Several subtypes of voltage-operated calcium channels have been identified, including the L-, N-, P-, and Q-type channels (1,2). ω-Conotoxin GVIA (ω-CTX) is a 27-amino-acid peptide that was isolated from the venom of the fish-hunting cone snail Conus geographus(3). ω-CTX potently and selectively inhibits calcium entry through N-type calcium channels, which are located on neuronal tissue in the central nervous system and periphery (4,5). It is well established that in sympathetic nerve terminals, the calcium entry that regulates norepinephrine release is predominantly through N-type calcium channels (6,7).
Pruneau and Angus (8) reported that i.v. administration of ω-CTX (3 μg/kg) caused hypotension, tachycardia, and a selective sympatholytic action on the baroreceptor-heart rate reflex (baroreflex) in conscious rabbits. In conscious rats, a similar effect of ω-CTX on cardiovascular parameters was observed (9), and in anesthetized guinea-pigs, i.v. ω-CTX causes hypotension without affecting pressor responses to exogenous norepinephrine (10). In contrast, L-type calcium channel antagonists such as diltiazem, verapamil, and nicardipine have a hypotensive effect in anesthetized guinea-pigs but also inhibit norepinephrine-induced pressor responses. This indicates that the L-type calcium channel antagonists act at the postsynaptic vascular smooth muscle (10). In rat isolated mesenteric artery, ω-CTX is a potent inhibitor of responses to sympathetic nerve stimulation, whereas even at very high concentrations, it has no effect on postsynaptic L-type calcium channel-mediated responses that are sensitive to blockade by felodipine (11).
In a more recent in vivo study (12), i.v. administration of a threefold higher dose of ω-CTX (10 μg/kg) in conscious rabbits caused a comparable decrease in blood pressure, tachycardia, and attenuation of the upper sympathetically mediated heart rate plateau of the baroreflex to that described previously. However, this higher dose of ω-CTX also had a significant vagolytic action on the baroreflex but no effect on the vagally mediated Bezold-Jarisch-like reflex (12). Further, in rabbit isolated right atria, ω-CTX inhibited sympathetic, but not vagal, responses to electrical nerve stimulation (12). Therefore the contrasting effect of ω-CTX on the vagally mediated reflexes in vivo may be due to an action on the afferent or central pathway of the baroreflex. In contrast, the i.v. route, intracerebroventricular administration of ω-CTX in conscious rabbits, results in a very slowly developing hypotensive response and selectively sympatholytic action on the baroreflex, with the peak effect occurring after 48 h (13).
Short-term i.v. administration of ω-CTX causes an increase in plasma renin in rats and rabbits (9,12). Thus the renin-angiotensin system may play a role in buffering the hypotensive effect of ω-CTX and in the eventual recovery of blood pressure. During hypotensive episodes in rats and rabbits, such as those induced by ganglion block or hemorrhage, increased levels of vasopressin and angiotensin II are important in maintaining blood pressure (14,15). As angiotensin-converting enzyme (ACE) is equivalent to kininase II, one of the enzymes responsible for the degradation of bradykinin (16), altered levels of endogenous kinins may contribute to the cardiovascular effects of ACE inhibitors (17). Angiotensin II also inhibits vagal efferent activity (18); therefore an increase in circulating angiotensin after ω-CTX may contribute to its vagolytic action on the baroreflex.
The aims of our study were to examine the direct effect of i.v. ω-CTX on the baroreflex without the confounding influences of activation of the renin-angiotensin system. First, the effects of ω-CTX on vagally mediated reflexes were assessed in conscious rabbits pretreated with the β-adrenoceptor antagonist propranolol to obviate sympathetically mediated heart rate responses. Second, the role of the renin-angiotensin system in the responses to i.v. ω-CTX were investigated by using the ACE inhibitor enalaprilat, the angiotensin II AT1-receptor antagonist losartan, and the bradykinin B2-receptor antagonist HOE-140. Before these experiments being performed, appropriate doses of enalaprilat, losartan, and HOE-140 were determined. Effects of i.v. ω-CTX on several autonomic reflexes were assessed-the baroreflex to allow an estimation of both sympathetic and vagal cardiac activity, as well as the nasopharyngeal and Bezold-Jarisch-like reflexes to test the bradycardia mediated via the efferent vagus. Some of this work was published in abstract form (19).
MATERIALS AND METHODS
The study was approved by the University of Melbourne animal ethics committee in accordance with the guidelines of the National Health and Medical Research Council of Australia.
