. Bottom: when bicuculline methiodide (BMI, 25-50 PM) was applied to bath simultaneously with another application of angiotensin, the barrage of IPSPs was no longer evident. These features suggest that ANG II is activating a GABAergic input.
ever, it is intriguing that the ANG II-induced depolarization is significantly potentiated when extracellular [ Ca2+] is removed. This may imply that influx of extracellular [ Ca2+] may negatively regulate the functional properties of ANG II receptors. In neuronal culture the number of functional binding sites for ANG II is substantially reduced in the presence of the [Ca2+] ionophore A23187 alone, but not in [Ca2+] -free medium (30). Whether the time course of this development is comparable with our findings remains to be determined. It appears that the influx of extracellular [Ca2+], but not mobilization of intracellular [Ca”+], has a negative influence on ANG II receptor functions. The potentiation of the ANG II-induced response in the absence of extracellular [Ca”+] may also be due to inactivation of [Ca”+] -dependent peptidases that catabolize ANG II (5, 10). The routine use of divalent cation chelating agents such as EDTA or ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) and neptidase inhibitors (e.g. bacitracin)
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ANGIOTENSIN
ANG
DEPOLARIZES
II
RAT
SUPRAOPTIC
Fig. 6. ANG II-evoked responses are attenuated by the nonpeptide AT, antagonist DuP753. Top: typical delayed membrane depolarization after bolus application (bar) of Ile5ANG II (20 PM), recorded from a spontaneously discharging supraoptic neuron. In this instance, ANG II-induced membrane depolarization and associated discharge of action potentials is followed by a prolonged membrane hyperpolarization. Middle: bath application of DuP753 (5 FM) had little influence on spontaneous firing but markedly attenuated ANG II-induced response. Bottom: 15 min after termination of DuP753 application (dashes), a partial recovery of ANG II-induced response was achieved.
5 ClM. DuP753
ANG
II
I ANG
(n=ll)
II
20 mV
1 min
II
to lengthen the biological half-life of the peptides in neuropeptide-binding studies support this interpretation. The introduction of nonpeptide antagonists has served to define at least two types of angiotensin receptors in the adrenal gland where DuP753 has a preference for receptors in the adrenal cortex (now designated ATI), whereas the structurally dissimilar PD 123 177 (formerly EXP655) binds preferentially to the ANG II receptors in the adrenal medulla (now designated ATB) (3). Binding studies have identified both types of receptors in brain, each with a distinct regional distribution (7, 15, 28, 33). Of particular interest is the selective displacement of 1251-labeled ANG II binding in the area of rat supraoptic nucleus by the AT1 antagonist DuP753 (33). The present electrophysiological findings provide the functional correlate for the existence of AT1 ANG II receptors on supraoptic neurons. What are possible sources of the ligand for these angiotensin receptors ? An endogenous origin for angiotensin II is suggested by various reports of angiotensinlike immunoreactivity in the magnocellular neurons and
ANG
RI337
CELLS
ANG
II
+DuP753 (5-20pM)
Fig. 7. Histogram summarizes the significant (P < 0.001) attenuation ANG II-induced depolarization in 11 neurons during bath-applied DuP753 (5-20 PM). Values are means t SD, * P < 0.001.
of
supraopticoneurohypophysial pathway (16, 26). One might speculate that ANG II is released locally from axon collaterals or soma-dendritic sites in a manner similar to that proposed for oxytocin or vasopressin (27). An exogenous origin is the angiotensin immunoreactive afferents to supraoptic neurons. Some of these can be traced to the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis, two circumventricular structures (12, 16). Both of these areas are also sites of intense angiotensin binding (7, 18, 25, 33) and therefore may be important interface zones whereby circulating hormones (e.g., angiotensin) influence the central nervous system. In the rat, evidence for a transmitter role for angiotensin in the efferent pathways from SF0 was obtained from in vivo observations whereby the increase in excitability of vasopressin- and oxytocin-secreting magnocellular neurons is reversibly attenuated in the presence of a peptide angiotensin antagonist, saralasin (12). The SF0 is a key structure in mediating the response of magnocellular neurons to circulating angiotensin (6) and its neurons demonstrate a sensitivity to circulating angiotensin (8). Hence this circumventricular organ contains neurons which may both receive a peripheral (i.e., circulating) message mediated by angiotensin and, in turn, use angiotensin to convey the message at central levels to activate a number of neurons, including the magnocellular neurosecretory cells. We thank Dr. Margaret Sullivan for her critical reading of the manuscript. This study was supported by the Canadian Medical Research Council and Canadian Heart and Stroke Foundation. Present address of M. I. Phillips: Dept. of Physiology, Univ. of Florida College of Medicine, JHMHC, Box J-274, Gainsville, FL 32610. Address for reprint requests: L. Renaud, Neurology/Neurosciences, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada KlY 4E9. Received
13 January
1992; accepted
in final
form
19 May
1992.
