European Journal of Pharmacology, 215 (1992) 83-91

83

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52404

Pressor response to posterior hypothalamic administration of carbachol is mediated by muscarinic M 3 receptor J o h n R. M a r t i n Department of Pharmacology, Kirkscille College of Osteopathic Medicine, Kirkst,ille, MO 63501, U.S.A. Received 21 January 1992, accepted 28 January 1992

Unilateral microinjection of the acetylcholine receptor agonist carbachol into the posterior hypothalamic nucleus evokes a pressor response in the conscious, freely moving rat. To further localize this response 3.3 or 5.5 nmol of carbachol was microiniected in a volume of 50 nl directly into and outside the region of the posterior hypothalamic nucleus. Administration of carbachol outside the posterior hypothalamic nucleus failed to evoke a change in blood pressure indicating that the carbachol-induced pressor response is mediated from the posterior hypothalamic nucleus. Since posterior hypothalamic administration of atropine completely blocks the carbachol-induced increase in blood pressure and atropine blocks the three pharmacologically identified muscarinic receptor subtypes, methylatropine and progressively more selective muscarinic antagonists were administered into the posterior hypothalamic nucleus prior to 5.5 nmol of carbachol. Microiniection of the M1/M2/M 3 muscarinic antagonist methylatropine (0.19-12.5 nmol), the M I / M 3 antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine; 0.9-3.6 nmol), the M a antagonist pirenzepine (9.5-38 nmol), the M 2 antagonist methoctramine (5.5-44 nmol), or the M 3 antagonist p-F-HHSiD (para-fluoro-hexahydro-sila-difenidol; 2.1-8.3 nmol) inhibited the peak increase in mean arterial pressure and the area under the curve of the change in mean arterial pressure versus time plot in a dose-dependent manner. Log IDs0s calculated for the antagonists from the dose-response curves were found to correlate significantly with the log Kis of the antagonists for the muscarinic M 3 receptor subtype. These results demonstrate that the increase in mean arterial pressure evoked by microinjection of carbachol into the posterior hypothalamic nucleus is mediated by the muscarinic M 3 receptor. Posterior hypothalamic nucleus; Carbachol; Muscarinic receptor antagonists; (Rat)

1. Introduction Electrical stimulation of the posterior hypothalamic nucleus of the anesthetized rat is known to evoke an increase in mean arterial pressure (Eferakeya and Bufiag, 1974; Faiers et al., 1976; Yardley and Hilton, 1985). This nucleus contains choline acetyltransferase (Uchimura et al., 1975), the enzyme primarily responsible for the synthesis of acetylcholine suggesting that acetylcholine may serve as a neurotransmitter in this brain region. It is not surprising, then, that unilateral administration of acetylcholinesterase inhibitors directly into the posterior hypothalamic nucleus can elicit an increase in mean arterial pressure in the conscious, freely moving rat (Buccafusco and Brezenoff, 1979). In addition, the microinjection of the acetylcholine receptor agonist carbachol into the posterior hypothalamic

Correspondence to: J.R. Martin, Department of Pharmacology, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, MO 63501, U.S.A. Tel. 1.816.626 2384, fax 1.816.626 2483.

nucleus of the awake rat also evokes an increase in blood pressure (Buccafusco and Brezenoff, 1979; Martin et al., 1991). However, since electrical stimulation of hypothalamic areas lateral and caudal to the posterior hypothalamic nucleus can also evoke increases in mean arterial pressure (Faiers et al., 1976), it is possible that the increase in mean arterial pressure induced by administration of the acetylcholinesterase inhibitors or carbachoi into the posterior hypothalamic nucleus may have been due to diffusion, as a result of the use of relatively large volumes, into hypothalamic regions surrounding the posterior hypothalamic nucleus. This was examined by the administration of carbachol in a 50 nl volume into the posterior hypothalamic nucleus and surrounding regions. Administration of an adequate amount of atropine into the posterior hypothalamic nucleus prior to carbachol is known to inhibit the carbachol-induced increase in blood pressure (Buccafusco and Brezenoff, 1979; Martin et al., 1988). These results established the involvement of the acetylcholine muscarinic receptor in the mediation of the increase in blood pressure. Phar-