Animals and measurements
New Zealand White rabbits of either sex (mean, 2.43 ± 0.03 kg; n = 57) were used in the study. On each experimental day, the central ear artery and marginal ear vein were cannulated under local anesthesia [0.5% lignocaine hydrochloride (Xylocaine); Astra, Sydney, Australia]. The ear artery catheter was connected to a CDX pressure transducer (Cobe, Lakewood, CO, U.S.A.) for the measurement of phasic and mean arterial pressure (MAP), which was recorded on a Grass polygraph (model 7D; Grass, Quincy, MA, U.S.A.). A rate meter (model 173; Baker Medical Research Institute, Melbourne, Australia) was triggered by the phasic arterial pressure for the measurement of heart rate (HR). Rabbits rested quietly in a polycarbonate rabbit box (Nalgene; Nalge, Rochester, NY, U.S.A.) for ∼45 min before experiments commenced.
Preliminary experiments to determine antagonist doses
Preliminary experiments were performed in conscious autonomically blocked rabbits (2.64 ± 0.06 kg, n = 24; most of these rabbits were used in the main study; time control experiments were performed in 3 rabbits in a randomized manner) to determine the appropriate doses of enalaprilat, losartan, and HOE-140 to cause significant long-lasting antagonism of ACE, angiotensin AT1, and bradykinin B2 receptors, respectively. Either ganglion block or cardiac block was maintained throughout the experimental protocol to obviate reflex changes in cardiovascular parameters that may be caused by the agonists/antagonists.
Cardiac block was attained with propranolol (0.5 mg/kg, i.v. bolus + 2.4 mg/kg/h, i.v. infusion) plus methscopolamine (50 μg/kg, i.v. bolus + 50 μg/kg/h, i.v. infusion). These doses abolish changes in HR to several reflex stimuli (20). Angiotensin I and bradykinin curves before and after antagonist or vehicle, as well as angiotensin II curves before and after vehicle, were assessed in the presence of cardiac block. Two methods were used to maintain ganglion block for the experimental period.
- Mecamylamine (4 mg/kg, i.v. bolus + 2.5 mg/kg/h, i.v. infusion) was shown previously to be sufficient to completely abolish the nasopharyngeal reflex, the Bezold-Jarisch-like reflex, and the reflex bradycardia induced by pressor doses of i.v. norepinephrine (21). Isoprenaline curves before and after propranolol were performed in the presence of mecamylamine infusion.
- Mecamylamine, 10 mg/kg, i.v., was administered over a 10-min period in 10 ml dextran (Dextran 40; 10% in saline) with a further 4 mg/kg, i.v., given after 4 h (22). The nasopharyngeal reflex was tested routinely throughout the experiment to ascertain that ganglion block was maintained. Angiotensin II curves before and after losartan were constructed in the presence of ganglion block with this method. The reason for this was that whereas losartan dissolves well in saline alone, it precipitates out of solution when mixed with either the cardiac- or ganglion-blocking drugs for infusion.
Control agonist curves were performed ∼20 min after establishment of ganglion or cardiac block. Isoprenaline doses (0.0125-204.8 μg/kg, i.v.) were administered before and 20 min after propranolol (0.5 mg/kg, i.v. bolus + 2.4 mg/kg/h, i.v. infusion; n = 6). Angiotensin I curves (0.01-20.48 μg/kg, i.v.) were constructed before (control) and 30, 90, and 180 min after enalaprilat administration (1 mg/kg, i.v. bolus + 1.5 mg/kg/h, i.v. infusion; n = 5). Angiotensin II curves (1-3,000 ng/kg, i.v.) were performed before (control) and 30, 120, and 210 min after losartan (4 mg/kg, i.v. bolus + 2 mg/kg/h, i.v. infusion; n = 1; or 6 mg/kg + 3 mg/kg/h, i.v.; n = 3). Bradykinin curves (0.01-30 μg/kg, i.v.) were assessed before (control) and 30, 120, and 240 min after 10 (n = 1), 30 (n = 1) or 100 μg/kg (n = 4) HOE-140 administered as an i.v. bolus (23,24). Angiotensin I, II, and bradykinin curves also were performed by following the same protocols as earlier but without the administration of enalaprilat, losartan, and HOE-140, respectively, to determine whether there was any change in the curves over time (n = 2-3).
Main study protocol
The effects of i.v. ω-CTX or vehicle were assessed on MAP, HR, the baroreflex, and nasopharyngeal and Bezold-Jarisch-like reflexes in the absence or presence of propranolol or inhibitors of the renin-angiotensin system. There were five treatment groups: (a) no pretreatment (Control; n = 12); (b) propranolol (0.5 mg/kg, i.v. bolus + 2.4 mg/kg/h, i.v. infusion; n = 12); (c) enalaprilat (1 mg/kg, i.v. bolus + 1.5 mg/kg/h, i.v. infusion; n = 11); (d) losartan (6 mg/kg, i.v. bolus + 3 mg/kg/h, i.v. infusion; n = 10); and (e) HOE-140 (100 μg/kg, i.v. bolus; n = 12).
