Angiotensin-sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III

130

Brain Research, 410 (1987) 130-134 Elsevier

BRE 22223

Angiotensin-sensitive neurons in the rat paraventricular nucleus: relative potencies of angiotensin II and angiotensin III Joseph W. Harding and Dominik Felix Division of Animal Physiology, Bern (Switzerland)

(Accepted 20 January 1987) Key words: Angiotensin II; Angiotensin III; Brain; Electrophysiology; Iontophoresis; Spontaneously hypertensive rat

Angiotensin-activated neurons were examined using microiontophoretic methods in the paraventricular nucleus (PNV) of the rat. In all cases angiotensin III (AIII) was more potent than angiotensin II (All). This greater sensitivity to AIII was manifested by lower thresholds, shorter latencies, and higher spike frequencies/amplitudes of applied current. The superior potency of AIII was further exaggerated in the spontaneously hypertensive rat (SHR) compared with normotensive Wistar Kyoto (WKY) rats. Postactivity for both AII and AIII was greatly prolonged in SHR. This appeared specific since no prolongation in acetylcholine postactivity was seen in SHR. These data support the notion that AIII may be the centrally active form of angiotensin and are consistent with an obligatory conversion of AII to AIII prior to activation. The selective enhancement of postactivity observed in SHR following angiotensin application suggests a possible defect in signal termination.

The brain angiotensin system, which plays a prominent role in body fluid homeostasis and cardiovascular regulation, has become one of the most completely characterized neuropeptide systems 9,12,13. This characterization has included the identification of appropriate substrates, enzymes, and receptors, the tracing of angiotensinergic pathways, and the correlation of angiotensin-dependent function with brain angiotensin 5. In most instances studies have proceeded with the belief that the ultimate angiotensin effector in the brain is A I I . However, the results of a number of recent investigations, including this one, are more consistent with the idea that A I I I , and not A I I , may represent the active form of brain angiotensin 1,2.4,7,10,11,14,15. The major purpose of the present study was to compare the response characteristics of individual rat PVN neurons to iontophoretically applied A I I and A I I I in normotensive and hypertensive animals and test the hypothesis that A I I I represents the centrally active form of angiotensin. The experiments were performed on 36 neurons

from the PVN of adult S H R and W K Y rats. Four female S H R and 4 female W K Y rats 4 - 6 months old were used in this study. The blood pressure recorded was 191 + 6 mm Hg for S H R and 119 _+ 4 mm Hg for W K Y rats. They were anesthetized with i.p. doses of thiopentane sodium (50 mg/kg). The animals were mounted in a stereotaxic flame and the cerebral cortex was exposed. Body temperature was continuously monitored and maintained at 37 °C with a heating pad. Extracellular action potentials were recorded through the 2 M NaCl-containing central barrel of a 5-barrel glass micropipette, tip diameter approximately 4/xm. The firing rate of each neuron was counted by a ratemeter (Nuclear Enterprises N E 4667) and displayed continuously on a UV-oscillograph (Bell and Howell 5-137). The outer channels of the micropipettes contained the compounds to be ejected microiontophoretically: (1) acetylcholine, 0.5 M, p H 3.0-3.5, (2) [IleS]-AII, prepared as a 10 -3 M solution in distilled water, final p H 3.5, (3) [IleS] desAsp-AII ( A I I I ) , prepared as a 10 -3 M solution in distilled water, final p H 4.5, and (4) [Sarl,IleS]-AII,

Correspondence: J.W. Harding. Present address: Dept. of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164, U.S.A.