84 macological studies have revealed the existence of three muscarinic receptor subtypes which have been designated as M~, M 2, and M s (De Jonge et al., 1986; Doods et al., 1987). Atropine binds with high affinity to all three of the pharmacologically identified muscarinic receptors (Doods et al., 1987; Michel and Whiting, 1988) while agents which were beneficial to the phar~ macological characterization of the muscarinic receptor subtypes have somewhat selective affinity for these receptors. Of these agents pirenzepine has the highest affinity for the M I ( H a m m e r et al., 1980; Waelbroeck et al., 1988), methoctramine for the M 2 (Melchiorre et al., 1986; Michel and Whiting, 1988), p - F - H H S i D for the M s (Lambrecht et al., 1988) and 4-DAMP has essentially equal affinity for the Mj and M s (Barlow et al., 1980; Doods et al., 1987; Michel and Whiting, 1988; Kromer et al., 1991) muscarinic receptors. Although it is now agreed that 4-DAMP blocks the muscarinic M~ and M 3 receptor subtypes, this compound was originally classified as a muscarinic M 2 receptor antagonist. Based on this original classification, earlier studies suggested that the pressor response e v o k e d by i n t r a c e r e b r o v e n t r i c u l a r injection of physostigmine was mediated in part by muscarinic M 2 receptors located in the posterior hypothalamic nucleus (Xiao and Brezenoff, 1988). This suggestion was based on the finding that direct administration of 4D A M P into the posterior hypothalamic nucleus prior to intracerebroventricular injection of physostigmine could partially block the increase in blood pressure. Since it is now recognized (because of the relative selectivity of the muscarinic receptor antagonists) that several of these antagonists should be used to characterize the receptor subtype responsible for a particular response, five different muscarinic receptor antagonists were used in the present study. Methylatropine, 4DAMP, pirenzepine, methoctramine or p - F - H H S i D was administered into the posterior hypothalamic nucleus prior to the microinjection of carbachol into this same region. Potency of the ability of these agents to inhibit the carbachol-induced increase in blood pressure was compared to the relative affinity of these compounds for each of the pharmacologically identified muscarinic receptor subtypes.

2. Materials and methods

2.2. Surgical preparation After two to three days housing upon arrival, the rats were anesthetized with pentobarbital sodium (50 m g / k g i.p.). The left femoral artery was isolated and catheterized with medical grade polyvinyl chloride (PVC) tubing (0.2 mm ID x 0.5 mm OD) connected to S-54-HL PVC tubing (0.5 mm ID × 1.5 mm OD) for direct arterial pressure measurement. The catheter was tunnelled subcutaneously to the nape of the neck where it was anchored with suture and exited. The rats were then placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL). The scalp was incised, exposing the skull. A burr hole, which exposed the superior sagittal sinus, was made in the skull overlying the posterior hypothalamic nucleus. After cutting the dura mater adjacent to the superior sagittal sinus, a 23-gauge stainless steel guide cannula, previously melded to a plastic pedestal, was lowered through the cut and into the brain tissue. On the basis of the atlas of Paxinos and Watson (1982) and earlier studies (Martin et al. 1988, 1989, 1991), the coordinates, with respect to bregma, A - 3 . 8 mm, L - 0 . 5 mm, H - 5 . 7 mm, and 0 = 0 ° were used for the guide cannula to direct a 30-gauge stainless steel injection cannula to the posterior hypothalamic nucleus. Other animals were prepared in which the guide cannula was targeted to an area lateral or caudal to the posterior hypothalamic nucleus using the coordinates A - 3 . 8 m m , L - 1 . 5 m m , H - 5 . 7 r a m , a n d 0 = 0 °,and A - 5 . 1 ram, L - 0 . 5 mm, H - 5 . 7 ram, and 0 = 0 °, respectively. The guide cannula was anchored to the skull using mounting screws and dental caulk. Each animal was allowed at least two days recovery before being used for their first experiment. The initial experiment consisted of the administration of carbachol for the purpose of obtaining a control pressor response. Subsequent experiments consisted of the administration of carbachol, McN-A-343, or the microinjection of an antagonist prior to the administration of carbachol. After the initial experiment, each animal was allowed at least two days recovery between subsequent experiments. Each animal was used for no more than four experiments. The animals were not allowed access to food or water during the experimental trials. Each experiment was done during the light cycle at an ambient temperature of 21 _+ I°C. The arterial catheters were kept patent by flushing with 0.3 ml of heparinized saline (200 U / m l ) the day following surgical preparation, and then prior to and following each experiment.

2.1. Animals 2.3. Microinjections Male Sprague-Dawley rats (Hilltop, Scottdale, PA) weighing from 270 to 330 g were used in all experiments. All animals were housed separately on a 12-h l i g h t / d a r k cycle with food and water provided ad libiturn.