When MAP and HR had stabilized after administration of propranolol, enalaprilat, losartan, or HOE-140 (20-30 min), autonomic reflexes were tested. The nasopharyngeal reflex was induced by blowing ∼20 ml cigarette smoke (Peter Jackson; Phillip Morris Ltd., Melbourne, Australia) through a plastic tube near the nostrils of the rabbit over a 10- to 12-s period. When cardiovascular parameters had returned to baseline, the Bezold-Jarisch-like reflex was elicited by serotonin (5-HT; 1-30 μg/kg, i.v. bolus, given over 4-6 s). The baroreceptor-HR reflex was then assessed by using the steady-state method produced by alternate step-wise increases and decreases in MAP (25,26). This was performed by intravenous injections of 1-150 μl of phenylephrine (250 μg/ml) and 5-500 μl of sodium nitroprusside (1 mg/ml) to achieve graded changes in MAP of ±5-35 mm Hg from baseline. Doses were chosen randomly; however, an increase in MAP (with phenylephrine) was always followed by a decrease in MAP (with sodium nitroprusside) to prevent a shift in resting parameters. ω-CTX (10 μg/kg, i.v. bolus) or vehicle (saline; 0.9% NaCl) was then administered, and MAP and HR monitored for 1 h. The nasopharyngeal, Bezold-Jarisch-like and baroreceptor-HR reflexes were then retested.
Drugs used and their sources were angiotensin I (Auspep, Parkville, Australia), angiotensin II (Auspep), bradykinin (Auspep), enalaprilat (a gift from Merck, Rahway, NJ, U.S.A.), HOE-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin; a gift from Hoechst, Frankfurt, Germany), isoprenaline [(−)-isoproterenol (+)-bitartrate salt; Sigma, St. Louis, MO, U.S.A.], losartan (a gift from Merck), mecamylamine hydrochloride (a gift from Merck), methscopolamine bromide (Upjohn, Rydalmere, Australia), L-phenylephrine hydrochloride (Sigma), propranolol hydrochloride (Sigma), 5-HT (5-hydroxytryptamine creatinine sulfate; Sigma), sodium nitroprusside (David Bull Laboratories, Melbourne, Australia), and ω-conotoxin GVIA [synthesized by J. P. Flinn et al. (27), Department of Pharmacology, University of Melbourne, Australia].
Enalaprilat, HOE-140, isoprenaline, losartan, mecamylamine, methscopolamine, and propranolol were made up fresh daily in saline. Phenylephrine, 5-HT, and sodium nitroprusside were diluted in saline and stored at −20°C. Angiotensin I, angiotensin II, bradykinin, and ω-conotoxin GVIA were stored in aliquots (10−3 or 10−4M) at −20°C and diluted daily in saline as required.
Data are presented as mean ± 1 standard error of the mean (SEM). Responses to isoprenaline, angiotensin I, angiotensin II, bradykinin, 5-HT, cigarette smoke, phenylephrine, and sodium nitroprusside were measured as peak changes in MAP or HR or both from resting baseline. Average MAP and HR values presented in text and tables have been rounded to the nearest whole number. Average SEM within animals was calculated from two-way analysis of variance (ANOVA) by using the pooled estimate of error from the residual mean square as (error mean square/number of animals)0.5 after subtracting the sum of squares "between animals" and "between times" from the "total" sum of squares for each parameter (22). This error bar is located on the mean lines for resting MAP and HR and the Bezold-Jarisch-like reflex. Agonist dose-response curves after administration of antagonist were tested within group by split-plot ANOVA for three groups (28). The baroreceptor-HR reflex curves were analyzed by fitting the MAP and HR changes to a sigmoidal logistic equation, which is characterized by (a) HR range (beats/min) between upper and lower plateaus of the curve; (b) median blood pressure [MAP50 (mm Hg)]; (c) average gain (beats/min/mm Hg); and (d) lower HR plateau (25). Comparisons of cardiovascular and baroreflex parameters (a) before and 1 h after ω-CTX or vehicle, and (b) before and 20 min after administration of propranolol, enalaprilat, losartan, or HOE-140, were made within treatment group by Student's t test for paired data. Baseline (0 min) values of MAP and HR, or reflex parameters before or after ω-CTX were compared between groups by one-way ANOVA with Tukey post hoc test where required. MAP and HR were measured every 5 min for 1 h after administration of ω-CTX or vehicle and were tested within and between groups by repeated-measures ANOVA with Green-house-Geisser correction for correlation (29), calculated by means of the statistical program SuperANOVA 1.11 for Macintosh. The time to the peak change in MAP or HR after ω-CTX was established for each rabbit and compared between groups by one-way ANOVA with Tukey post hoc test where appropriate. Responses to 5-HT, 1-30 μg/kg, were tested within group before and 1 h after ω-CTX or vehicle by split-plot ANOVA for two groups. Probability values <0.05 were taken as statistically significant.
