- Email: [email protected]
Haemodynamic and humoral effects of ω-conotoxin GVIA in normotensive and spontaneously hypertensive rats
European Journal of Pharmacology, 211 (1992) 329-335
2
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52291
Haemodynamic and humoral effects of ¢o-conotoxin GVIA in normotensive and spontaneously hypertensive rats Didier Pruneau and Pierre B~lichard Centre de Recherche, Laboratoires Fournier S.C.A., 50 Rue de Dijon, 21121-FontaineLes Dijon, France
Received 29 October 1991, revised MS received 19 November 1991, accepted 26 November 1991
oJ-Conotoxin GVIA, a 27-amino acid peptide, has been shown to be a potent and selective inhibitor of N-type voltage-op ated calcium channels (VOCCs). A single intravenous dose of 10/~g/kg conotoxin slowly lowered blood pressure by 41.3 +, and 73.3 + 4.6 mm Hg in conscious Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) respectively with~ changing the heart rate. Plasma renin activity was significantly increased after conotoxin. In anaesthetized rats, conotoxin (3 a 10/~g/kg) lowered blood pressure and heart rate and produced a marked increase in renal vascular conductances. Barorecep heart rate reflex experiments using methoxamine and sodium nitroprusside before and after treatment with conotoxin show that conotoxin almost totally abolished the sympathetic component of the reflex without affecting the vagal tone to the heart both rat strains. Because conotoxin does not affect directly the vasculature and heart contractile properties, we suggest that 1 control of presynaptic calcium influx and of neurotransmitter release mostly depends on conotoxin-sensitive N-type VOCCs the peripheral sympathetic system of the rat. ~o-Conotoxin; Sympathetic nerves; Hypertension; Vasodilatation
1. Introduction The release of neurotransmitter is triggered by an influx of calcium ions in nerve terminals through voltage-operated calcium channels (VOCCs). Although both L- and N-type VOCCs coexist in a number of neuronal preparations (Nowycky et al., 1985; Miller, 1987; Tsien et al., 1988; Bean, 1989), N-type channels seem to play a dominant role in depolarisation-coupled noradrenaline release (Hirning et al., 1988; Lipscombe et al., 1989). w-Conotoxin G V I A (CTX), a 27-amino acid peptide isolated from Conus geographus venom, was shown to bind to neuronal VOCCs with an affinity in the sub-picomolar range (Barhanin et al., 1988; Hayakawa et al., 1990). Thus CTX selectively inhibits Ca 2+ influx in dorsal ganglion, sensory, sympathetic and hippocampal neurons of vertebrates without affecting Ca 2+ movements in smooth, skeletal and cardiac muscle (McCleskey et al., 1987). In accordance with these data we have recently shown that CTX produced a marked inhibition of sympathetic neurotransmission in rat small mesenteric arteries (Pruneau
Correspondence to: D. Pruneau, Centre de Recherche, Laboratoires Fournier S.C.A., 50 Rue de Dijon, 21121-Fontaine les Dijon, France. Tel. 33.80.44.75.39, fax 33.80.56.56.64.
and Angus, 1990a) and in the pithed rat (Pruneau a Angus, 1990b). In the conscious rabbit intravenc (i.v.) administration of C T X lowered blood presst and blocked the sympathetic component of the baro~ ceptor-heart rate reflex (Pruneau and Angus, 1990 We concluded that CTX-sensitive channels play a n jot role in controlling the sympathetic tone towards t vasculature and the heart. Since increased sympathe nervous activity has been proposed, among other fi tots, to explain the increased total peripheral res tance observed in spontaneously hypertensive n (SHR) (Okamoto et al., 1967; Touw et al., 191 Lokhandwala and Eikenburg, 1983; Lee et al., 198 we now describe the haemodynamic effects of CTX S H R and in their control Wistar-Kyoto rats (WK" Relative inhibition of sympathetic and parasyrnpathe nervous tone to the heart by CTX was assessed usi full baroreceptor-heart rate reflex curves for bc strains. Because the release of renin from jux glomerular ceils is also a process depending on calcit and on sympathetic tone (Keeton and Campbell, 19~ we examined the changes in plasma renin activ (PRA) following administration of CTX. A preliminary account of these experiments presented at the British Pharmacological Society Association Fran~aise des Pharmacologistes joint me ing, April 25-27, 1991.
330 2. Materials and methods
2.1. General Male WKY and SHR (16-20 weeks) obtained from Iffa Credo Laboratories (L'Arbresle, France) were used. For chronic experiments the rats were anaesthetized with diethyl oxide and were implanted with catheters introduced into the left carotid artery and the right jugular vein. The tips of catheters were exteriorized behind the head. The next day, the arterial catheter was connected to a blood pressure transducer (Statham P23 XL or P50), the rat being free-moving in its cage. Blood pressure was obtained through a Gould Transducer amplifier (model 13-4615-50) and the signal was recorded on a Gould polygraph (model 8188-402). Heart rate was derived from the arterial pressure pulse (Gould Biotach amplifier, model 13-4615-66). After a 15-min stabilization period, animals were randomly assigned to receive i.v. CTX (10 /xg/kg) or the corresponding vehicle. Sixty minutes later, arterial blood samples (3 ml) were collected in refrigerated (4°C) polystyrene tubes containing EDTA at 20% (20 txl) and PRA was determined using an angiotensin I 125I RIA kit (CA 533, Baxter, Maurepas, France). For baroreceptor heart rate reflex experiments, rats maintained under diethyl oxide anaesthesia were implanted with a catheter into the left femoral artery and a dual catheter into the right jugular vein as previously described (Head and McCarty, 1987). The day following the surgery, arterial blood pressure and heart rate were recorded simultaneously as outlined above. Alternate i.v. injections of 5-200 ~1 of methoxamine (3.75-150 txg/kg) and nitroprusside (3.75-150 p~g/kg) were given to induce steady state increases and decreases in mean arterial pressure (5-50 mm Hg) in each animal (Head and McCarty, 1987). This procedure was repeated 30 min after i.v. injection of CTX (10/xg/kg). For haemodynamic experiments, the rats were anaesthetized with sodium pentobarbitone (50 mg/kg i.p.). Arterial blood pressure was recorded continuously from the left common carotid artery via a Statham P23 XL pressure transducer and heart rate was derived from the pulse pressure. The penile vein was cannulated for drug administration. Blood velocity Doppler flow probes (Valpey Fisher, Hopkinton, MA) were placed around the upper abdominal aorta, the superior mesenteric artery and the left renal artery. The abdomen was closed to avoid hypothermia and the probe wires were connected to a pulsed Doppler flowmeter (Model VF-1, Valpey Fisher, Hopkinton, MA). Velocity values, which are proportional to corresponding blood flows (Haywood et al., 1981; Wright et al., 1987), were recorded on a Gould polygraph (Model 8188-G3302-06). After a 15-min stabilization period, CTX (3 or 10 tzg/kg) or its vehicle was injected and
the cardiovascular parameters were measured for min. Some SHR were treated with (+)-propranolol mg/kg i.v.) 10 min before CTX (10/xg/kg).
2.2. Drugs The drugs and suppliers were as follows: o)-co otoxin GVIA (CTX, Peninsula Lab.), (_+)-propranok sodium nitroprusside and methoxamine hydrochloric (Sigma Chemical Co.). Stock solutions of CTX we stored frozen and diluted in saline the day of tl experiments. All drugs were administered in a volun of 0.5 ml/kg. Control animals received saline (13 ml/kg).