On the day of an experiment, an animal was connected to a pressure transducer (COBE, Lakewood, CO) via the arterial catheter. The pressure transducer was connected to a polygraph (Grass Model 7D) for

85

for determining agonist-induced changes in blood pressure. Areas under the curves were calculated using the trapezoidal rule. Data were pooled for each treatment group, calculated by A N O V A , and are presented as means _+ S.E. Differences between groups were tested for statistical significance by N e w m a n - K e u l s multiplerange test (P < 0.05 was considered significant).

continuous monitoring of blood pressure. Following an equilibration period of approximately 30 min, the animal was microinjected with carbachol (1.7-5.5 nmol) or McN-A-343 (31.5-63 nmol), or an antagonist 30 min prior to microinjection of 5.5 nmol of carbachol. The microinjections were made through a 30-gauge stainless steel injection cannula made to extend 2 m m beyond the end of the guide cannula so that the microinjections were made at a level of approximately H - 7.7 mm. The injection cannula was connected to a 5 pA Hamilton syringe with polyethylene (PE-20) tubing. The injection cannula was filled by backfilling with the appropriate volume of injectate. The syringe itself was filled with distilled water, and an air bubble was left between the water and injection solution. The injection cannula was lowered through the guide cannula and into the brain just prior to injection of the antagonist or agonist. Blood pressure was monitored for 30-60 min following microinjection of an agonist.

2.6. Drugs Carbamylcholine chloride (carbachol; Sigma Chemical Co., St. Louis, MO) and McN-A-343 (Research Biochemicals Inc., Natick, MA) were dissolved in 0.9% saline. Methylatropine bromide (Sigma), 4-DAMP (4diphenylacetoxy-N-methylpiperidine; Research Biochemicals Inc.), pirenzepine (Research Biochemicals Inc.), and methoctramine (Research Biochemicals Inc.) were dissolved in distilled water, p - F - H H S i D (para-fluoro-hexahydro-sila-difenidol; Research Biochemicals Inc.) was dissolved in 95% ethanol. Carbachol and McN-A-343 were administered in a volume of 50 nl over 30-60 s. Because of the solubilities of the antagonists these compounds were administered in volumes of 100-800 nl over 1-3 min (methylatropine was administered in 100 hi, 4-DAMP in 200 nl, pirenzepine in 100-200 nl, methoctramine in 200-800 nl, and p-FH H S i D in 100 nl). All dosages are expressed as the salt.

2. 4. Histology Each rat was killed after completion of the experiments by anesthesia with urethane (1.6 g / k g i.p.) followed by transcardial perfusion with phosphatebuffered Formalin. The brains were removed and kept in Formalin for at least one week at which time each brain was transferred to a 30% sucrose solution until histological preparation. Frozen brain sections (75/.~m thick) were cut through the posterior hypothalamic area using a freezing microtome. These sections were mounted onto slides and stained with cresyl violet.

3. Results

2.5. Statistical methods

3.1. Carbachol-induced increase in blood pressure and localization of area from which this effect is mediated

Percent inhibition of the control carbachol-induced pressor response was calculated for each animal administered an antagonist. The mean arterial pressure at the time of agonist microinjection was used as the baseline mean arterial pressure. Mean arterial pressures at various times following agonist microinjection were compared to the baseline mean arterial pressure

Microinjection of 5.5 nmol of carbachol in a volume of 50 nl directly into the posterior hypothalamic nucleus of a conscious rat evokes an increase in blood pressure as shown in fig. 1. This increase in blood pressure was dose-dependent as evident from the means of the peak increases in mean arterial pressure for 1.7, 3.3 and 5.5 nmol of carbachol (fig. 2; F(1,30) =

200 EE

100 0

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200 I 100

TIME (MIN)

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. . . .

15 ~ . . . .

20 ~ '

25 '

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i . . . .

30 i

. . . .

35 I

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1 p g CCh ~n 50 nl

Fig. 1. Microinjection of 5.5 nmol (1 p.g) of carbachol (CCh) in a volume of 50 nl into the posterior hypothalamic nucleus of the conscious, freely moving rat induces an increase in blood pressure as shown in this chart recording (AP, arterial pressure; MAP, mean arterial pressure).