Agonist dose-response curves
Intravenous bolus doses of isoprenaline caused tachycardia in conscious rabbits. After administration of propranolol (0.5 mg/kg + 2.4 mg/kg/h, i.v.), there was an ∼300-fold shift of the isoprenaline curve to the right (n = 6; data not shown). Bolus i.v. doses of angiotensin I had a pressor effect in the conscious rabbit (Fig. 1). The dose-response curves were shifted to the right by ∼300-fold in the presence of the ACE inhibitor, enalaprilat (1 mg/kg + 1.5 mg/kg/h, i.v.; n = 5; Fig. 1). There was no significant difference between the angiotensin I curves performed 30, 90, and 180 min after the commencement of enalaprilat infusion (p = 0.91), demonstrating that the degree of ACE inhibition was maintained.
Angiotensin II also caused pressor responses in conscious rabbits (Fig. 1). Losartan (4 mg/kg + 2 mg/kg/h, i.v.) caused only a 10-fold shift of the angiotensin II curves to the right (n = 1; data not shown), whereas the higher dose of losartan (6 mg/kg + 3 mg/kg/h, i.v.) resulted in a 30-fold rightward shift of the angiotensin II curves (n = 3; Fig. 1). This inhibition was maintained, as there was no difference between the angiotensin II curves performed at 30, 120, and 210 min in the presence of losartan infusion (p = 0.89). Bolus i.v. doses of bradykinin caused depressor responses in conscious rabbits (Fig. 1). After i.v. administration of 100 μg/kg HOE-140, the response to bradykinin ≤10 μg/kg was virtually abolished in the curves performed after 30 and 120 min (n = 4; Fig. 1). After 240 min, the effect of HOE-140 was beginning to wear off, but there was still a >1,000-fold rightward shift of the bradykinin curve (Fig. 1). The lower doses of HOE-140 (10 and 30 μg/kg, i.v.) were not sufficient to cause long-lasting shift (for ≤4 h) of the bradykinin curves (data not shown). Time control curves to angiotensin I, angiotensin II, and bradykinin were reproducible (n = 2-3; data not shown).
Short-term effects of antagonist pretreatment
In each treatment group, resting MAP and HR were measured before and 20 min after administration of the particular antagonist. In the control group, there was no change from the baseline values of MAP and HR, 71 ± 2 mm Hg and 185 ± 6 beats/min (p > 0.05; n = 12), respectively. In the propranolol treatment group, there was no change in MAP from 74 ± 2 mm Hg, but a small increase in HR of 5 ± 2 beats/min from 211 ± 6 beats/min (p = 0.016; n = 12). A decrease in MAP of 5 ± 1 mm Hg from 70 ± 2 and 77 ± 3 mm Hg was observed in both the enalaprilat (p = 0.003; n = 11) and losartan (p = 0.005; n = 10) groups, respectively. Concurrent increases in HR were 21 ± 5 and 15 ± 5 beats/min from 222 ± 7 and 225 ± 8 beats/min in the enalaprilat (p = 0.0016) and losartan (p = 0.017) groups, respectively. In the HOE-140 group, there were increases in both MAP, from 78 ± 2 to 81 ± 2 mm Hg (p = 0.043), and HR from 231 ± 8 to 244 ± 9 beats/min (p = 0.0014; n = 12).
Effect of ω-CTX administration on MAP and HR
After administration of ω-CTX (10 μg/kg, i.v. bolus) in the control group, there was a slowly developing hypotensive response with the peak decrease in MAP of 13 ± 3 mm Hg from 72 ± 2 mm Hg after 33 ± 3 min, with a corresponding tachycardia of 80 ± 20 beats/min from 174 ± 8 beats/min (Fig. 2; n = 6). Similar changes and time to peak change (p > 0.05) were observed in the propranolol pretreatment group (n = 6), with MAP decreasing from 71 ± 2 to 56 ± 2 mm Hg after 38 ± 1 min and the maximal increase in HR of 84 ± 8 beats/min from 216 ± 8 beats/min observed 34 ± 4 min after ω-CTX administration (Fig. 2). Note that the rabbits in the control group had a lower mean baseline HR than those in the other treatment groups (p < 0.05; Figs. 2 and 3).
In the enalaprilat pretreatment group (n = 6), a larger and more rapid change in MAP of −19 ± 4 mm Hg from 65 ± 1 mm Hg was observed within 18 ± 2 min in response to ω-CTX compared with the control group (p < 0.01; Fig. 3), with a smaller tachycardia (p = 0.0001) of 222 ± 9 to 243 ± 6 beats/min. This larger change in MAP was despite the lower 0-min baseline caused by ACE inhibition. In the presence of the angiotensin II AT1-receptor antagonist, losartan, ω-CTX administration resulted in a change in MAP of −22 ± 3 mm Hg from 70 ± 4 mm Hg (n = 5) after 22 ± 4 min, with HR increasing by 46 ± 10 beats/min from 202 ± 13 beats/min after 21 ± 5 min (Fig. 3). MAP decreased from 77 ± 2 to 54 ± 3 mm Hg by 24 ± 2 min after ω-CTX in the HOE-140 group (n = 6). The change in HR had reached a plateau ∼30 min after ω-CTX at 46 ± 10 beats/min above the baseline of 230 ± 9 beats/min; however, the peak change in HR of 58 ± 8 beats/min was observed after 13 ± 4 min (Fig. 3). There was a significant difference in the time to peak change in MAP (p = 0.0001) and HR (p = 0.0008) across all treatment groups. The time to peak MAP decrease was more rapid in the enalaprilat and losartan groups (p < 0.05), but not quite statistically different in the HOE-140 group (p > 0.05), compared with the control group. The changes in cardiovascular parameters after ω-CTX administration remained stable for the duration of the experimental day (3-4 h) in all treatment groups. No change in MAP or HR occurred during the hour after vehicle administration in each treatment group (n = 5-6; data not shown).