2.3. Calculations and statistical analysis Mean arterial pressure (MAP) was calculated fro systolic arterial pressure (SAP) and diastolic arteri pressure (DAP) as MAP = DAP + ( S A P - D A P ) / The Doppler signal (in kHz) from the upper abdomin aortic probe was considered a good estimate of cardi~ output (CO). Renal blood flow (RBF) and mesentel blood flow (MBF) were measured from the corr sponding Doppler probes. Total peripheral condu tance (TPC), renal (RVC) and mesenteric (MVC) w cular conductances were then calculated from the cc responding values of MAP and flows. The values a reported as means _+ S.E.M. Comparisons of each p rameter between CTX- and vehicle-treated anim~ were made by one-way (basal values) or two-way anal sis of variance with repeated measurements follow~ by Student's t-test for comparison of the means. In the baroreceptor-heart rate reflex experimer the relationship between changes in MAP and he~ rate was used to fit the data points as previous, described (Head and McCarty, 1987). The resultb sigmoid curves were characterised by five parameteJ heart rate range (beats (b)/min) between upper al lower plateau, median blood pressure (BPs0 in m Hg), average gain (b/min per mm Hg), and values the lower and upper heart rate plateau (Head al McCarty, 1987). The significance of changes in the curve parameters was assessed by one-way analysis variance. Statistical significance was taken as P < 0.(
3. Results
Figure 1 shows the effects of CTX (10 /xg/kg) , MAP and heart rate in WKY and SHR. Basal M] was significantly higher in SHR (167 + 4 mm Hg) thz in WKY (122 + 2 mm Hg). Ten minutes after CTX, t] blood pressure in SHR was lowered by 73 _ 5 mm t and remained significantly reduced over 60 min (fig. i In WKY, blood pressure was decreased maximally
5 WKY
SHR
4oo
HR
12
30o
b/rain
---~ O~ ~-
20o 240 [
MAP
i -= ~
16o 120
4i 2 0i
/
mm Hg
6i
Fig. 2. Effect of C T X or its vehicle on plasma renin activity (PRA] the conscious normotensive (WKY) and spontaneously hypertens rat (SHR). P R A was measured 1 h after i.v. C T X or its vehk (black bars) Vehicle, n = 6; (hatched bars) CTX, 1 0 / ~ g / k g , n = 6 * Denotes significant difference (P < 0.05) from control.
C~ ''~%Lx- ; = u =~" ; 2"= -
ao
I
i
0
20
i
40
i
60
0
~
i
20
minutes
i
J
40
60
minutes
Fig. 1. Effect of C T X or its vehicle on m e a n arterial blood pressure (MAP) and heart rate (HR) in the conscious normotensive (WKY; left part) and spontaneously hypertensive rat (SHR; right part). ~o-Conotoxin or its vehicle was given i.v. as a bolus at time 0 min. (*) Vehicle, n = 6 ; ( o ) C T X , 10 /xg/kg, n = 6-7. * Denotes significant
differences(P < 0.05) from control,
41 + 4 mm Hg 20 min after CTX injection (fig. 1). Although the heart rate tended to increase early after the injection in both rat strains, it was not significantly modified by CTX (fig. 1). The basal PRA values did not differ between SHR and WKY (fig. 2). One hour after CTX (10 /zg/kg) P R A was increased by 83 and 80% in WKY and SHR, respectively (fig. 2). Haemodynamic variables before the administration of CTX or its vehicle to anaesthetized SHR and WKY did not differ in each strain (table 1). Mean blood pressure was higher in SHR than in WKY whereas MBF was significantly lower (table 1). After CTX (3 and 10 # g / k g ) , heart rate and mean blood pressure were dose dependently reduced over 30 rain in both SHR and WKY (fig. 3). The time course of the effects
of CTX revealed a slow start of the action, with maximal response 10-20 min after injection (fig. Variations (%) of total peripheral and regional v a s e lar conductances after CTX or its vehicle are ilh trated in fig. 4. In SHR, RVC were increased 79 + 15 and by 107 + 32% 30 min after CTX 3 and /zg/kg respectively. TPC increased by 43 + 8% after /~g/kg CTX whereas MVC were not significan changed (fig. 4). The CO fell in both SHR and WI~ after CTX, however the analysis of variance reveal no statistical differences between vehicle- and C T treated rats. Aortic blood velocity was maximally ] duced from 5.6 + 0.4 to 4.1 + 0.4 kHz in SHR and fro 5.8 + 0.6 to 4.5 + 0.5 kHz in WKY. Renal condt tances of WKY were maximally increased by 59 _q and 81 + 14% after CTX (3 and 10 /zg/kg) (fig. whereas TPC were only slightly elevated after Cq (fig. 4). The basal heart rate (388 + 14 b / m i n ) in fc anaesthetized SHR was decreased to 294 + 9 b / m i n min after (_+)-propranolol. In these animals, heart r~ remained unchanged 30 min after 10 /zg/kg Cq
TABLE 1 Baseline values of haemodynamic variables before administration of C T X or its vehicle to anaesthetized normotensive (WKY) and spontaneou hypertensive rats (SHR). Values are means_+S.E.M, and were compared by one-way analysis of variance; one vascular conductance unit is 100 k H z / m m Hg; abbreviations see Materials and methods section; n = 5 - 6 a n i m a l s / g r o u p . Variable
HR(b/min) MAP(mmHg) CO(kHz) MBF(kHz) RBF(kHz) TPC(units) MVC(units) RVC(units)
WKY
SHR
Vehicle
CTX (3 ~ g / k g )
CTX (10 p,g/kg)
Vehicle
CTX (3/.~g/kg)
CTX (10/~g/kg)
396 + 1 3 113 _+ 6 5.5_+ 0.5 4.1-+ 0.8 2.4_+ 0.4 5.0_+ 0.5 3.8_+ 0.8 2.3_+ 0.4
434 + 1 3 116 _+ 3 4.9_+ 0.6 3.1_+ 0.1 3.5_+ 0.2 4.2_+ 0.5 2.7_+ 0.2 2.9_+ 0.3
421 _+10 108 _+ 4 5.8_+ 0.6 3.6+ 0.5 2.4± 0.3 5.3_+ 0.6 3.3_+ 0.4 2.1_+ 0.3
398 _+15 151 _+ 4 4,9_+ 0.3 2,2_+ 0.l 2,5_+ 0.2 3.3_+ 0.2 1,4_+ 0.1 1.7_+ 0.2
393 _+7 145 _+4 5.2_+0.4 2.0_+0.2 3.1_+0.4 3.6-+0.2 1.4_+0.1 2.2_+0.3
379 _+10 149 + 4 5.6_+ 0.4 2.1_+ 0.2 2.7_+ 0.3 3.8_+ 0.4 1.4+ 0.1 1.8-+ 0.3
332 4so
WKY
SHR
WKY
SHR
120 I
HR
*
*
80
}
350
RVC
b/rain
%
40 0
250 160
120
MAP m m Hg
~
z
.
.
120
~o ~ |
rPC .
.
*
,~
*
*
%
; 40
~.
40 L
Fig. 3. Effect of CTX or its vehicle on mean arterial blood pressure (MAP) and heart rate (HR) in anaesthetized normotensive (WKY) and spontaneously hypertensive rats (SHR). CTX or its vehicle was injected at time 0 min. (e) Vehicle, n = 6; (A) CTX, 3/zg/kg, n = 6; (©) CTX, 10 /xg/kg, n = 6. * Indicates significant differences (P < 0.05) from control.
0
80 [
0 h
(298 + 6 b / m i n ) . R e s t i n g M A P (151 + 8 m m H g ) w a s slightly r e d u c e d by ( + ) - p r o p r a n o l o l to 137 + 7 mm Hg --
Results from baroreceptor-heart
rate reflex experi-
m e n t s in S H R a n d W K Y a r e i l l u s t r a t e d in fig. 5. Average MAP-heart r a t e s i g m o i d a l c u r v e s for t h e combination of vasodilator and vasoconstrictor stimuli i n d i c a t e d a l o w e r h e a r t r a t e r a n g e a n d a h i g h e r BPso for S H R (171 + 8 b / m i n a n d 169 + 10 m m H g ) t h a n for W K Y (287 + 10 b / m i n a n d 130 + 6 m m H g ) . C T X (10/zg/kg) reduced MAP without changing heart rate (fig. 5). T h e t a c h y c a r d i a in r e s p o n s e to a fall in b l o o d pressure following the injection of sodium nitroprusside was m a r k e d l y r e d u c e d a f t e r C T X in b o t h s t r a i n s
. o
.
. . 1 ominutes 2o
,00
~"
300
. 3o
.
. o
. 1 ominutes 2o
3
Fig. 4. Changes (%) in renal (RVC), total peripheral (TPC) a mesenteric (MVC) vascular conductances induced by CTX in ana, thetized normotensive (WKY) and spontaneously hypertensive r', (SHR). CTX or its vehicle was given i.v. at time 0 rain. (o) Vehic n = 5 to 6; (zx) CTX, 3/zg/kg, n = 5-6; (©) CTX, 10/zg/kg, n = to 6. * Indicates significant differences (P < 0.05) from control.