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R1338
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Tamura, C. S., and R. C. Speth. Effects of angiotensin II on phosphatidylinositide hydrolysis in rat brain. Neurochem. Int. 17:
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Unger, T., E. Badoer, D. Ganten, R. E. Lang, and R. Rettig. Brain angiotensin: pathways and pharmacology. Circulation
475-479,
77, Suppl.
1990.
1: l-40,
1988.
Wamsley, J. K., W. F. Herblin, M. E. Alburges, and M. Hunt. Evidence for the presence of angiotensin II type-1 receptors in brain. Bruin Res. BULL. 25: 397-400, 1990. 34. Yamashita, H., K. I. Nenaga, M. Kawata, and Y. Sano. Phasically firing neurones in the supraoptic nucleus of the rat hypothalamus: immunocytochemical and electrophysiological studies. Neurosci. Lett. 37: 87-92, 1983. 35. Yang, C. R., C. W. Bourque, and L. P. Renaud. Dopamine D2 receptor activation depolarizes rat supraoptic neurones in hypothalamic explants. J. Physiol. Lond. 443: 405-419, 1991. 33.
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R. YANG,
M. IAN PHILLIPS,
Unit, Ottawa
R., M. Ian Phillips,
Civic Hospital,
and Leo P. Renaud.
NERVOUS SYSTEM is now perceived as a site for the synthesis of angiotensin (32), and a substantial body of evidence supports the notion that angiotensin II (ANG II) may have a neurotransmitter role in select neural circuits that mediate body fluid homeostasis and cardiovascular regulation. For instance, angiotensin-like immunoreactivity is present within neurons and pathways connecting principal autonomic and cardiovascular regulatory regions of the brain stem, hypothalamus, and forebrain (16), with a high density of angiotensin binding sites distributed along the lamina terminalis (7, 18, 25), an important site for regulation of body fluid homeostasis (13). Within this area neurons are influenced by systemic, central, and local administration of angiotensin to elicit behavioral (e.g., drinking), autonomic (e.g., elevation in arterial pressure), and neuroendocrine (e.g., release of neurohypophysial hormones) responses (23). At the level of the magnocellular neurosecretory neurons, extracellular electrophysiological data obtained during exogenous application of ANG II suggest that they possess functional ANG II receptors whose activation results in an increase in firing rate, an action that is reversibly blocked by the angiotensin peptide antagonist saralasin (12, 20, 21). To date, however, there are scant intracellular data as to the actual transmembrane events that accompany the actions of angiotensin on these mammalian magnocellular neurosecretory cells. The recent development of nonpeptide ANG
THE CENTRAL
$2.00
Copyright
rat supraoptic
AND LEO P. RENAUD Ottawa,
Angiotensin II receptor activation depolarizes rat supraoptic neurons in vitro. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1333-R1338, 1992.-Functional studies indicate that hypothalamic magnocellular neurosecretory neurons are a target for angiotensin. The present investigation used intracellular recordings to characterize the nature and type of angiotensin II receptors on rat supraoptic nucleus neurons maintained in superfused hypothalamic explants. Of 68 cells transiently exposed to either Va15- or Ile5-angiotensin II (maximum peak concentration 1-25 PM), 34 responded with a gradual membrane depolarization (l-15 mV) that peaked in 2.2 t 0.4 (SD) min and was accompanied by a 17.6 t 4.8% reduction of input resistance. Responses persisted (and were actually enhanced) in media containing tetrodotoxin (0.5-1.0 PM) and/or nominally zero calcium, indicating a direct postsynaptic action. In 19 responsive cells, the mean reversal potential for the angiotensin-induced response was -26.4 t 2 mV. Bath application of the nonpeptide type- 1 angiotensin receptor antagonist DuP753 (5-20 PM) reversibly blocked the angiotensin-induced depolarization in all of 11 cells tested. By contrast, equimolar applications of the type-2 antagonist PD123177 were ineffective in all seven angiotensin-responsive cells tested. These observations provide novel evidence for the existence of functional type-l receptors on rat supraoptic nucleus neurons. The reversal potential for the angiotensin-induced response suggests mediation through a nonselective cationic conductance. angiotensin; receptors; electrophysiology; supraoptic nucleus
0363-6119/92
depolarizes
0 1992
Ontario
Kl Y 4E9, Canada