Baroreceptor-heart rate reflex
Baroreflex curve parameters for each of the treatment groups are shown in Table 1. The curves for the control and propranolol treatment groups before and after ω-CTX administration are shown in Fig. 2, and baroreflex curves obtained 1 h after administration of either vehicle or ω-CTX for the other groups are represented in Fig. 4 (for clarity, the curves before vehicle or before ω-CTX are not shown). In the propranolol treatment group, the HR range of the baroreflex before ω-CTX administration was 118 ± 11 beats/min (n = 6), ∼65% of the range of the control group (Table 1; Fig. 2). As described earlier, the dose of propranolol used was shown in preliminary experiments to shift isoprenaline dose-response curves by ∼300-fold in conscious rabbits. Therefore sympathetically mediated HR responses were blocked by the β-adrenoceptor antagonist, leaving just the vagal component of the barocurve. Because of the lower resting MAP in the enalaprilat and losartan groups, baroreflex curves were left-shifted compared with the control group (Fig. 4).
ω-CTX administration resulted in changes in baroreflex-curve parameters in all treatment groups (Table 1; Figs. 2 and 4). In all groups 1 h after peptide administration, MAP was significantly lower and HR significantly higher than baseline (p < 0.05). There was also a significant increase in the lower HR plateau of the curves, with a decrease in the HR range in all treatment groups (Table 1). After i.v. ω-CTX, the HR range decreased to 69 ± 3 and 69 ± 7 beats/min in the control (p = 0.0003, n = 6) and propranolol groups (p = 0.013; n = 6), respectively, which reflects a 61% reduction in the HR range compared with the pre-ω-CTX value in the control group (Fig. 2). In the presence of enalaprilat (n = 6), losartan (n = 5), or HOE-140 (n = 6), the HR range was significantly smaller (p = 0.0024) after ω-CTX, with values ∼60% of those after ω-CTX in the control and propranolol groups (i.e., representing a decrease in baroreflex HR range of ≥76% compared with the pre-ω-CTX value in the control group (Fig. 4, right; Table 1). Significant decreases in MAP50 after ω-CTX administration compared with baseline were observed only in the losartan (p = 0.009) and HOE-140 (p = 0.036) treatment groups. There was a significant decrease in the average gain (in beats/min/mm Hg) of the baroreflex curves after ω-CTX administration in all treatment groups (p < 0.05; Table 1).
After vehicle administration, there were no significant changes in baroreflex parameters (p > 0.05; Table 1; Fig. 4) in the propranolol (n = 6), enalaprilat (n = 5), and losartan (n = 5) treatment groups. Small but significant changes in HR (p = 0.044) and average gain (p = 0.045) were observed after vehicle in the control group (n = 6; Table 1). In the HOE-140 group (n = 6), vehicle administration resulted in significant changes in MAP (p = 0.008), HR (p = 0.002), MAP50 (p = 0.017), and the lower HR plateau (p = 0.011; Table 1).
In all treatment groups, cigarette smoke blown near the nostrils of rabbits resulted in profound bradycardia (Fig. 5). The administration of vehicle had no effect on the bradycardic response to nasopharyngeal stimulation in any treatment group (data not shown). After i.v. administration of ω-CTX, 10 μg/kg, resting HR was increased in all groups, as described earlier. In the presence of ω-CTX, the nasopharyngeal reflex-induced bradycardia was unaffected in the control, propranolol, and losartan treatment groups. However, there was a significant attenuation of the bradycardic response in the enalaprilat and HOE-140 groups (Fig. 5) of 173 ± 10 to 104 ± 20 beats/min (p = 0.033; n = 6) and 207 ± 5 to 137 ± 19 beats/min (p = 0.008; n = 6), respectively.