86 40 35' 30 ~

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2s: 20' 15'

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10'

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CCh

--o--

McN-A-343

evoked an increase in blood pressure which was significantly greater than that evoked by administration lateral or caudal to the posterior hypothalamic nucleus. In addition, the change in blood pressure evoked by administration of carbachol outside the posterior hypothalamic nucleus did not differ from the change evoked by the administration of 50 nl of 0.9% saline microinjected either directly into, or outside the posterior hypothalamic nucleus. In contrast to carbachol, the administration of 31.5 or 63 nmol of the selective M 1 agonist McN-A-343 directly into the posterior hypothalamic nucleus failed to induce a significant, or a dosedependent increase in blood pressure (fig. 2). R e p e a t e d administration of 5.5 nmol of carbachol directly into the posterior hypothalamic nucleus with 2 - 3 days between consecutive administrations revealed no significant changes in either the time course of the response or the area under the curves calculated for the first 30 min following carbachol administration (fig. 3).

T__oT /

I

0 10-,o

10 -9

lO'S

10-~

DOSE OF AGONIST (mol)

Fig. 2. D o s e - r e s p o n s e curves depicting the peak increase in mean arterial pressure (MAP) evoked by 1.7 nmol (9 animals), 3.3 nmol (10 animals) or 5.5 nmol (13 animals) of carbachol (CCh), or 31.5 or 63 nmol (6 animals each dose) of the selective M~ agonist McN-A-343 following microinjection into the posterior hypothalamic nucleus.

15.70, P < 0.001). Examination of brain slices from these animals showed that the tip of the injection cannula was in the posterior hypothalamic nucleus. Microinjections of 50 nl of carbachol (3.3 or 5.5 nmol) made approximately 0.5 m m lateral or caudal to the border of the posterior hypothalamic nucleus failed to evoke a significant change in blood pressure. This is in constrast to the microinjection of the same amounts of carbachol in 200 or 500 nl volumes which evoked the same increase in blood pressure whether administered directly into, 0.5 m m lateral, or 0.5 mm caudal to the posterior hypothalamic nucleus. Thus the microinjection of 3.3 or 5.5 nmol of carbachol in a volume of 50 nl directly into the posterior hypothalamic nucleus --C-First 652 t 159

--m-A U C :

3.2. Inhibition of the carbachol-induced pressor response by muscarinic antagonists Microinjection of 0.39-12.5 nmol of the muscarinic receptor antagonist methylatropine directly into the posterior hypothalamic nucleus prior to carbachol resuited in a dose-dependent inhibition of the peak increase in blood pressure induced by 5.5 nmol of carbachol (fig. 4A; F(1,24)= 18.77, P < 0.001). This inhibition was also evident in the time course of the pressorresponse to carbachol as shown by a dose-dependent inhibition of the area under the time course curves

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Fig. 3. Changes in m e a n arterial pressure (MAP) induced by repeated microinjections of 5.5 nmol of carbachol (CCh) into the posterior hypothalamic nucleus. Each animal (n = 7) received four microinjections of carbachol with at least 48 h elapsing between consecutive injections. T h e time course curves were not significantly different from each other: The area under the curves (AUC) for the 30 min immediately following microinjection of carbachol, also were not significantly different from each other.

87 TABLE 1

a c c u m u l a t e d for the first 30 m i n following microinjection of carbachol (fig. 4B; F ( 1 , 2 4 ) = 7.59, P < 0.05). F r o m these dose r e s p o n s e curves, m e t h y l a t r o p i n e was calculated to have a log IDs0 of - 8 . 7 4 for i n h i b i t i o n of the p e a k increase in m e a n arterial p r e s s u r e a n d a log IDs0 of - 9 . 2 4 for i n h i b i t i o n of the area u n d e r the curve i n d u c e d by 5.5 n m o l of carbachol. M i c r o i n j e c t i o n of 4 - D A M P (0.9-3.6 n m o l ) into the posterior h y p o t h a l a m i c n u c l e u s prior to carbachol dose d e p e n d e n t l y i n h i b i t e d the peak increase in blood pressure evoked by 5.5 n m o l of carbachol (fig. 4A; F(1,23) = 13.7, P < 0.001). This i n h i b i t i o n was also dose-dep e n d e n t for the i n h i b i t i o n of the area u n d e r the curve (fig. 4B; F(1,23) = 10.09, P < 0.01). T h e log IDs0s calc u l a t e d for 4 - D A M P from these d o s e - r e s p o n s e curves were -8.63 for i n h i b i t i o n of peak increase in blood pressure, a n d - 8 . 7 7 for i n h i b i t i o n of the area u n d e r the curve. P i r e n z e p i n e ( 9 . 5 - 3 8 n m o l ) a d m i n i s t e r e d directly into the posterior h y p o t h a l a m i c n u c l e u s prior to carbachol also i n h i b i t e d the increase in blood pressure i n d u c e d by 5.5 n m o l of carbachol. T h e i n h i b i t i o n of the peak increase in m e a n arterial p r e s s u r e was d o s e - d e p e n d e n t (fig. 4A; F(1,23) = 26.33, P < 0.001) as was the inhibition of the area u n d e r the curve (fig. 4B; F ( 1 , 2 3 ) = 19.21, P < 0.001). T h e log IDs0s calculated from the d o s e - r e s p o n s e curves for the i n h i b i t i o n of the carbac h o l - i n d u c e d p e a k increase in blood p r e s s u r e was - 7 . 4 5 a n d for the i n h i b i t i o n of the area u n d e r the curve was - 7.62. T h e c a r b a c h o l - i n d u c e d pressor response, both the time course a n d the area u n d e r the curve, was also i n h i b i t e d by m e t h o c t r a m i n e ( 5 . 5 - 4 4 nmol). This inhibition was d o s e - d e p e n d e n t for the i n h i b i t i o n of the peak increase in m e a n arterial pressure (fig. 4A; F ( 1 , 3 1 ) =