II antagonists permits differentiation between two types of angiotensin receptors in the adrenal gland (3). In the rat brain, at least two types of binding sites for angiotensin are demonstrated (7, 28, 33), and the hypothalamic magnocellular neurons have one of these subtypes (11, 33). However, no intracellular electrophysiological correlate of angiotensin on magnocellular neurosecretory neurons has been reported. In the present study, we used intracellular recordings obtained from neurons of rat supraoptic nucleus (SON) maintained in superfused hypothalamic explants to evaluate transmembrane events and receptor types involved in responses to exogenously applied angiotensin II. The data indicate that a portion of the vasopressinand oxytocin-secreting neurons in the rat supraoptic nucleus appear to contain type-l angiotensin receptors whose activation induces membrane depolarization and an increase in membrane conductance, possibly mediated through nonselective cationic channels. A preliminary report has been presented (24). METHODS Experiments used acutely prepared hypothalamic explants of adult (200-250 g) male Long-Evans rats (35). Within 4 min of decapitation, the brain was removed and a horizontal explant of basal forebrain containing the supraoptic nucleus was dissected and pinned to the Sylgard base of a temperature-regulated (33°C) chamber. Each explant was superfused with oxygenated (95% 02-5% CO,) artificial cerebral spinal fluid (aCSF) delivered by gravity at a rate of 0.9-1.3 ml/min. The standard aCSF (pH 7.4) contained (in mM) 126 NaCl, 3 KCl, 1.3 MgC12, 25.9 NaHCO,, 2.4 CaCl,, and 10 glucose and had a freezing point osmolality of 290-295 mosmol/kg. Modifications for different experiments are specified in the text. Levels of aCSF were adjusted with cotton wicks to minimize electrode capacitance. Recordings commenced after 3 h of equilibration. Both [Ile”] ANG II and [Val”]ANG II (Peninsula, Belmont, CA) were shown to be effective in exciting rat neurons in previous electrophysiological studies (2, 9, 20, 21) and thus they were used as ANG II agonists in the present study. Nonpeptide antagonists were DuP753 (Losartan, 2-n-butyl-4-chloro-5hydroxymethyl-l-[2’-(l~-tetrazole-5-yl)biphenyl-4-y methyl] imidazole, potassium salt; Refs. 3, 15, 19, 29) and PD123177 (hydrochloride salt of EXP655, l-(4-amino-3-methylphenyl) methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-lH-imidazole[4,5-Clpyridine-6-carboxylic acid; Refs. 3, 15, 33; both are gifts from Du Pont). Both the agonists and the antagonists were prepared as concentrated stock in aCSF and stored in siliconecoated (Sigmacote, Sigma) vials at -25°C until use, when appropriate dilutions were achieved. All syringes used to deliver ANG II were also silicone-coated. Drugs were either bath applied or injected as a lo- to 40-~1 bolus over 2-4 s into the superfusion line proximal to the explant by a timer-controlled pump. As described elsewhere (35), the final concentration of the drug reaching the supraoptic nucleus was an estimate based on the volume and concentration of the injected drug and the volume of aCSF overlying the nucleus. The values quoted in the The
American
Physiological
Society
R1333
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R1334
ANGIOTENSIN
DEPOLARIZES
text refer to the transient peak concentration; effective concentrations reaching a cell are likely to be much lower because of diffusion and metabolism within the tissue. Intracellular recordings were obtained through micropipettes containing a mixture of potassium acetate (3-4 M) and potassium chloride (0.05 M) at pH 7.0. Signals were processed with an Axoclamp-2A amplifier. A Ag-AgCl electrode connected to the bath solution via a KCl-agar bridge served as reference. Bridge balance was maintained throughout the experiments by eliminating the time-independent component of the voltage response occurring at the onset and offset of rectangular current pulses applied through the recording electrode. Resting membrane potentials were recorded as the difference between the potential inside the cell and that in the extracellular space. Input resistance was determined from the slope of voltage-current (V-I) plots obtained from membrane voltage deflections after delivery of a series of 100-ms depolarizing and hyperpolarizing current pulses. Amplified signals were digitized by a Neuro-Data (DR-384) pulse-code modulator, sampled on-line using pCLAMP software (Axon Instruments), and recorded on video tapes for analysis off-line. In experiments using nominally calcium-free media, the effectiveness of calcium removal was assessed by the disappearance of a [Ca”+]-dependent postburst afterhyperpolarization (AHP) evoked by a l-s depolarizing current pulse (26). Group data are expressed as means t SD and all pairwise comparisons of the group data were made using the Student’s t test. RESULTS