The administration of i.v. bolus doses of 5-HT (1-30 μg/kg) resulted in dose-dependent bradycardia (Fig. 6) and a corresponding decrease in MAP. In the control and propranolol treatment groups, there was no significant effect of ω-CTX on the change in HR in response to 5-HT. However, a significantly smaller bradycardia to 5-HT was observed after ω-CTX in the enalaprilat (p = 0.002), losartan (p = 0.028), and HOE-140 (p = 0.026) groups (Fig. 6) when the whole dose-response line before and after ω-CTX was compared by split-plot ANOVA. In all groups, there was a significantly smaller change in MAP after the administration of ω-CTX, as the pre-5-HT baseline MAP was lower. However, in all groups, the peak depressor response to 30 μg/kg, i.v., 5-HT was to a similar minimal level before and after ω-CTX (p > 0.05; data not shown). The administration of vehicle had no effect on the reflex (e.g., Fig. 6, upper right), with the exception of the losartan group, in which smaller changes in both HR and MAP were observed to each dose of 5-HT (p < 0.05; data not shown).
Acute N-type calcium channel blockade in conscious rabbits results in hypotension, tachycardia, and attenuation of both the sympathetic and vagal components of the baroreflex, determined by HR response to changes in arterial pressure. We show here that endogenous angiotensin II buffers the hypotensive response to ω-CTX because ACE inhibition or angiotensin AT1-receptor antagonism enhances the magnitude and rate of decrease in blood pressure. Curiously, bradykinin B2-receptor antagonism also revealed a greater hypotensive effect of ω-CTX. In all treatment groups, there was an increase in the lower HR plateau and decrease in the HR range of the baroreflex-HR-blood pressure relation after ω-CTX, indicating an attenuation of both the vagal and sympathetic components, respectively. However, contrasting effects on the Bezold-Jarisch-like and nasopharyngeal reflexes, both mediated through the efferent vagus, were observed in the groups of rabbits treated with inhibitors of the renin-angiotensin system.
Preliminary experiments were performed to determine the appropriate doses of antagonist to ensure adequate and sustained inhibition for the duration of the ∼4-h protocol. These experiments were considered essential, as most reports in the literature failed to quantify the antagonism against a full agonist dose-response curve. The lower dose of losartan tested (4 mg/kg + 2 mg/kg/h, i.v.) was reported to block two bolus doses of angiotensin II in conscious rabbits (17). There was no mention of the degree of shift of the angiotensin II curve by losartan, nor that the infusion was adequate to maintain the degree of shift over time. However, in our study, this dose of losartan only right-shifted an angiotensin II dose-pressor response curve by ∼10-fold. Therefore to ensure adequate AT1-receptor inhibition in our experiments, a higher dose of 6 mg/kg + 3 mg/kg/h, i.v. (30) was used, as it shifted angiotensin II-pressor response curves by 30-fold in vivo. A recent report by Tomoda et al. (24) found that HOE-140 caused a degree of inhibition of bradykinin dose-response curves over a 4-h period in conscious rabbits similar to the results obtained in our study. They also decided that 100 μg/kg, i.v., HOE-140 was an appropriate dose, as a 10-fold higher dose did not cause substantially more blockade of the bradykinin curves (24).
Pretreatment with the antagonists resulted in changes in blood pressure and HR over the 20-min stabilization period at the commencement of the experiment. In the enalaprilat and losartan groups, there was a decrease in blood pressure, suggesting that circulating angiotensin II contributes to resting vascular tone in the conscious rabbit. The corresponding increase in HR was most likely due to baroreflex compensation. Bradykinin also appears to be involved in the maintenance of blood pressure, as the administration of the bradykinin B2-receptor antagonist HOE-140 caused small but significant increases in blood pressure and HR. Similar effects on blood pressure and HR were observed in spontaneously hypertensive rats (31), but no change in resting parameters was reported after HOE-140 in another study in conscious rabbits (24). The small increase in heart rate (5 beats/min) that occurred after propranolol administration may have been due to baroreflex compensation (vagal withdrawal) to maintain blood pressure.
In conscious rabbits and anesthetized rats, i.v. administration of ω-CTX causes an increase in peripheral vascular conductance, particularly in the renal and hindquarter vascular beds, which results in a decrease in blood pressure (8,9,12). In the rabbit, withdrawal of vagal tone to the heart was believed to cause the tachycardia in response to ω-CTX, as the baroreflex resets HR to a higher level (8,12,32). This has been confirmed by the results found in the propranolol treatment group of our study. The dose of propranolol (a β-adrenoceptor antagonist) was sufficient to shift isoprenaline dose-response curves by ∼300-fold and attenuate the upper sympathetically mediated HR plateau of the baroreflex curve. In the presence of propranolol, the decrease in blood pressure and, more important, increase in HR in response to ω-CTX were of a magnitude and time course similar to that observed in the control group. These results indicate that ω-CTX is not causing direct stimulation via β-adrenoceptors to increase HR, and therefore, the observed tachycardia is indeed due to reflex withdrawal of cardiac vagal tone as a consequence of the hypotensive action of ω-CTX. Similarly, ω-CTX does not affect resting HR in the pithed rat (33).