(fig. 5). I n c o n t r a s t , t h e b r a d y c a r d i a in r e s p o n s e m e t h o x a m i n e r e m a i n e d u n a f f e c t e d by C T X in S H a n d W K Y (fig. 5). A f t e r C T X , t h e h e a r t r a t e chanl a n d t h e a v e r a g e g a i n w e r e r e d u c e d in b o t h S H R al W K Y w h e r e a s t h e l o w e r p l a t e a u w a s n o t significanl c h a n g e d ( s e e fig. 5).
WKY
~
,,
12o
o
those in untreated SHR (data not shown).
t
is0[-
MVC
b u t C T X l o w e r e d M A P f u r t h e r to 83 + 4 m m H g . U n d e r t h e s e c o n d i t i o n s , t h e c h a n g e s in v a s c u l a r c o n d u c t a n c e s p r o d u c e d by C T X w e r e n o t d i f f e r e n t f r o m
-
SHR
m ~"
z
200
100 0
!
i
J
i
|
i
m
50
100
150
200
100
150
200
250
MAP
(rnm Hg)
MAP (mm Hg)
Fig. 5. Average baroreceptor reflex curves relating mean arterial blood pressure (MAP) to heart rate (HR), from conscious uormotensive (WK and spontaneously hypertensive rats (SHR) treated with CTX (10 p.g/kg i.v., • and solid line) or its vehicle (e and solid line), n = 5 in e~ group. Error bars are S.E.M. on upper and lower plateaus and on average resting values for MAP and HR.
3~ 4. Discussion This study showed that CTX lowered MAP without changing heart rate and produced peripheral vascular dilation in SHR and WKY. If CTX acts through blockade of N-type VOCCs as previously demonstrated (Nowycky, 1985; McCleskey et al., 1987; Hirning et al., 1988), then these channels might represent a new target to reduce blood pressure. A single dose of 10 /zg/kg CTX reduced blood pressure with a slow start of action in both SHR and WKY and the blood pressure-lowering effect of CTX was maintained over 30 min. Such a slowly developing and sustained hypotensive action of CTX was previously observed in the conscious rabbit (Pruneau and Angus, 1990c) and can be explained on the basis of slow receptor-binding kinetics (Barhanin et al., 1988; Hayakawa et al., 1990). It also indicates that CTX may be relatively resistant to endogenous peptidases in mammals. The hypotensive response to CTX in the rat results from peripheral vasodilation since renal and total peripheral vascular conductances were markedly increased after CTX. AIthough regional conductance measurements were performed in anaesthetized animals, it is noteworthy that CTX increased preferentially renal vascular conductances as previously observed in the conscious rabbit (Pruneau and Angus, 1990c). This could be related to a more pronounced vasoconstrictor tone in the renal bed as compared to the mesenteric and hindquarter beds (Iriuchijima and Sakata, 1985). A negative inotropic action leading to a decrease in cardiac output is unlikely since we have observed that CTX was devoid of action on contractile force in isolated eletrically stimulated guinea-pig atria (personal observation). Thus, the slight reduction of cardiac output produced by CTX was probably due to withdrawal of the sympathetic tone to the heart, Electrophysiological studies have demonstrated a lack of CTX-sensitive sites in smooth muscle cells (McCleskey et al., 1987) and CTX has been shown to be devoid of interactions with dihydropyridine-sensitive postsynaptic calcium channels, classified as L-type (Tsien et al., 1988). In this regard, vasoconstrictor responses to depolarisation or to noradrenaline in isolated rat small arteries (Pruneau and Angus, 1990a) and in the rat tail artery (Clasbrummel et al., 1989) were insensitive to CTX. Furthermore, in the pithed rat, the blood pressure response to exogenous noradrenaline was unaffected by CTX (Pruneau and Angus, 1990b). Assuming that CTX reduced the Ca 2+ influx triggering the release of noradrenaline from the sympathetic nerve terminals, we proposed that inhibition of the sympathetic tone to the vasculature accounts for the haemodynamic effects of CTX. Functionally, CTX inhibited contractile responses to nerve stimulation in isolated arteries (Clasbrummel et al., 1989; Pruneau
and Angus, 1990a) and markedly reduced the press~ responses to electrical stimulation of the spinal cord i the pithed rat (Pruneau and Angus, 1990b). CTX w~ also shown to reduce noradrenaline release and corr~ sponding responses in non-vascular isolated prepar~ tions (Maggi et al., 1988; De Luca et al., 1990). CoJ versely, a number of studies on a variety of ner~ terminals have shown that dihydropyridines do n~ block Ca 2+ influx and Ca2+-dependent secretion lear ing to the conclusion that presynaptic calcium channe are not of the L-type (Hirning et al., 1988; Stanley an Atrakchi, 1990). Together, these results provide ev dence for a major role of CTX-sensitive N-type chal nels in neurotransmitter release and control of tt autonomic nervous system to the vasculature. An interaction of CTX with central vasomotor are~ is unlikely to underlie the blood pressure lowerir effects of the toxin. When injected i.c.v, in mice, CT provokes shaking, paralysis and death, effects whk are not observed after i.p. administration (McClesk~ et al., 1987). Similarly, intrathecal administration , CTX in rats produces tremors and movements of tt hindlimbs (Maggi et al., 1990). In conscious rats (pr, sent study) and rabbits (Pruneau and Angus, 1990c) r death occurred and we did not observe any unusu behavior after a single dose of CTX (3 or 10/zg/k~ These observations suggest that CTX does not cross crosses poorly the blood-brain barrier after i.v. admiJ istration in mammals. PRA was markedly increased in SHR and WKY 1 after CTX. Renin release from juxtaglomerular cells induced by the direct action of circulating hormon~ but is also dependent upon reflex mechanisms ar renal blood flow (Keeton and Campbell, 1984). Even CTX reduces the sympathetic tone to the kidney, renal baroreceptor-mediated renin release in respon: to the lowering of blood pressure could occur (Keet~ and Campbell, 1980). Furthermore, the release of ren is dependent on the intracellular calcium level so that reduction of calcium influx by CTX might actual promote the release of renin. However, further studi, are needed to understand the mechanisms l e a d i n g an increase of PRA after CTX. The possibility of an increased contribution of tl sympathetic nervous system to the elevated blood pre sure of the SHR has been the subject of extensi, controversy. Chemosympathectomy produced throut administration of guanethidine sulfate or 6-hydrox dopamine lowered blood pressure with a greater amp tude in SHR than in WKY (Sakaguchi et al., 198 whereas hexamethonium, a blocker of ganglionic tran mission produced an equivalent fall in arterial pressu (expressed in %) of SHR and WKY (Touw et a 1980). In our study, CTX reduced MAP and increas~ vascular conductances to a greater extent in SHR th~ in control WKY (see Results). These results are cor
334 patible with an increased c o n t r i b u t i o n of the sympathetic nervous system to the m a i n t e n a n c e of vascular resistances in S H R as suggested by others (Brody et al., 1980; Lee et al., 1987) although r e m o d e l l i n g of the arteries, characterized by a n increased w a l l / l u m e n ratio can explain the amplified vasodilator responses seen in S H R (Folkow, 1982).