The data base was derived from stable intracellular recordings in 68 supraoptic neurons whose resting membrane potentials ranged between -55 and -63 mV and whose action potentials exceeded 70 mV and input resistances ranged between 75 and 300 MO. All cells demonstrated two features typical of recordings from supraoptic neurons, i.e., activity-dependent action potential broadening and the presence of an “A’‘-notch, signifying a transient outward K+-current when depolarized from holding potentials more negative than -70 mV (26) (Fig. 1). Of the 68 cells tested, 34 cells (50%) demonstrated membrane depolarization (range l-15 mV) after bolus infusions of either Ile” or VaPANG II at transient maximum concentrations estimated between 10 and 25 PM. A typical response, illustrated in Fig. 1, was a gradual membrane depolarization that peaked in 2.2 t 0.4 min and subsided after 5-7 min. Successive responses to ANG II could be reproduced provided that consecutive applications were at least 10 min apart in the case of small (53 mV) depolarizations or at least 30 min apart in the case of larger depolarizations.
RAT
SUPRAOPTIC
CELLS
Tests applied at shorter intervals yielded responses of diminished magnitude. There was virtually no difference in the magnitude of the membrane depolarization or conductance change among cells responding to either VaPANG II or Ile5ANG II; therefore the data on these parameters in this group were pooled. However, sequential testing of both peptide analogues on 27 cells revealed apparent differences in responsivity: four responded to both peptides with membrane depolarization, five responded to only one of the two peptides, and 18 showed no response to either peptide. Five cells were tested by bath application of ANG II. The lowest effective concentration of ANG II to induce a depolarization of 3 mV was 1 pM. ANG II-responsive neurons included five neurons whose spontaneous activity demonstrated clear phasic discharges. This firing pattern is frequently associated with magnocellular neurosecretory neurons that are immunoreactive to vasopressin (4, 34). After ANG II applications, all cells demonstrated prolongation in the duration of each phasic discharge. In another six cells, whose spontaneous firing appeared to be of a continuous nature and might therefore be either vasopressin or oxytocin secreting (26), exposure to ANG II produced an increase in firing without a change in pattern. ANG II did not transform continuous firing neurons into phasic firing cells. Cells in both categories were among the neurons not responding to ANG II (Fig. 2). Measurements derived from the slope of V-I plots indicated that a mean decrease of 17.6 t 4.8% in membrane resistance accompanied the ANG II-induced depolarization. The mean reversal potential for the ANG II-induced response in 19 cells was -26.4 t 2 mV, as extrapolated from the intercept of the linear range (-60 to -120 mV) of the V-I plots obtained in normal aCSF at resting membrane potentials and during drug-induced membrane depolarizations (Fig. 2A). In five cells tested in media containing tetrodotoxin (TTX, 1.0 ,uM), the ANG II-induced depolarizations and reduction of input resistance persisted, with a mean reversal potential of -30 t 2 mV for the peptide-induced response that differed little from the control. However, in two of these five neurons, marked increases in the ANG II-induced membrane depolarization were observed, and they were associated with a greater reduction in their slope resistance from 12 and 15% in control media to 35 and 38% in TTX, respectively. However, their reversal
ANGII -
Fig. 1. Top trace: intracellular current clamp recording from a supraoptic neuron (resting membrane potential -62 mV) after a brief bolus infusion (bar) of Ile5ANG II into the perfusion media sufficient to attain a transient maximum concentration of 10 PM. Note gradual onset of prolonged membrane depolarization. Full spike amplitude is attenuated by chart recorder. Downward &flec-
~IIIIIMI~~
I 0.1 nA 2 min
current pulses (100 ms, -0.05 nA, bottom trace) delivered to monitor input resistance; these are reduced by 22% during the drug-induced depolarization.