131 prepared as a 10-3 M solution in distilled water, final p H 4.5. All peptides were applied with cationic currents typically for 1 min intervals. Compensation current was used to prevent any direct current effects. The recording electrode solution also contained Fast green FCF which was iontophoretically deposited subsequent to recording and used to mark the electrode site. The position of electrode placement was histologically determined after each experiment. These examinations indicated that electrode placement in successful experiments (ones in which angiotensin-sensitive cells were found) was generally in the area of the PVN. Angiotensin-sensitive cells were found both in the parvocellular and magnocellular portion of the PVN. N o distinction could be seen in the response characteristics of cells in these two regions. Since the purpose of this study was to directly compare the relative potencies of iontophoretically applied A I I and A I I I , it was first necessary to determine the actual rate of microiontophoretic release of A I I and A I I I for a given amount of ejection current. Preliminary in vitro experiments established these

A

ACh6OnA

relationships by adding [3H]AII and [3H]AIII (Amersham) to pipettes containing A I I and A I I I and measuring the ejection of this preparation into tubes containing Ringer's solution. These experiments demonstrated that the release of both A l l and A I I I was linear with respect to the amount of ejecting current up to at least 60 nA. In addition, the actual rate of release for A l l and A I I I from 6 different micropipettes was compared, thus allowing us to directly relate the quantity of ejection current to the amount of each peptide released. Although considerable interpipette variability was found, the mean rate of A l l and A I I I release was very similar (AII: 41.5 + 4.8 fmol/nA/min; A I I I : 45.1 + 8.0 fmol/nA/min; mean + S.E.M., n = 6). A detailed description of this quantitative comparison of A l l and A I I I iontophoresis can be found elsewhere 6. Because of the above-mentioned interpipette variability, experiments done on hypertensive animals had to be carried out in the same manner using the same electrode. Only those experiments that included successful recordings from both S H R and W K Y animals are reported. Thirty-six angiotensin-sensitive neurons were de-

t3

ANGE12nA

ANG'n'12nA

40

50

60

20

1 min

C

40 -

15 .........

40 I

S A R I . ILE 8 - ANGll"

I

........ 100

Fig. 1. Response of rat paraventricular neurons to iontophoreticalty applied angiotensin II (All), angiotensin III (AIII), the competitive antagonist [Sarl,Iles]-AII, and acetylcholine (ACh). Experimental details are described in the text. The frequency (J) of response is expressed as spikes/s. A: this cell responded to ACh, AII, and AIII. An ejection of 12 nA of AIII produced a robust response while 12 nA of All yielded no response. However, 40 nA of AII exhibited a significant response which was characterized by a long latency. B: in this cell 60 nA of AII and 20 nA of AIII produced similar responses. The latency for AIII is clearly shorter than for AIII. C: this cell responded to All, AIII, and ACh. The antagonist [Sart,Iles]-AII transiently blocked the response to both AII and AIII while having no effect on ACh responsiveness. This blockade was totally reversible.

132 tected in the p a r a v e n t r i c u l a r nucleus of the rat. These neurons, which were activated by angiotensin, were universally m o r e sensitive to A I I I than to A I I . This o b s e r v a t i o n is illustrated b e l o w w h e r e either the same quantity of p e p t i d e p r o d u c e d variable responses (Fig. 1A) or different quantities p r o d u c e d similar responses (Fig. 1B). T h e specific antagonist, [Sar 1,IleS]-AII, was c a p a b l e o f blocking the response of both A I I and A I I I (Fig. 1C). A s seen in T a b l e I, the e n h a n c e d sensitivity to A I I I is manifested by several response characteristics including: a lower threshold, a higher m a x i m u m res p o n s e / n A of applied current, and a s h o r t e r latency (also Fig. 1B). T h e r e s p o n s e threshold for A I I I , which was always lower than for A I I , a v e r a g e d a b o u t 40% of the A I I value. B o t h S H R and W K Y rats exhibited the same t h r e s h o l d differences. The s u p e r i o r p o t e n c y of A I I I was also evident when a second p a r a m e t e r , the m a x i m u m response/ n A of a p p l i e d current ( m R / n A ) , was c o m p a r e d . The potential p r o b l e m s o f r e c e p t o r saturation or the response frequency limitations of the cell influencing the value of m R / n A were circumvented by p e r f o r m ing the calculation at several levels of current application. In no case were significantly different values d e t e r m i n e d for high and low current application. D r a m a t i c differences in the m R / n A were seen for A I I and A I I I with A I I I ' s m R / n A being higher (Table I). T h e difference in p o t e n c y b e t w e e n A I I and A I I I was even clearer when cell to cell and e l e c t r o d e variations were eliminated. Since all cells were tested with both A I I and A I I I in the same e l e c t r o d e , a ratio