It was observed recently that the excitatory junctional p o t e n t i a l s (e.j.p.) g e n e r a t e d t h r o u g h electrical nerve stimulation of resistance mesenteric arteries of S H R were m a r k e d l y e n h a n c e d c o m p a r e d to W K Y (Van H e l d e n a n d Woolridge, 1990). Since we have d e m o n strated that C T X abolished e.j.p, in rat resistance arteries at low c o n c e n t r a t i o n s ( P r u n e a u a n d A n g u s , 1990a), we p r o p o s e d that C T X inhibits electrical signailing a n d the release of n o r a d r e n a l i n e from nerve e n d i n g s in the vasculature, b o t h effects resulting in blockade of sympathetic tone. W e also addressed the issue of the specificity of C T X for the sympathetic n e r v o u s system to the heart since it has b e e n d e m o n s t r a t e d that C T X was able to reduce cholinergic a n d n o n - a d r e n e r g i c n o n - c h o l i n e r g i c n e u r o t r a n s m i s s i o n in n o n - v a s c u l a r p r e p a r a t i o n s (Maggi et al., 1988; D e Luca et al., 1990). B a r o r e c e p t o r - h e a r t rate reflex m e a s u r e m e n t s provide v a l u a b l e i n f o r m a t i o n c o n c e r n i n g the relative effects of a drug on cardiac sympathetic a n d vagal tones ( H e a d a n d McCarty, 1987). W e d e m o n s t r a t e d that, in S H R a n d W K Y , C T X almost abolished the sympathetic c o m p o n e n t of the b a r o r e c e p t o r - h e a r t rate reflex w i t h o u t affecting the vagal c o m p o n e n t (fig. 5). This c o n c l u s i o n is b a s e d on the lack of c h a n g e in the b r a d y c a r d i a c p l a t e a u a n d it has b e e n shown that the vagus c o n t r i b u t e d more to the reflex
bradycardia than did the sympathetic component in response to a n increase in blood p r e s s u r e ( H e a d a n d McCarty, 1987). W e had o b t a i n e d a similar result with C T X in the rabbit, using two differents m e t h o d s ( P r u n e a u a n d A n g u s , 1990c). W i t h d r a w a l of sympathetic tone to the heart by C T X was also shown in a n a e s t h e t i z e d rats p r e t r e a t e d with p r o p r a n o l o l since C T X did n o t f u r t h e r r e d u c e the h e a r t rate after /3blockade (see Results). Thus, as suggested by Maggi et al. (1988), CTX-sensitive V O C C s are n o t u n i f o r m l y r e p r e s e n t e d o n m a m m a l i a n a u t o n o m i c n e r v e endings. I n the rat cardiovascular system, nerve t e r m i n a l s in which t r a n s m i t t e r release d e p e n d s entirely u p o n C T X sensitive V O C C s seem to be c o n f i n e d to the sympathetic n e r v o u s system,
Acknowledgements We thank Rose-Marie Frank and Bruno Loillier for excellent technical assistance. The authors also gratefully acknowledge Dr. G.A. Head (Baker Medical Research Institute, Prahran, Australia) for supplying the software allowingthe analysis of baroreceptor heart rate reflex experimental data.
References Barhanin, J., A. Schmid and M. Lazdunski, 1988, Properties structure and interaction of the receptor for oJ-conotoxin, polypeptide active on Ca2+ channels, Biochem. Biophys. R Commun. 150, 1051. Bean, B.P., 1989, Classes of calcium channels in vertebrate cel Ann. Rev. Physiol. 51,367. Brody, M.J., J.R. Haywood and K.B. Touw, 1980, Neural mect nisms in hypertension, Ann. Rev. Physiol. 42, 441. Clasbrummel, B., H. Osswald and P. Illes, 1989, Inhibition of no1 drenaline release by ~o-conotoxinGVIA in the rat tail artery, 1 J. Pharmacol. 96, 101. De Luca, A., C.G. Li, M.J. Rand, J.J. Reid, P. Thaina and H. Wong-Dusting, 1990, Effects of o)-conotoxinGVIA on autonon neuroeffector transmission in various tissues, Br. J. Pharmac 101,437. Folkow,B., 1982, Physiological aspects of primary hypertensk Physiol. Rev. 62, 347. Hayakawa,N., T. Morita, T. Yamaguchi, H. Mitsui, K.J. Mori, Saisu and T. Abe, 1990, The high affinity receptor for o)-cc otoxin represents calcium channels different from those sensiti to dihydropyridines in mammalian brain, Biochem. Biophys. R Commun, 173, 483. Haywood,J.R., R.A. Schaffer, C. Fastenow, G.D. Fink and M Brody, 1981, Regional blood flow measurement with puls Doppler flowmeter in conscious rat, Am. J. Physiol. 241, H273 Head, G.A. and R. McCarty, 1987, Vagal and sympathetic comr nents of the heart rate range and gain of the baroreceptor-he~ rate reflex in conscious rats, J. Auton. Nerv. Syst. 21,203. Hirning, L.D., A.P. Fox, E.W. McCleskey, B.M. Olivera, S.A. Thay, R.J. Miller and R.W. Tsien, 1988, Dominant role of N-type Ca: channels in evoked release or norepinephrine from sympathe neurons, Science 239, 57. Iriuchijima, J. and S. Sakata, 1985, Regional difference in symlc thetic vasoconstrictor tone in conscious rats, Jap. J. Pharmac 35, 1051. Keeton, T.K. and W.B. Campbell, 1980, The pharmacologic alt( ation of renin release, Pharmacol. Rev. 32, 81. Keeton, T.K. and W.B. Campbell, 1984, Control of renin release a its alteration by drugs, in: Cardiovascular Pharmacology, ed. M Antonaccio (Raven Press, New York)p. 65. Lee, R.M.K.W., C.R. Triggle, D.W.T. Cheung and M.D. Coughl 1987, Structural and functional consequence of neonatal syml; thectomy on the blood vessels of spontaneously hypertensive ra Hypertension 10, 328. Lipscombe, D., S. Kongsamut and R.W. Tsien, 1989, a-Adrenerl inhibition of sympathetic neurotransmitter release mediated modulation of N-type calcium channel gating, Nature 340, 639 Lokhandwala,M.F. and D.C. Eikenburg, 1983, Presynaptic recept( and alterations in norepinephrine release in spontaneously hyp( tensive rats, Life Sci. 33, 1527. Maggi, C.A., R. Pattachini, P. Santicioli, l.Th. Lippe, S. Giuliani, Geppetti, E. Del Bianco, S. Selleri and A. Meli, 1988, The eft( of omega conotoxin GVIA, a peptide modulator of the N-ty voltage sensitive channels, on motor responses produced by ac vation of efferent and sensory nerves in mammalian smoc muscle, Nauyn-Schmiedeb. Arch. Pharmacol. 338, 107. Maggi, C.A., S. Giuliani, P. Santicioli, M. Tramontana and A. M( 1990, Effect of omega conotoxin on reflex responses mediated activation of capsaicin-sensitive nerves of the rat urinary blade and peptide release from the rat spinal cord, Neuroscience ' 243. McCleskey,E.W., A.P. Fox, D.H. Feldman, L.J. Cruz, B.M. Olive R.W. Tsien and D. Yoshikami, 1987, to-Conotoxin: Direct a persistent blockade of specific types of calcium channels in nc rons but not muscle, Proc. Natl. Acad. Sci. U.S.A. 84, 4327.
Miller, R.J., 1987, Multiple calcium channels and neuronal function, Science 235, 440. Nowycky, M.C., A.P. Fox and R.W. Tsien, 1985, Three types of neuronal calcium channels with different calcium agonist sensitivity, Nature 316, 440. Okamoto, K., S. Nosaka, Y. Yamori and M. Matsumoto, 1967, Participation of neural factors in the pathogenesis of hypertension in the spontaneously hypertensive rat, Jap. Heart J. 8, 1 6 8 . Pruneau, D. and J.A. Angus, 1990a, to-Conotoxin GVIA is a potent inhibitor of sympathetic neurogenic responses in rat small mesenteric arteries, Br. J. Pharmacol. 100, 180. Pruneau, D. and J.A. Angus, 1990b, Apparent vascular to cardiac sympatholytic selectivity of to-conotoxin GVIA in the pithed rat, Eur. J. Pharmacol. 184, 127. Pruneau, D. and J.A. Angus, 1990c, to-Conotoxin GVIA, the N-type calcium channel inhibitor, is sympatholytic but not vagolytic: Consequences for hemodynamics and autonomic reflexes in conscious rabbits, J. Cardiovasc. Pharmacol. 16, 675. Sakaguchi, A., J.E. LeDoux and D.J. Reis, 1983, Sympathetic nerves and adrenal medulla: Contributions to cardiovascular-condi-
tioned emotional responses in spontaneously hypertensive r~ Hypertension 5, 728. Stanley, E.F. and A.H. Atrakchi, 1990, Calcium currents recorc from a vertebrate presynaptic nerve terminal are resistant to dihydropyridine nifedipine, Proc. Natl. Acad. Sci. U.S.A. 9683. Touw, K.B., J.R. Haywood, R.A. Schaffer and M.J. Brody, 19 Contribution of the sympathetic nervous system to vascular re: tance in conscious young and adult spontaneously hypertens rats, Hypertension 2, 408. Tsien, R.W., D. Lipscombe, D.V. Madison, K.R. Bley and A.P. F 1988, Multiple types of neuronal calcium channels and tll selective modulation, Trends Neurosci. 11,431. Van Helden, D.F. and S. Woolridge, 1990, Role of nerves in hyp tension, Nature 348, 118. Wright, C.E., J.A. Angus and P.I. Korner, 1987, Vascular amplil properties in renovascular hypertension in conscious rabb hindquarter responses to constrictor and dilator stimuli, Hyl0 tension 9, 122.