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ANGIOTENSIN
DEPOLARIZES
RAT
SUPRAOPTIC
R1335
CELLS
Fig. 2. Left: extrapolations of the linear range of voltage-current (V-I) plots (-120 to -60 mV) obtained from supraoptic neurons (SON) under control conditions (open symbols) and during membrane depolarizations follow a bolus infusion of Va15ANG II (maximum peak concentration, 10 PM; closed symbols) showed a reversal potential of -27 mV from the ANG II-induced depolarization. ANG II depolarized this cell by 7 mV and induced a reduction of slope resistance of 8%. Right: V-I plots from another neuron recorded in control artificial cerebrospinal fluid (aCSF) containing tetrodotoxin (1 PM) before (open symbols) and after (closed symbols) bolus infusion of Va15ANG II (maximum concentration, 10 PM) reveals a large drug-induced membrane depolarization (25 mV) and a 38% reduction of slope resistance, with a virtually identical reversal potential (-28 mV). Suppression of sodium spikes by tetrodotoxin permitted voltage readings in the depolarized range. Regression lines through linear range are fitted by eye.
potentials remained similar to those obtained in control media (Fig. 2B). In five cells tested in control aCSF where the mean ANG II-induced membrane depolarization was small (1.5 t 0.2 mV), the same ANG II test in media containing nominally zero [Ca2+10 and 6 mM Mg2+/Mn2+ induced a significantly greater depolarization (10.6 t 1.1 mV) (Figs. 3 and 4). Reversal potentials remained at -30 mV (not illustrated). Two other patterns of response were detected in a minority of instances. Two neurons displayed a weak membrane hyperpolarization with no consistent change in membrane resistance when tested with Ile5ANG II. In three other cells, ANG II applications were associated with an increase in spontaneous inhibitory postsynaptic potentials (IPSPs) blocked in each instance by bath application of 25-50 PM bicuculline (Fig. 5). Evidently there are y-aminobutyric acid-ergic inputs to supraoptic neurons that are activated by ANG II. To address the type of receptor that might mediate these ANG II-induced responses in supraoptic neurons, cells were tested in the presence of nonpeptide angio-
A
CONTROL
ANG - II
tensin antagonists recently reported to distinguish between two types of ANG II receptors in the adrenal gland and in the brain (3, 15, 19, 29). In 11 cells responding to ANG II, bath application of the nonpeptide ANG II type-l antagonist DuP753 (5-20 PM) had no appreciable influence on membrane potential or spontaneous firing. For this reason we chose to use this concentration range of antagonist. The ANG II-induced response in all 11 cells was blocked during DuP753 application, with partial recovery of the ANG II response evident some 15-20 min after return to control media (Fig. 6). We did not seek the lowest effective antagonist concentration. Figure 7 summarizes the actions of DuP753 on their ANG II-induced depolarizations. In three neurons in which bath-applied DuP753 (5 FM) was evaluated for its specificity of action, in each instance membrane depolarizations evoked by ANG II were reversibly attenuated, whereas those evoked by bolus application of glutamate (50 PM) were unaffected (data not illustrated). Bath application of the ANG II type-2 receptor antagonist PDl23177 (5-20 PM) induced small changes in
B
I 2s
5 mV
I(2nA
ANG II -
O[Ca++]. + 6 mM Mn”
0.5 mill
Fig. 3. Removal of extracellular calcium enhances action of angiotensin. A: application of IlesANG II (solid bar; final concentration, 20 PM) induces no apparent membrane response in normal aCSF (control). B: left, in normal aCSF, a [ Ca2+],-dependent postburst after hyperpolarization (arrow; AHP) is elicited by intracellular injection of a depolarizing pulse (1 s, 0.1 nA); right, 15 minutes after application of zero [Ca2+], media containing 6 mM Mg2+/Mn2+, the same intracellular injection of depolarizing pulse no longer evoked an AHP indicating effective removal of [Ca2+10. C: in the absence of extracellular [Ca2+],,, reapplication of the ANG II onto this previously unresponsive cell induced a prolonged membrane depolarization and action potential discharge. Calibration bars in C also apply to A.