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Log Kis used for the correlation with the log lDsos determined in the present study and their sources. Antagonist PZ 4-DAMP MTT p-F-HHSiD ATR

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20.78, P < 0.001) b u t not for the area u n d e r the curve (fig. 4B; F ( 1 , 2 2 ) = 2 . 5 2 , P > 0 . 1 ) . However, b e c a u s e t h e r e was a significant d o s e - d e p e n d e n t effect on the m e a n s of the area u n d e r the curve ( F ( 1 , 1 ) = 431.02; P < 0.05) a log lDs0 was calculated for the i n h i b i t i o n of the area u n d e r the curve by m e t h o c t r a m i n e which was - 7 . 3 8 . A log ID50 for the i n h i b i t i o n of the peak increase in m e a n arterial p r e s s u r e was also calculated a n d f o u n d to be - 7 . 3 0 . A d m i n i s t r a t i o n of p - F - H H S i D (2.1-8.3 n m o l ) into the posterior h y p o t h a l a m i c n u c l e u s prior to carbachol also i n h i b i t e d the increase in blood pressure. T h e i n h i b i t i o n of the p e a k increase in blood pressure was d o s e - d e p e n d e n t (fig. 4A; F(1,22) = 21.08, P < 0.001) as was the i n h i b i t i o n of the area u n d e r the curve (fig. 4B; F(1,22) = 8.78, P < 0.01). T h e log IDs0 for the inhibition of the peak increase in the m e a n arterial pressure was - 8 . 2 4 a n d area u n d e r the curve was - 8 . 3 7 .

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Mi

a Kromer et al., 1991. b Michel and Whiting, 1988. c Lambrecht et al., 1989. d Averaged from Doods et al., 1987; Michel and Whiting, 1988; and Kromer et al., 1991. CAveraged from Doods et al., 1987; Michel and Whiting, 1988; Eglen et al., 1990; and Kromer et al., 1991. fAveraged from Doods et al., 1987; and Michel and Whiting, 1988.

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Log K i

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OF ANTAGONIST

I0-' (tool)

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10 7 (tool)

Fig. 4. Several muscarinic antagonists inhibited the carbachol-induced increase in blood pressure in a dose-dependent manner as determined from the linear portions of the dose-response curves. The muscarinic antagonists inhibited the peak increase in mean arterial pressure as shown in panel A, and the area under the curve determined over the first 30 min following microinjection of carbachol as shown in panel B. All points consist of eight or nine animals, except for the 44 nmol dose of methoctramine and the 12.5 nmol dose of methylatropine both of which consist of six animals.

88

None of the antagonists had any significant effect on mean arterial pressure during the 30 rain prior to the administration of carbachol.

that the log lDs.s for the five antagonists used in this study correlated significantly with the log K~s pubfished for these antagonists at the muscarinic M 3 receptor (fig. 5). There was a lack of a significant correlation between the log IDs0s and the log Kis of these antagonists for the M~ and the M 2 muscarinic receptors (fig. 5).

3.3. Correlation of the log IDsos for the muscarinic antagonists to their log Kis A correlation analysis was carried out between the log IDs0s calculated in this study and the published log K~s for the antagonists used in this study (the log K i for atropine was used for methylatropine). The log K~s used in the present study, and the sources of these log K~s are shown in table 1. This analysis showed A

4. Discussion Microinjection of the cholinergic agonist carbachol into the posterior hypothalamic nucleus is known to

M,

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