2
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52291
Haemodynamic and humoral effects of ¢o-conotoxin GVIA in normotensive and spontaneously hypertensive rats Didier Pruneau and Pierre B~lichard Centre de Recherche, Laboratoires Fournier S.C.A., 50 Rue de Dijon, 21121-FontaineLes Dijon, France
Received 29 October 1991, revised MS received 19 November 1991, accepted 26 November 1991
oJ-Conotoxin GVIA, a 27-amino acid peptide, has been shown to be a potent and selective inhibitor of N-type voltage-op ated calcium channels (VOCCs). A single intravenous dose of 10/~g/kg conotoxin slowly lowered blood pressure by 41.3 +, and 73.3 + 4.6 mm Hg in conscious Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) respectively with~ changing the heart rate. Plasma renin activity was significantly increased after conotoxin. In anaesthetized rats, conotoxin (3 a 10/~g/kg) lowered blood pressure and heart rate and produced a marked increase in renal vascular conductances. Barorecep heart rate reflex experiments using methoxamine and sodium nitroprusside before and after treatment with conotoxin show that conotoxin almost totally abolished the sympathetic component of the reflex without affecting the vagal tone to the heart both rat strains. Because conotoxin does not affect directly the vasculature and heart contractile properties, we suggest that 1 control of presynaptic calcium influx and of neurotransmitter release mostly depends on conotoxin-sensitive N-type VOCCs the peripheral sympathetic system of the rat. ~o-Conotoxin; Sympathetic nerves; Hypertension; Vasodilatation
1. Introduction The release of neurotransmitter is triggered by an influx of calcium ions in nerve terminals through voltage-operated calcium channels (VOCCs). Although both L- and N-type VOCCs coexist in a number of neuronal preparations (Nowycky et al., 1985; Miller, 1987; Tsien et al., 1988; Bean, 1989), N-type channels seem to play a dominant role in depolarisation-coupled noradrenaline release (Hirning et al., 1988; Lipscombe et al., 1989). w-Conotoxin G V I A (CTX), a 27-amino acid peptide isolated from Conus geographus venom, was shown to bind to neuronal VOCCs with an affinity in the sub-picomolar range (Barhanin et al., 1988; Hayakawa et al., 1990). Thus CTX selectively inhibits Ca 2+ influx in dorsal ganglion, sensory, sympathetic and hippocampal neurons of vertebrates without affecting Ca 2+ movements in smooth, skeletal and cardiac muscle (McCleskey et al., 1987). In accordance with these data we have recently shown that CTX produced a marked inhibition of sympathetic neurotransmission in rat small mesenteric arteries (Pruneau
Correspondence to: D. Pruneau, Centre de Recherche, Laboratoires Fournier S.C.A., 50 Rue de Dijon, 21121-Fontaine les Dijon, France. Tel. 33.80.44.75.39, fax 33.80.56.56.64.
and Angus, 1990a) and in the pithed rat (Pruneau a Angus, 1990b). In the conscious rabbit intravenc (i.v.) administration of C T X lowered blood presst and blocked the sympathetic component of the baro~ ceptor-heart rate reflex (Pruneau and Angus, 1990 We concluded that CTX-sensitive channels play a n jot role in controlling the sympathetic tone towards t vasculature and the heart. Since increased sympathe nervous activity has been proposed, among other fi tots, to explain the increased total peripheral res tance observed in spontaneously hypertensive n (SHR) (Okamoto et al., 1967; Touw et al., 191 Lokhandwala and Eikenburg, 1983; Lee et al., 198 we now describe the haemodynamic effects of CTX S H R and in their control Wistar-Kyoto rats (WK" Relative inhibition of sympathetic and parasyrnpathe nervous tone to the heart by CTX was assessed usi full baroreceptor-heart rate reflex curves for bc strains. Because the release of renin from jux glomerular ceils is also a process depending on calcit and on sympathetic tone (Keeton and Campbell, 19~ we examined the changes in plasma renin activ (PRA) following administration of CTX. A preliminary account of these experiments presented at the British Pharmacological Society Association Fran~aise des Pharmacologistes joint me ing, April 25-27, 1991.
330 2. Materials and methods
2.1. General Male WKY and SHR (16-20 weeks) obtained from Iffa Credo Laboratories (L'Arbresle, France) were used. For chronic experiments the rats were anaesthetized with diethyl oxide and were implanted with catheters introduced into the left carotid artery and the right jugular vein. The tips of catheters were exteriorized behind the head. The next day, the arterial catheter was connected to a blood pressure transducer (Statham P23 XL or P50), the rat being free-moving in its cage. Blood pressure was obtained through a Gould Transducer amplifier (model 13-4615-50) and the signal was recorded on a Gould polygraph (model 8188-402). Heart rate was derived from the arterial pressure pulse (Gould Biotach amplifier, model 13-4615-66). After a 15-min stabilization period, animals were randomly assigned to receive i.v. CTX (10 /xg/kg) or the corresponding vehicle. Sixty minutes later, arterial blood samples (3 ml) were collected in refrigerated (4°C) polystyrene tubes containing EDTA at 20% (20 txl) and PRA was determined using an angiotensin I 125I RIA kit (CA 533, Baxter, Maurepas, France). For baroreceptor heart rate reflex experiments, rats maintained under diethyl oxide anaesthesia were implanted with a catheter into the left femoral artery and a dual catheter into the right jugular vein as previously described (Head and McCarty, 1987). The day following the surgery, arterial blood pressure and heart rate were recorded simultaneously as outlined above. Alternate i.v. injections of 5-200 ~1 of methoxamine (3.75-150 txg/kg) and nitroprusside (3.75-150 p~g/kg) were given to induce steady state increases and decreases in mean arterial pressure (5-50 mm Hg) in each animal (Head and McCarty, 1987). This procedure was repeated 30 min after i.v. injection of CTX (10/xg/kg). For haemodynamic experiments, the rats were anaesthetized with sodium pentobarbitone (50 mg/kg i.p.). Arterial blood pressure was recorded continuously from the left common carotid artery via a Statham P23 XL pressure transducer and heart rate was derived from the pulse pressure. The penile vein was cannulated for drug administration. Blood velocity Doppler flow probes (Valpey Fisher, Hopkinton, MA) were placed around the upper abdominal aorta, the superior mesenteric artery and the left renal artery. The abdomen was closed to avoid hypothermia and the probe wires were connected to a pulsed Doppler flowmeter (Model VF-1, Valpey Fisher, Hopkinton, MA). Velocity values, which are proportional to corresponding blood flows (Haywood et al., 1981; Wright et al., 1987), were recorded on a Gould polygraph (Model 8188-G3302-06). After a 15-min stabilization period, CTX (3 or 10 tzg/kg) or its vehicle was injected and
the cardiovascular parameters were measured for min. Some SHR were treated with (+)-propranolol mg/kg i.v.) 10 min before CTX (10/xg/kg).
2.2. Drugs The drugs and suppliers were as follows: o)-co otoxin GVIA (CTX, Peninsula Lab.), (_+)-propranok sodium nitroprusside and methoxamine hydrochloric (Sigma Chemical Co.). Stock solutions of CTX we stored frozen and diluted in saline the day of tl experiments. All drugs were administered in a volun of 0.5 ml/kg. Control animals received saline (13 ml/kg).