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ANGIOTENSIN
DEPOLARIZES
RAT
SUPRAOPTIC
CELLS
CONTROL
(n=5) *
ACSF
ANG II
o[ca++](-j
Fig. 4. Histogram of data from 5 neurons demonstrating < 0.001) increase of the net depolarization by ANG [Ca2+10. Values are means t SD.
a significant II in absence
(P of
membrane potential in both depolarized and hyperpolarized directions (2 mV) with no detectable change in input resistance. PD123177 did not alter the response to ANG II in any of the seven cells tested. DISCUSSION
The present study provides novel intracellular data to indicate that rat supraoptic nucleus neurosecretory neurons contain functional angiotensin AT1 receptors whose activation results in membrane depolarization. These results are consistent with previous reports of a predominantly excitatory action from iontophoretically applied ANG II on rat and cat supraoptic neurons (20,21). Moreover, if (in the majority of instances) the firing patterns of these cells are a true reflection of their ability to synthesize either vasopressin or oxytocin (4, 34), then the present data would indicate that a population of both cell types are likely to contain type-l ANG II receptors (12). As noted earlier, not all cells responded to both forms of ANG II used in this study. This may reflect further heterogeneity of the ANG II receptor subtypes. Although the ionic mechanism mediating the excitatory actions of ANG II on supraoptic neurons remains to be detailed, a linear V-I relationship and a mean reversal potential of -26 mV for the ANG II response suggests activation of a voltage-independent nonselective cationic conductance. This is similar to their response to dopamine (35) and hypertonic media (1) and bears a close resemblance to the cationic conductance that has been demonstrated in invertebrate bursting neurons and glandular secretory cells (22). ANG II also induces membrane depolarization in hippocampal (9) and sympathetic ganglia neurons (2), although the ionic basis of these responses remains to be defined. A prerequisite for the activation of nonselective cationic conductance is the mobilization of intracellular calcium (22). Stimulation of ANG II receptors on a variety of cell types will promote hydrolysis of inositol triphosphate, and a concomitant rise in intracellular calcium (14, 17, 19, 29). While such mobilization of cytosolic [Ca2+] may itself activate a calcium-dependent nonselective cation conductance, the details surrounding the intracellular messengers induced by ANG II in central neurons remain controversial (31). The depolarizing response to ANG II suggests a direct activation of ANG II receptors on SON neurons, since this response persists after synaptic blockade through removal of extracellular TCa2+l or addition of TTX. How-
ANG II + BMI
4J
10 mV 0.5 min
Fig. 5. Top: 5 traces of control baseline voltage recordings from a supraoptic neuron (3 M KAc-filled micropipette) held at -60 mV with current injection (-0.01 nA) display transient membrane fluctuations, some of which are presumably inhibitory postsynaptic potentials. A&& dZe: traces obtained during application of Va15ANG II (10 PM) reveal a prominent increase in magnitude and frequency of inhibitory postsynaptic potentials (IPSPs). IPSPs did not summate so there was no apparent membrane hyperpolarization. Bottom: when bicuculline methiodide (BMI, 25-50 PM) was applied to bath simultaneously with another application of angiotensin, the barrage of IPSPs was no longer evident. These features suggest that ANG II is activating a GABAergic input.