of m R / n A for A I I I / A I I could be calculated. W h e n this was done A I I I a p p e a r e d to be 3.3 times m o r e potent than A I I in W K Y rats and 5.6 times m o r e p o t e n t in S H R , a difference that was significantly different (Table I). A final m e a s u r e of responsiveness was the postactivity that occurs after the injecting current is terminated. Following the application of angiotensins, dramatic differences in postactivity were a p p a r e n t in S H R and W K Y rats. B o t h A I I and A I I I y i e l d e d nearly 5 times the postactivity in S H R as in W K Y (Table I). H o w e v e r , no difference in postactivity was evident in S H R or W K Y rats in response to acetylcholine. These d a t a indicate that A I I I is much m o r e effective at stimulating P V N neurons than A I I and strongly support the hypothesis that A I I I is the centrally active form of angiotensin. W e e x a m i n e d several (5 o r less) angiotensin-sensitive cells in the subfornical organ, lateral s e p t u m , and nucleus tractus solitarius, in addition to the PVN. In each case these cells exhibited a quantitatively similar difference in A I I I / A I I responsiveness, as witnessed in the P V N , suggesting that the o b s e r v e d difference may be universal t h r o u g h o u t the brain. A closer e x a m i n a t i o n and c o m p a r i s o n of the response characteristics of P V N neurons to A l l and A I I I reveal two additional points that could help to clarify the relationship of A l l , A I I I , and central angiotensin receptors. First, the latency of the A I I I responses was consistently s h o r t e r than that of A I I , even where the m a x i m u m responses were equal. This

TABLE I Response characteristics o f neurons o f the rat paraventricular nucleus to iontophoretically applied substances

ND, not determined. AH WKY

Threshold(nA)* Max. response/nA** (spike/s/nA) AIII/AII+'~ Latency++ Postactivity+++(s)

All1 SHR

28.0 + 1.1 (20) §

31.5 + 1.4 (20)

0.19 + 0.03 (17)

0.12 + 0.01 (17) 27.27 +-0.8 (22) 51.0+_3.1 (20)

20.6+ 1.1 (19) 13.25+0.9 (17)

ACh

WKY

SHR

12.4 + 1.3 (24) 0.51 + 0.05 3.3 __+0.5 9.1 +0.6 11.9+0.5

11.4_+1.0 (26)

(17) 0.55 + 0.05 (13) 5.6 + 0.4 (22) 13.45 +_0.7 (20) 51.8+__1.9

(18) (14) (20) (20)

WKY

SHR

ND

ND

ND 25.2 + 0.7(12) 28.8+-1.6(9)

ND 32.3 + 2.0 27.3+0.1

* Threshold - - AIII << AII, P < 0.01; SHR - WKY, P > 0.05; ** Max. response/nA - - AIII >> AII, P < 0.01; SHR --- WKY P > 0.05; ++ Latency - - AIII << AII, P < 0.01; SHR = WKY, P < 0.05; § All data expressed as mean + S.E.M.; ~ AIII/AII was calculated by dividing the mR/hA for AIII by the nR/nA for AII for each cell; + AIIUAII SHR > WKY, P < 0.05; +++ Postactivity AIII = A I I , P > 0.05; SHR >> WKY, P < 0.01; for ACh SHR = WKY, P > 0.05.