2.3. Calculations and statistical analysis Mean arterial pressure (MAP) was calculated fro systolic arterial pressure (SAP) and diastolic arteri pressure (DAP) as MAP = DAP + ( S A P - D A P ) / The Doppler signal (in kHz) from the upper abdomin aortic probe was considered a good estimate of cardi~ output (CO). Renal blood flow (RBF) and mesentel blood flow (MBF) were measured from the corr sponding Doppler probes. Total peripheral condu tance (TPC), renal (RVC) and mesenteric (MVC) w cular conductances were then calculated from the cc responding values of MAP and flows. The values a reported as means _+ S.E.M. Comparisons of each p rameter between CTX- and vehicle-treated anim~ were made by one-way (basal values) or two-way anal sis of variance with repeated measurements follow~ by Student's t-test for comparison of the means. In the baroreceptor-heart rate reflex experimer the relationship between changes in MAP and he~ rate was used to fit the data points as previous, described (Head and McCarty, 1987). The resultb sigmoid curves were characterised by five parameteJ heart rate range (beats (b)/min) between upper al lower plateau, median blood pressure (BPs0 in m Hg), average gain (b/min per mm Hg), and values the lower and upper heart rate plateau (Head al McCarty, 1987). The significance of changes in the curve parameters was assessed by one-way analysis variance. Statistical significance was taken as P < 0.(
3. Results
Figure 1 shows the effects of CTX (10 /xg/kg) , MAP and heart rate in WKY and SHR. Basal M] was significantly higher in SHR (167 + 4 mm Hg) thz in WKY (122 + 2 mm Hg). Ten minutes after CTX, t] blood pressure in SHR was lowered by 73 _ 5 mm t and remained significantly reduced over 60 min (fig. i In WKY, blood pressure was decreased maximally
5 WKY
SHR
4oo
HR
12
30o
b/rain
---~ O~ ~-
20o 240 [
MAP
i -= ~
16o 120
4i 2 0i
/
mm Hg
6i
Fig. 2. Effect of C T X or its vehicle on plasma renin activity (PRA] the conscious normotensive (WKY) and spontaneously hypertens rat (SHR). P R A was measured 1 h after i.v. C T X or its vehk (black bars) Vehicle, n = 6; (hatched bars) CTX, 1 0 / ~ g / k g , n = 6 * Denotes significant difference (P < 0.05) from control.
C~ ''~%Lx- ; = u =~" ; 2"= -
ao
I
i
0
20
i
40
i
60
0
~
i
20
minutes
i
J
40
60
minutes
Fig. 1. Effect of C T X or its vehicle on m e a n arterial blood pressure (MAP) and heart rate (HR) in the conscious normotensive (WKY; left part) and spontaneously hypertensive rat (SHR; right part). ~o-Conotoxin or its vehicle was given i.v. as a bolus at time 0 min. (*) Vehicle, n = 6 ; ( o ) C T X , 10 /xg/kg, n = 6-7. * Denotes significant
differences(P < 0.05) from control,
41 + 4 mm Hg 20 min after CTX injection (fig. 1). Although the heart rate tended to increase early after the injection in both rat strains, it was not significantly modified by CTX (fig. 1). The basal PRA values did not differ between SHR and WKY (fig. 2). One hour after CTX (10 /zg/kg) P R A was increased by 83 and 80% in WKY and SHR, respectively (fig. 2). Haemodynamic variables before the administration of CTX or its vehicle to anaesthetized SHR and WKY did not differ in each strain (table 1). Mean blood pressure was higher in SHR than in WKY whereas MBF was significantly lower (table 1). After CTX (3 and 10 # g / k g ) , heart rate and mean blood pressure were dose dependently reduced over 30 rain in both SHR and WKY (fig. 3). The time course of the effects
of CTX revealed a slow start of the action, with maximal response 10-20 min after injection (fig. Variations (%) of total peripheral and regional v a s e lar conductances after CTX or its vehicle are ilh trated in fig. 4. In SHR, RVC were increased 79 + 15 and by 107 + 32% 30 min after CTX 3 and /zg/kg respectively. TPC increased by 43 + 8% after /~g/kg CTX whereas MVC were not significan changed (fig. 4). The CO fell in both SHR and WI~ after CTX, however the analysis of variance reveal no statistical differences between vehicle- and C T treated rats. Aortic blood velocity was maximally ] duced from 5.6 + 0.4 to 4.1 + 0.4 kHz in SHR and fro 5.8 + 0.6 to 4.5 + 0.5 kHz in WKY. Renal condt tances of WKY were maximally increased by 59 _q and 81 + 14% after CTX (3 and 10 /zg/kg) (fig. whereas TPC were only slightly elevated after Cq (fig. 4). The basal heart rate (388 + 14 b / m i n ) in fc anaesthetized SHR was decreased to 294 + 9 b / m i n min after (_+)-propranolol. In these animals, heart r~ remained unchanged 30 min after 10 /zg/kg Cq
TABLE 1 Baseline values of haemodynamic variables before administration of C T X or its vehicle to anaesthetized normotensive (WKY) and spontaneou hypertensive rats (SHR). Values are means_+S.E.M, and were compared by one-way analysis of variance; one vascular conductance unit is 100 k H z / m m Hg; abbreviations see Materials and methods section; n = 5 - 6 a n i m a l s / g r o u p . Variable
HR(b/min) MAP(mmHg) CO(kHz) MBF(kHz) RBF(kHz) TPC(units) MVC(units) RVC(units)
WKY
SHR
Vehicle
CTX (3 ~ g / k g )
CTX (10 p,g/kg)
Vehicle
CTX (3/.~g/kg)
CTX (10/~g/kg)
396 + 1 3 113 _+ 6 5.5_+ 0.5 4.1-+ 0.8 2.4_+ 0.4 5.0_+ 0.5 3.8_+ 0.8 2.3_+ 0.4
434 + 1 3 116 _+ 3 4.9_+ 0.6 3.1_+ 0.1 3.5_+ 0.2 4.2_+ 0.5 2.7_+ 0.2 2.9_+ 0.3
421 _+10 108 _+ 4 5.8_+ 0.6 3.6+ 0.5 2.4± 0.3 5.3_+ 0.6 3.3_+ 0.4 2.1_+ 0.3
398 _+15 151 _+ 4 4,9_+ 0.3 2,2_+ 0.l 2,5_+ 0.2 3.3_+ 0.2 1,4_+ 0.1 1.7_+ 0.2
393 _+7 145 _+4 5.2_+0.4 2.0_+0.2 3.1_+0.4 3.6-+0.2 1.4_+0.1 2.2_+0.3
379 _+10 149 + 4 5.6_+ 0.4 2.1_+ 0.2 2.7_+ 0.3 3.8_+ 0.4 1.4+ 0.1 1.8-+ 0.3
332 4so
WKY
SHR
WKY
SHR
120 I
HR
*
*
80
}
350
RVC
b/rain
%
40 0
250 160
120
MAP m m Hg
~
z
.
.
120
~o ~ |
rPC .
.
*
,~
*
*
%
; 40
~.
40 L
Fig. 3. Effect of CTX or its vehicle on mean arterial blood pressure (MAP) and heart rate (HR) in anaesthetized normotensive (WKY) and spontaneously hypertensive rats (SHR). CTX or its vehicle was injected at time 0 min. (e) Vehicle, n = 6; (A) CTX, 3/zg/kg, n = 6; (©) CTX, 10 /xg/kg, n = 6. * Indicates significant differences (P < 0.05) from control.
0
80 [
0 h
(298 + 6 b / m i n ) . R e s t i n g M A P (151 + 8 m m H g ) w a s slightly r e d u c e d by ( + ) - p r o p r a n o l o l to 137 + 7 mm Hg --
Results from baroreceptor-heart
rate reflex experi-
m e n t s in S H R a n d W K Y a r e i l l u s t r a t e d in fig. 5. Average MAP-heart r a t e s i g m o i d a l c u r v e s for t h e combination of vasodilator and vasoconstrictor stimuli i n d i c a t e d a l o w e r h e a r t r a t e r a n g e a n d a h i g h e r BPso for S H R (171 + 8 b / m i n a n d 169 + 10 m m H g ) t h a n for W K Y (287 + 10 b / m i n a n d 130 + 6 m m H g ) . C T X (10/zg/kg) reduced MAP without changing heart rate (fig. 5). T h e t a c h y c a r d i a in r e s p o n s e to a fall in b l o o d pressure following the injection of sodium nitroprusside was m a r k e d l y r e d u c e d a f t e r C T X in b o t h s t r a i n s
. o
.
. . 1 ominutes 2o
,00
~"
300
. 3o
.
. o
. 1 ominutes 2o
3
Fig. 4. Changes (%) in renal (RVC), total peripheral (TPC) a mesenteric (MVC) vascular conductances induced by CTX in ana, thetized normotensive (WKY) and spontaneously hypertensive r', (SHR). CTX or its vehicle was given i.v. at time 0 rain. (o) Vehic n = 5 to 6; (zx) CTX, 3/zg/kg, n = 5-6; (©) CTX, 10/zg/kg, n = to 6. * Indicates significant differences (P < 0.05) from control.