In the presence of propranolol, the portion of the baroreflex remaining was the vagally mediated component, the HR range being ∼65% of that observed in the control group. The sympathetically mediated reflex tachycardia in response to the hypotension caused by sodium nitroprusside was attenuated, whereas the lower vagally mediated HR plateau was unaffected. After administration of 10 μg/kg ω-CTX, the HR range of the curve further decreased to a level similar to that obtained after ω-CTX in the control group. There was also an increase in the lower HR plateau of the curve. Therefore, ω-CTX does have a vagolytic action on the baroreceptor-HR reflex. This result is in contrast to the observations of Pruneau and Angus (8); they found that a lower dose of ω-CTX (3 μg/kg, i.v.) resulted in only attenuation of the sympathetic component of the baroreflex. This dose may have been on the threshold required to affect baroreflex-related vagal activity in the rabbit. Central administration of ω-CTX also has a selectively sympatholytic action on the baroreflex; however, it takes 48 h for the peak effects to occur in conscious rabbits (13).
Despite the maintenance of the hypotension and tachycardia after i.v. ω-CTX for the duration of the experimental day (8,12), the return of blood pressure and HR toward pre-ω-CTX levels is apparent after 24 h (12,32). The administration of i.v. ω-CTX results in increased plasma renin activity in rats and rabbits (9,12), indicating a possible role for angiotensin II in the recovery of blood pressure. In our study, compared with the control group, the administration of ω-CTX resulted in a larger and more rapid decrease in blood pressure in rabbits treated with the ACE inhibitor enalaprilat (peak after only 18 min) or the angiotensin AT1-receptor antagonist, losartan (peak after 22 min). In all other treatment groups, the peak change in blood pressure was not observed until 25-40 min after ω-CTX was administered. Therefore angiotensin II appears to have a homeostatic role in buffering (attenuating and slowing) the hypotensive effect of ω-CTX. Similarly, increased levels of vasopressin and angiotensin are important in maintaining blood pressure in rats and rabbits during hypotension induced by ganglion block or hemorrhage (14,15,34). It may also be that vasopressin has a role in buffering the hypotensive response to i.v. ω-CTX, but this was not tested in our study. The increases in HR (due to reflex vagal withdrawal as N-type calcium channel blockade prevents sympathetically mediated increases) in the hour after ω-CTX administration in the enalaprilat, losartan, and HOE-140 groups were ∼50% less than that observed in the control group. However, these smaller increases were from a raised resting HR compared with the control group. So although the differences in baseline cloud interpretation of this finding, it may indicate an influence of these renin-angiotensin system antagonists on this reflex phenomenon.
As well as inhibiting the formation of angiotensin II, ACE inhibitors prevent the degradation of bradykinin (16), and evidence suggests that the accumulation of endogenous kinins may contribute to the cardiovascular actions of ACE inhibitors (17,35). The decrease in blood pressure after ω-CTX in the enalaprilat group peaked ∼5 min earlier than that in the losartan group (see earlier). The difference between the two groups may be due to accumulation of bradykinin in the presence of ACE inhibition, but not in the presence of AT1-receptor blockade. The bradykinin B2-receptor antagonist HOE-140 was used to test this hypothesis. However, after HOE-140 treatment, the ω-CTX-induced decrease in blood pressure was also surprisingly enhanced. Therefore from these results, bradykinin may similarly play a role in buffering the hypotensive effect of ω-CTX. Bradykinin is an endothelium-dependent vasodilator peptide that has also been shown to act on B2 receptors to cause or facilitate release of norepinephrine from cultured rat sympathetic neurons (36), rat isolated heart (37,38), and guinea-pig cardiac sympathetic neurons (39). Central administration of bradykinin has been demonstrated to cause a B2-receptor-mediated increase in both blood pressure and HR, consistent with sympathetic stimulation, in conscious rats (40). Further, bradykinin can stimulate release of catecholamines from adrenal chromaffin cells by activation of presynaptic B2 receptors, and certain bradykinin antagonists also display nonspecific stimulatory effects in this tissue (41). So in our study, pretreatment with the B2-receptor antagonist HOE-140 may have inhibited an endogenous bradykinin-induced increase in catecholamine release, thus allowing a greater decrease in blood pressure after ω-CTX administration. It is intriguing to note that the rate and range of decrease in blood pressure after N-type calcium channel blockade is enhanced to a similar degree by antagonism of any one of angiotensin AT1 receptors, bradykinin B2 receptors, or angiotensin II formation (causing endogenous bradykinin accumulation). Perhaps the enhancement of the sympathetic effects of angiotensin II and bradykinin at sympathetic ganglia and nerve endings, and at the level of the chromaffin cell, are mutually effective, so that blockade of one attenuates the outcome of both. This hypothesis begs the analysis of complex interactions, especially amplification of multiple signals, giving rise to organ/tissue responses, and warrants further investigation.