ever, it is intriguing that the ANG II-induced depolarization is significantly potentiated when extracellular [ Ca2+] is removed. This may imply that influx of extracellular [ Ca2+] may negatively regulate the functional properties of ANG II receptors. In neuronal culture the number of functional binding sites for ANG II is substantially reduced in the presence of the [Ca2+] ionophore A23187 alone, but not in [Ca2+] -free medium (30). Whether the time course of this development is comparable with our findings remains to be determined. It appears that the influx of extracellular [Ca2+], but not mobilization of intracellular [Ca”+], has a negative influence on ANG II receptor functions. The potentiation of the ANG II-induced response in the absence of extracellular [Ca”+] may also be due to inactivation of [Ca”+] -dependent peptidases that catabolize ANG II (5, 10). The routine use of divalent cation chelating agents such as EDTA or ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) and neptidase inhibitors (e.g. bacitracin)
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ANGIOTENSIN
ANG
DEPOLARIZES
II
RAT
SUPRAOPTIC
Fig. 6. ANG II-evoked responses are attenuated by the nonpeptide AT, antagonist DuP753. Top: typical delayed membrane depolarization after bolus application (bar) of Ile5ANG II (20 PM), recorded from a spontaneously discharging supraoptic neuron. In this instance, ANG II-induced membrane depolarization and associated discharge of action potentials is followed by a prolonged membrane hyperpolarization. Middle: bath application of DuP753 (5 FM) had little influence on spontaneous firing but markedly attenuated ANG II-induced response. Bottom: 15 min after termination of DuP753 application (dashes), a partial recovery of ANG II-induced response was achieved.
5 ClM. DuP753
ANG
II
I ANG
(n=ll)
II
20 mV
1 min
II
to lengthen the biological half-life of the peptides in neuropeptide-binding studies support this interpretation. The introduction of nonpeptide antagonists has served to define at least two types of angiotensin receptors in the adrenal gland where DuP753 has a preference for receptors in the adrenal cortex (now designated ATI), whereas the structurally dissimilar PD 123 177 (formerly EXP655) binds preferentially to the ANG II receptors in the adrenal medulla (now designated ATB) (3). Binding studies have identified both types of receptors in brain, each with a distinct regional distribution (7, 15, 28, 33). Of particular interest is the selective displacement of 1251-labeled ANG II binding in the area of rat supraoptic nucleus by the AT1 antagonist DuP753 (33). The present electrophysiological findings provide the functional correlate for the existence of AT1 ANG II receptors on supraoptic neurons. What are possible sources of the ligand for these angiotensin receptors ? An endogenous origin for angiotensin II is suggested by various reports of angiotensinlike immunoreactivity in the magnocellular neurons and
ANG
RI337
CELLS
ANG
II
+DuP753 (5-20pM)
Fig. 7. Histogram summarizes the significant (P < 0.001) attenuation ANG II-induced depolarization in 11 neurons during bath-applied DuP753 (5-20 PM). Values are means t SD, * P < 0.001.
of
supraopticoneurohypophysial pathway (16, 26). One might speculate that ANG II is released locally from axon collaterals or soma-dendritic sites in a manner similar to that proposed for oxytocin or vasopressin (27). An exogenous origin is the angiotensin immunoreactive afferents to supraoptic neurons. Some of these can be traced to the subfornical organ (SFO) and the organum vasculosum of the lamina terminalis, two circumventricular structures (12, 16). Both of these areas are also sites of intense angiotensin binding (7, 18, 25, 33) and therefore may be important interface zones whereby circulating hormones (e.g., angiotensin) influence the central nervous system. In the rat, evidence for a transmitter role for angiotensin in the efferent pathways from SF0 was obtained from in vivo observations whereby the increase in excitability of vasopressin- and oxytocin-secreting magnocellular neurons is reversibly attenuated in the presence of a peptide angiotensin antagonist, saralasin (12). The SF0 is a key structure in mediating the response of magnocellular neurons to circulating angiotensin (6) and its neurons demonstrate a sensitivity to circulating angiotensin (8). Hence this circumventricular organ contains neurons which may both receive a peripheral (i.e., circulating) message mediated by angiotensin and, in turn, use angiotensin to convey the message at central levels to activate a number of neurons, including the magnocellular neurosecretory cells. We thank Dr. Margaret Sullivan for her critical reading of the manuscript. This study was supported by the Canadian Medical Research Council and Canadian Heart and Stroke Foundation. Present address of M. I. Phillips: Dept. of Physiology, Univ. of Florida College of Medicine, JHMHC, Box J-274, Gainsville, FL 32610. Address for reprint requests: L. Renaud, Neurology/Neurosciences, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada KlY 4E9. Received
13 January
1992; accepted
in final
form
19 May
1992.
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R1338
ANGIOTENSIN
DEPOLARIZES
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