133 p h e n o m e n o n may occur simply because A I I has a lower affinity for the central receptor than A I I I 1'2 and as such, more peptide is needed to produce an electrical response, or it may indicate that an obligatory conversion of A I I to A I I I is required prior to receptor binding and neuronal activation. Recent studies from our laboratory appear to support the second explanation (Wright et al., unpublished). These studies have shown that the aminopeptidase B inhibitor bestatin, which can block the degradation of A I I I , is a potent dipsogenic agent alone as well as a potentiator for the dipsogenic effects of A I I and A I I I . Furthermore, the dipsogenic effects of bestatin are totally blockable using the specific angiotensin antagonist, [Sar 1,Thrs]-AII. On the other hand, the aminopeptidase A inhibitor amastatin, which blocks the conversion of A l l to A I I I , has little or no dipsogenic activity alone and depresses the maximum pressor response to intracerebroventricularly (i.c.v.) applied AII. A second interesting finding is that both A I I and A I I I responses are characterized by nearly identical postactivities for both S H R and W K Y rats. These data suggest that both A I I and A I I I have a c o m m o n active form whose rate of inactivation determines postactivity. Although in general both S H R and W K Y rats exhibited similar response characteristics to A l l and A I I I , two distinct differences could be noted. The most dramatic difference was the slow cessation of angiotensin-induced activation in SH rats after withdrawal of the ejection current of either A l l or A I I I . This greatly extended postactivity could not be attributed to a general change in passive properties of

the neuronal membrane since postactivity in response to acetylcholine in the same angiotensin-sensitive cells was not enhanced in SHR. These data are similar to an observation made by Felix and Schelling using SHR-stroke prone (SHR-sp) rats, where septal neurons exhibited increased postactivity following A l l microiontophoretic application 3. Of additional interest is the observation that the relative sensitivity to A I I I , as expressed by the A I I I / A I I ratio, increased in SHR. In summary, the results of this study highlight the probable importance of A I I I to the brain angiotensin system by demonstrating the superior potency of iontophoretically applied A I I I as compared with A I I . Furthermore, these findings suggest than an obligatory conversion of A l l to A I I I is required prior to activation. Finally, the dramatic difference in postactivity witnessed in S H R following both A l l and A I I I application is consistent .with other data that indicate that this hypertensive strain suffers from a defect in angiotensin signal termination.

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iontophoresis, J. Neurosci. Meth., 19 (1987)209-215. 7 Harding, J.W., Stone, L.P. and Wright, J.W., The distribution of angiotensin II binding sites in rodent brain, Brain Research, 205 (1981) 265-274. 8 Hicks, T.P., The history and development of microiontophoresis in experimental neurobiology, Prog. Neurobiol., 22 (1984) 185-240. 9 Johnson, A.K., Neurobiology of the periventricular tissue surrounding the anteroventral third ventricle (AV3V) and its role in behavior, fluid balance and cardiovascular control. In Smith, Galosy and Weiss (Eds.), Circulation, Neurobiology, and Behavior, Elsevier, Amsterdam, 1982, pp. 277-295. 10 Petersen, E.P., Camara, C.G., Abhold, R.H., Wright, J.W. and Harding, J.W., Characterization of angiotensin binding in the gerbil brain using [125I]-angiotensinIII as the radioligand, Brain Research, 321 (1984) 225-325. 11 Petersen, E.P., Camara, C.G., Abhold, R.H., Wright,

This work was supported by Grants T W 01112 and H L 32063 from the N I H and Grant 831145 from the American Heart Association and its Washington affiliate to J . W . H . , as well as Grant 3.626-0.84 from the Swiss National Science Foundation and assistance from the Stiftung zur F f r d e r u n g der wissenschaftlichen Forschung an der Universit/it Bern to D.F. We would especially like to thank Y. Liu for technical assistance and J. Conger for preparing the manuscript.

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F533-F543. 14 Wright, J.W., Morseth, S.L., Abhold, R.H. and Harding, J.W., Comparisons of angiotensin II- and Ill-induced pressor action and dipsogenicity in rats, Am. J. Physiol., 249 (1985) R514-R521. 15 Wright, J.W., Sullivan, M.J., Petersen, E.P. and Harding, J.W., Brain angiotensin II and III dipsogenicity in the rabbit, Brain Research, 358 (1985) 376-379.