In conscious rabbits, endogenous angiotensin II has a tonic action on the baroreflex, which is mediated via AT1 receptors. The administration of angiotensin II results in a resetting of the baroreflex curve to the right, with an increase in MAP50 and no change in HR or gain of the curve (18,42,43). The AT1-receptor antagonist losartan can reverse these changes in the baroreflex induced by exogenous angiotensin II (42) and moreover resets the baroreflex to the left when administered alone, indicating a tonic role for endogenous angiotensin II (42,43). Angiotensin II (and bradykinin as discussed earlier) may also act at sympathetic nerves to facilitate sympathetic neurotransmission (36,38,44). Endogenous angiotensin II modulates the sympathetic arm of the baroreflex and decreases baroreflex sensitivity via AT1 receptors in rabbits with heart failure (45). Central administration of bradykinin in conscious rats increases baroreflex sensitivity without changing blood pressure or HR (46); however, intravenous bradykinin or HOE-140 does not affect baroreflex parameters in conscious normotensive or hypertensive rats, respectively (46,47). The effects of angiotensin II or bradykinin-receptor blockade on the baroreflex were not assessed directly in our study. The decreases in the HR range of the baroreflex curves after ω-CTX in the control and propranolol groups were to similar absolute values. However, the HR range was attenuated by another 40% (compared with post-ω-CTX in the control group) in the enalaprilat, losartan, and HOE-140 groups, largely due to a further increase in the lower HR plateau of the baroreflex. Thus these antagonists appear to augment the vagolytic effects of ω-CTX on the baroreflex and suggest a possible interaction at the N-type calcium channel. Alternatively, preventing the tonic actions of endogenous angiotensin II or bradykinin or both may cause an additive effect on the baroreflex when combined with N-type calcium channel blockade. The attenuation of the HR range relates clearly to the decrease in the reflex tachycardia (lack of vagal withdrawal) seen with administration of ω-CTX in the presence of the renin-angiotensin system antagonists mentioned earlier, and further with the effects on the other vagally mediated reflexes discussed later.
The nasopharyngeal and Bezold-Jarisch-like reflexes are both characterized by bradycardia mediated by the efferent vagus (48,49). In both the control and propranolol treatment groups, ω-CTX did not affect either of these reflexes-a result that concurs with previous studies (8,12). Endogenous angiotensin II is believed to inhibit vagal tone to the heart via a central mechanism (18,44), with ACE inhibitors and losartan therefore having a potentiating effect on vagally mediated bradycardia (50). In contrast, Montemayor et al. (51) showed that the ACE inhibitor lisinopril had no effect on vagal reflexes in young rats but caused a decrease in angiotensin II-evoked reflex bradycardia in old rats that already had impaired responses. In our study, the HR responses to the nasopharyngeal and Bezold-Jarisch-like reflex were attenuated in some of the treatment groups. After ω-CTX administration, the nasopharyngeal reflex was inhibited by 30-40% in the presence of ACE inhibition or HOE-140, whereas the reflex bradycardia to i.v. 5-HT was attenuated in the enalaprilat, losartan, and HOE-140 treatment groups. As the nasopharyngeal and Bezold-Jarisch-like reflexes are both mediated via the efferent vagus, but via different afferent pathways, it seems more likely that the vagolytic effect is due to an interaction of ω-CTX with the efferent pathway of the reflexes. The mechanism(s) by which this weak vagolytic effect of ω-CTX is "unmasked" in the presence of the antagonists is unknown but may help account for the further attenuation of the HR range of the baroreflex described earlier. Further studies are clearly necessary to understand these effects, for instance, in an isolated atrium preparation. Previous studies in our laboratory showed that vagally mediated bradycardia in rat isolated right atria is much less sensitive to ω-CTX than is sympathetically mediated tachycardia (data not published). This is also the case in vivo, as the dose of ω-CTX required to attenuate the vagal component of the baroreflex (10 μg/kg, i.v.) is higher than that required to affect the sympathetically mediated component (3 μg/kg; 8). Therefore the presence of the antagonists of angiotensin II or bradykinin receptors may increase the potency of ω-CTX in inhibiting vagally mediated bradycardia.
In conclusion, the blockade of N-type calcium channels with i.v. ω-CTX in conscious rabbits causes hypotension, tachycardia due to vagal withdrawal, and both sympatholytic and vagolytic actions on the baroreflex. Endogenous angiotensin II and bradykinin have a role in attenuating and slowing the blood pressure decrease after ω-CTX administration, as the hypotensive response is enhanced in the presence of the ACE inhibitor, enalaprilat, the angiotensin AT1-receptor antagonist, losartan, and the bradykinin B2-receptor antagonist, HOE-140. There is also an abstruse interaction between N-type calcium channel blockade and the efferent vagus in the presence of inhibitors of the renin-angiotensin system, as the latter attenuate the bradycardic effects of the nasopharyngeal and Bezold-Jarisch-like reflexes. Further studies are necessary to understand the complexity of these responses and to characterize fully the role of the N-type calcium channel in the autonomic nervous system.
Acknowledgment: This work was supported by a grant from Glaxo Wellcome Australia Pty. Ltd.
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