(fig. 5). I n c o n t r a s t , t h e b r a d y c a r d i a in r e s p o n s e m e t h o x a m i n e r e m a i n e d u n a f f e c t e d by C T X in S H a n d W K Y (fig. 5). A f t e r C T X , t h e h e a r t r a t e chanl a n d t h e a v e r a g e g a i n w e r e r e d u c e d in b o t h S H R al W K Y w h e r e a s t h e l o w e r p l a t e a u w a s n o t significanl c h a n g e d ( s e e fig. 5).
WKY
~
,,
12o
o
those in untreated SHR (data not shown).
t
is0[-
MVC
b u t C T X l o w e r e d M A P f u r t h e r to 83 + 4 m m H g . U n d e r t h e s e c o n d i t i o n s , t h e c h a n g e s in v a s c u l a r c o n d u c t a n c e s p r o d u c e d by C T X w e r e n o t d i f f e r e n t f r o m
-
SHR
m ~"
z
200
100 0
!
i
J
i
|
i
m
50
100
150
200
100
150
200
250
MAP
(rnm Hg)
MAP (mm Hg)
Fig. 5. Average baroreceptor reflex curves relating mean arterial blood pressure (MAP) to heart rate (HR), from conscious uormotensive (WK and spontaneously hypertensive rats (SHR) treated with CTX (10 p.g/kg i.v., • and solid line) or its vehicle (e and solid line), n = 5 in e~ group. Error bars are S.E.M. on upper and lower plateaus and on average resting values for MAP and HR.
3~ 4. Discussion This study showed that CTX lowered MAP without changing heart rate and produced peripheral vascular dilation in SHR and WKY. If CTX acts through blockade of N-type VOCCs as previously demonstrated (Nowycky, 1985; McCleskey et al., 1987; Hirning et al., 1988), then these channels might represent a new target to reduce blood pressure. A single dose of 10 /zg/kg CTX reduced blood pressure with a slow start of action in both SHR and WKY and the blood pressure-lowering effect of CTX was maintained over 30 min. Such a slowly developing and sustained hypotensive action of CTX was previously observed in the conscious rabbit (Pruneau and Angus, 1990c) and can be explained on the basis of slow receptor-binding kinetics (Barhanin et al., 1988; Hayakawa et al., 1990). It also indicates that CTX may be relatively resistant to endogenous peptidases in mammals. The hypotensive response to CTX in the rat results from peripheral vasodilation since renal and total peripheral vascular conductances were markedly increased after CTX. AIthough regional conductance measurements were performed in anaesthetized animals, it is noteworthy that CTX increased preferentially renal vascular conductances as previously observed in the conscious rabbit (Pruneau and Angus, 1990c). This could be related to a more pronounced vasoconstrictor tone in the renal bed as compared to the mesenteric and hindquarter beds (Iriuchijima and Sakata, 1985). A negative inotropic action leading to a decrease in cardiac output is unlikely since we have observed that CTX was devoid of action on contractile force in isolated eletrically stimulated guinea-pig atria (personal observation). Thus, the slight reduction of cardiac output produced by CTX was probably due to withdrawal of the sympathetic tone to the heart, Electrophysiological studies have demonstrated a lack of CTX-sensitive sites in smooth muscle cells (McCleskey et al., 1987) and CTX has been shown to be devoid of interactions with dihydropyridine-sensitive postsynaptic calcium channels, classified as L-type (Tsien et al., 1988). In this regard, vasoconstrictor responses to depolarisation or to noradrenaline in isolated rat small arteries (Pruneau and Angus, 1990a) and in the rat tail artery (Clasbrummel et al., 1989) were insensitive to CTX. Furthermore, in the pithed rat, the blood pressure response to exogenous noradrenaline was unaffected by CTX (Pruneau and Angus, 1990b). Assuming that CTX reduced the Ca 2+ influx triggering the release of noradrenaline from the sympathetic nerve terminals, we proposed that inhibition of the sympathetic tone to the vasculature accounts for the haemodynamic effects of CTX. Functionally, CTX inhibited contractile responses to nerve stimulation in isolated arteries (Clasbrummel et al., 1989; Pruneau
and Angus, 1990a) and markedly reduced the press~ responses to electrical stimulation of the spinal cord i the pithed rat (Pruneau and Angus, 1990b). CTX w~ also shown to reduce noradrenaline release and corr~ sponding responses in non-vascular isolated prepar~ tions (Maggi et al., 1988; De Luca et al., 1990). CoJ versely, a number of studies on a variety of ner~ terminals have shown that dihydropyridines do n~ block Ca 2+ influx and Ca2+-dependent secretion lear ing to the conclusion that presynaptic calcium channe are not of the L-type (Hirning et al., 1988; Stanley an Atrakchi, 1990). Together, these results provide ev dence for a major role of CTX-sensitive N-type chal nels in neurotransmitter release and control of tt autonomic nervous system to the vasculature. An interaction of CTX with central vasomotor are~ is unlikely to underlie the blood pressure lowerir effects of the toxin. When injected i.c.v, in mice, CT provokes shaking, paralysis and death, effects whk are not observed after i.p. administration (McClesk~ et al., 1987). Similarly, intrathecal administration , CTX in rats produces tremors and movements of tt hindlimbs (Maggi et al., 1990). In conscious rats (pr, sent study) and rabbits (Pruneau and Angus, 1990c) r death occurred and we did not observe any unusu behavior after a single dose of CTX (3 or 10/zg/k~ These observations suggest that CTX does not cross crosses poorly the blood-brain barrier after i.v. admiJ istration in mammals. PRA was markedly increased in SHR and WKY 1 after CTX. Renin release from juxtaglomerular cells induced by the direct action of circulating hormon~ but is also dependent upon reflex mechanisms ar renal blood flow (Keeton and Campbell, 1984). Even CTX reduces the sympathetic tone to the kidney, renal baroreceptor-mediated renin release in respon: to the lowering of blood pressure could occur (Keet~ and Campbell, 1980). Furthermore, the release of ren is dependent on the intracellular calcium level so that reduction of calcium influx by CTX might actual promote the release of renin. However, further studi, are needed to understand the mechanisms l e a d i n g an increase of PRA after CTX. The possibility of an increased contribution of tl sympathetic nervous system to the elevated blood pre sure of the SHR has been the subject of extensi, controversy. Chemosympathectomy produced throut administration of guanethidine sulfate or 6-hydrox dopamine lowered blood pressure with a greater amp tude in SHR than in WKY (Sakaguchi et al., 198 whereas hexamethonium, a blocker of ganglionic tran mission produced an equivalent fall in arterial pressu (expressed in %) of SHR and WKY (Touw et a 1980). In our study, CTX reduced MAP and increas~ vascular conductances to a greater extent in SHR th~ in control WKY (see Results). These results are cor
334 patible with an increased c o n t r i b u t i o n of the sympathetic nervous system to the m a i n t e n a n c e of vascular resistances in S H R as suggested by others (Brody et al., 1980; Lee et al., 1987) although r e m o d e l l i n g of the arteries, characterized by a n increased w a l l / l u m e n ratio can explain the amplified vasodilator responses seen in S H R (Folkow, 1982).
It was observed recently that the excitatory junctional p o t e n t i a l s (e.j.p.) g e n e r a t e d t h r o u g h electrical nerve stimulation of resistance mesenteric arteries of S H R were m a r k e d l y e n h a n c e d c o m p a r e d to W K Y (Van H e l d e n a n d Woolridge, 1990). Since we have d e m o n strated that C T X abolished e.j.p, in rat resistance arteries at low c o n c e n t r a t i o n s ( P r u n e a u a n d A n g u s , 1990a), we p r o p o s e d that C T X inhibits electrical signailing a n d the release of n o r a d r e n a l i n e from nerve e n d i n g s in the vasculature, b o t h effects resulting in blockade of sympathetic tone. W e also addressed the issue of the specificity of C T X for the sympathetic n e r v o u s system to the heart since it has b e e n d e m o n s t r a t e d that C T X was able to reduce cholinergic a n d n o n - a d r e n e r g i c n o n - c h o l i n e r g i c n e u r o t r a n s m i s s i o n in n o n - v a s c u l a r p r e p a r a t i o n s (Maggi et al., 1988; D e Luca et al., 1990). B a r o r e c e p t o r - h e a r t rate reflex m e a s u r e m e n t s provide v a l u a b l e i n f o r m a t i o n c o n c e r n i n g the relative effects of a drug on cardiac sympathetic a n d vagal tones ( H e a d a n d McCarty, 1987). W e d e m o n s t r a t e d that, in S H R a n d W K Y , C T X almost abolished the sympathetic c o m p o n e n t of the b a r o r e c e p t o r - h e a r t rate reflex w i t h o u t affecting the vagal c o m p o n e n t (fig. 5). This c o n c l u s i o n is b a s e d on the lack of c h a n g e in the b r a d y c a r d i a c p l a t e a u a n d it has b e e n shown that the vagus c o n t r i b u t e d more to the reflex
bradycardia than did the sympathetic component in response to a n increase in blood p r e s s u r e ( H e a d a n d McCarty, 1987). W e had o b t a i n e d a similar result with C T X in the rabbit, using two differents m e t h o d s ( P r u n e a u a n d A n g u s , 1990c). W i t h d r a w a l of sympathetic tone to the heart by C T X was also shown in a n a e s t h e t i z e d rats p r e t r e a t e d with p r o p r a n o l o l since C T X did n o t f u r t h e r r e d u c e the h e a r t rate after /3blockade (see Results). Thus, as suggested by Maggi et al. (1988), CTX-sensitive V O C C s are n o t u n i f o r m l y r e p r e s e n t e d o n m a m m a l i a n a u t o n o m i c n e r v e endings. I n the rat cardiovascular system, nerve t e r m i n a l s in which t r a n s m i t t e r release d e p e n d s entirely u p o n C T X sensitive V O C C s seem to be c o n f i n e d to the sympathetic n e r v o u s system,
Acknowledgements We thank Rose-Marie Frank and Bruno Loillier for excellent technical assistance. The authors also gratefully acknowledge Dr. G.A. Head (Baker Medical Research Institute, Prahran, Australia) for supplying the software allowingthe analysis of baroreceptor heart rate reflex experimental data.
References Barhanin, J., A. Schmid and M. Lazdunski, 1988, Properties structure and interaction of the receptor for oJ-conotoxin, polypeptide active on Ca2+ channels, Biochem. Biophys. R Commun. 150, 1051. Bean, B.P., 1989, Classes of calcium channels in vertebrate cel Ann. Rev. Physiol. 51,367. Brody, M.J., J.R. Haywood and K.B. Touw, 1980, Neural mect nisms in hypertension, Ann. Rev. Physiol. 42, 441. Clasbrummel, B., H. Osswald and P. Illes, 1989, Inhibition of no1 drenaline release by ~o-conotoxinGVIA in the rat tail artery, 1 J. Pharmacol. 96, 101. De Luca, A., C.G. Li, M.J. Rand, J.J. Reid, P. Thaina and H. Wong-Dusting, 1990, Effects of o)-conotoxinGVIA on autonon neuroeffector transmission in various tissues, Br. J. Pharmac 101,437. Folkow,B., 1982, Physiological aspects of primary hypertensk Physiol. Rev. 62, 347. Hayakawa,N., T. Morita, T. Yamaguchi, H. Mitsui, K.J. Mori, Saisu and T. Abe, 1990, The high affinity receptor for o)-cc otoxin represents calcium channels different from those sensiti to dihydropyridines in mammalian brain, Biochem. Biophys. R Commun, 173, 483. Haywood,J.R., R.A. Schaffer, C. Fastenow, G.D. Fink and M Brody, 1981, Regional blood flow measurement with puls Doppler flowmeter in conscious rat, Am. J. Physiol. 241, H273 Head, G.A. and R. McCarty, 1987, Vagal and sympathetic comr nents of the heart rate range and gain of the baroreceptor-he~ rate reflex in conscious rats, J. Auton. Nerv. Syst. 21,203. Hirning, L.D., A.P. Fox, E.W. McCleskey, B.M. Olivera, S.A. Thay, R.J. Miller and R.W. Tsien, 1988, Dominant role of N-type Ca: channels in evoked release or norepinephrine from sympathe neurons, Science 239, 57. Iriuchijima, J. and S. Sakata, 1985, Regional difference in symlc thetic vasoconstrictor tone in conscious rats, Jap. J. Pharmac 35, 1051. Keeton, T.K. and W.B. Campbell, 1980, The pharmacologic alt( ation of renin release, Pharmacol. Rev. 32, 81. Keeton, T.K. and W.B. Campbell, 1984, Control of renin release a its alteration by drugs, in: Cardiovascular Pharmacology, ed. M Antonaccio (Raven Press, New York)p. 65. Lee, R.M.K.W., C.R. Triggle, D.W.T. Cheung and M.D. Coughl 1987, Structural and functional consequence of neonatal syml; thectomy on the blood vessels of spontaneously hypertensive ra Hypertension 10, 328. Lipscombe, D., S. Kongsamut and R.W. Tsien, 1989, a-Adrenerl inhibition of sympathetic neurotransmitter release mediated modulation of N-type calcium channel gating, Nature 340, 639 Lokhandwala,M.F. and D.C. Eikenburg, 1983, Presynaptic recept( and alterations in norepinephrine release in spontaneously hyp( tensive rats, Life Sci. 33, 1527. Maggi, C.A., R. Pattachini, P. Santicioli, l.Th. Lippe, S. Giuliani, Geppetti, E. Del Bianco, S. Selleri and A. Meli, 1988, The eft( of omega conotoxin GVIA, a peptide modulator of the N-ty voltage sensitive channels, on motor responses produced by ac vation of efferent and sensory nerves in mammalian smoc muscle, Nauyn-Schmiedeb. Arch. Pharmacol. 338, 107. Maggi, C.A., S. Giuliani, P. Santicioli, M. Tramontana and A. M( 1990, Effect of omega conotoxin on reflex responses mediated activation of capsaicin-sensitive nerves of the rat urinary blade and peptide release from the rat spinal cord, Neuroscience ' 243. McCleskey,E.W., A.P. Fox, D.H. Feldman, L.J. Cruz, B.M. Olive R.W. Tsien and D. Yoshikami, 1987, to-Conotoxin: Direct a persistent blockade of specific types of calcium channels in nc rons but not muscle, Proc. Natl. Acad. Sci. U.S.A. 84, 4327.
Miller, R.J., 1987, Multiple calcium channels and neuronal function, Science 235, 440. Nowycky, M.C., A.P. Fox and R.W. Tsien, 1985, Three types of neuronal calcium channels with different calcium agonist sensitivity, Nature 316, 440. Okamoto, K., S. Nosaka, Y. Yamori and M. Matsumoto, 1967, Participation of neural factors in the pathogenesis of hypertension in the spontaneously hypertensive rat, Jap. Heart J. 8, 1 6 8 . Pruneau, D. and J.A. Angus, 1990a, to-Conotoxin GVIA is a potent inhibitor of sympathetic neurogenic responses in rat small mesenteric arteries, Br. J. Pharmacol. 100, 180. Pruneau, D. and J.A. Angus, 1990b, Apparent vascular to cardiac sympatholytic selectivity of to-conotoxin GVIA in the pithed rat, Eur. J. Pharmacol. 184, 127. Pruneau, D. and J.A. Angus, 1990c, to-Conotoxin GVIA, the N-type calcium channel inhibitor, is sympatholytic but not vagolytic: Consequences for hemodynamics and autonomic reflexes in conscious rabbits, J. Cardiovasc. Pharmacol. 16, 675. Sakaguchi, A., J.E. LeDoux and D.J. Reis, 1983, Sympathetic nerves and adrenal medulla: Contributions to cardiovascular-condi-
tioned emotional responses in spontaneously hypertensive r~ Hypertension 5, 728. Stanley, E.F. and A.H. Atrakchi, 1990, Calcium currents recorc from a vertebrate presynaptic nerve terminal are resistant to dihydropyridine nifedipine, Proc. Natl. Acad. Sci. U.S.A. 9683. Touw, K.B., J.R. Haywood, R.A. Schaffer and M.J. Brody, 19 Contribution of the sympathetic nervous system to vascular re: tance in conscious young and adult spontaneously hypertens rats, Hypertension 2, 408. Tsien, R.W., D. Lipscombe, D.V. Madison, K.R. Bley and A.P. F 1988, Multiple types of neuronal calcium channels and tll selective modulation, Trends Neurosci. 11,431. Van Helden, D.F. and S. Woolridge, 1990, Role of nerves in hyp tension, Nature 348, 118. Wright, C.E., J.A. Angus and P.I. Korner, 1987, Vascular amplil properties in renovascular hypertension in conscious rabb hindquarter responses to constrictor and dilator stimuli, Hyl0 tension 9, 122.