Naunyn-Schmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 292, i - 7 (1976)
Pharmacology
9 by Springer-Verlag 1976
Hypothalamic Superfusion with Muscarinic Drugs Their Effects on Pressor Responses to Hypothalamic Stimulation* A. Philippu and N. Bohuschke ]{nstitut ffir Pharmakologie und Toxikologie der Universitfit Wtirzburg, Versbacher Landstrasse 9, D-8700 Wfirzburg, Federal Republic of Germany
Summary. The posterior hypothalamus of cats anaesthetized with pentobarbital sodium was superfused with artificial cerebrospinal fluid through a push-pull cannula and electrically stimulated with the noninsulated tip of the cannula. The effects of muscarinic drugs on the pressor response to stimulation of the hypothalamus were investigated. Superfusion with muscarine, oxotremorine or N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602) decreased the pressot responses to hypothalamic stimulation. Superfusion with methylatropine did not influence the pressor responses to hypothalamic stimulation; however, superfusion with methylatropine 60 min prior to and during superfusion with the muscarinic drugs abolished the inhibitory effects of muscarine and oxotremorine and temporarily reversed that of A H R 602 on the pressor responses. Superfusion of the posterior hypothalamus with arecoline enhanced the rise of blood pressure elicited by hypothalamic stimulation. When the hypothalamus was superfused with hexamethonium 60 min prior to and during superfusion with arecoline, arecoline reduced the pressor responses to electrical stimulation of the hypothalamus. Superfusion with methylatropine prior to and together with an ineffective concentration of arecoline increased the rise of blood pressure elicited by hypothalamic stimulation. From the drugs studied here only oxotremorine caused a fall of the "resting" arterial blood pressure; it was abolished by the intravenous injection of methylatropine. From these results it was concluded that superfusion of the posterior hypothalamus with muscarinic drugs impairs the pressor responses ro hypothalamic Send offprint requests to." A. Philippu at the above address. * This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.
stimulation. Drugs possessing both nicotinic and muscarinic properties either enhance or diminish the pressor responses according to their relative potencies on the two types of receptor.
Key words: Hypothalamus - Electrical stimulation Pressor responses - Muscarinic drugs - Methylatropine - Hexamethonium.
INTRODUCTION Recently, the posterior hypothalamus of cats was superfused with cholinergic agents through a double barrelled push-pull cannula and electrically stimulated with the cannula tip. It was shown that both 1,]dimethyl-4-phenylpiperazinium (DMPP) and carbachol caused a dose-dependent enhancement of the pressor responses to the electrical stimulation of the hypothalamus; furthermore, their effects were abolished when the hypothalamus was superfused with hexamethonium (Philippu et al., 1974a). Hence, it was concluded that the enhancement of the pressor responses was due to the nicotinic character of these agents. In the present paper the posterior hypothalamus was superfused with drugs acting predominantly on muscarinic receptors and their effects on the rise of blood pressure elicited by hypothalamic stimulation were studied. Preliminary results of this study were presented at the Meeting of the German Pharmacological Society in Graz, September 1974 (Philippu et al., 1974 b) and the Third Symposium of the International Society of Hypertension, Milan, September 1974 (Philippu, 1975).
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METHODS
A. Superfusion and Stimulation of the Hypothalamus with a Push-Pull Cannula Cats of either sex and weighing 1.6-2.2 kg were anaesthetized with pentobarbital sodium (36 mg/kg i.p.). Tracheotomy was performed and the head fixed in a stereotaxic instrument (la Pr6cision Cinematographique). The mean arterial blood pressure was monitored from the femoral artery with a Statham transducer connected to a Schwarzer recorder. For the intravenous application of drugs a catheter was inserted into the femoral vein. A push-pull eannula with its stilettes (Philippu et aI., 1973) was stereotaxically inserted into the posterior hypothalamus according to the stereotaxic atlas of Reinoso-Suarez (1961) (anterior 10 mm, lateral: 2 ram, vertical: 7 mm above zero point). The outer surface of the double barrelled cannula was insulated. The stilettes were removed and the posterior hypothalamus was superfused through the cannula with artificial cerebrospinal fluid (CSF) (Merlis, 1940). The CSF was saturated with a mixture of 95 ~o Oz and 5 ~o CO2; the pH of the saturated fluid was 7.2. Hypothalamic superfusion was carried out at the rate of 0.075 ml/min and the hypothalamus stimulated with the non-insulated tip of the cannula. The indifferent electrode was attached at the ipsilateral ear. Stimulation was carried out every 10 rain with 8 - 1 0 V, 40 Hz, 6 msec with square waves (H. Sachs Stimulator I). A stimulation period of 1 0 - 3 0 sec elicited a rise of the arterial blood pressure of 45 - 50 mm Hg. Optimal voltage and stimulation period were adjusted at the beginning of, and maintained throughout, the experiment. Superfusion with drugs was carried out only when the pressor responses to at least three stimulation periods were identical. The drugs were dissolved in CSF and snperfused for 60 min. In most experiments, the hypothalamus was subsequently superfused with drug-free CSF for another 60 min. At the end of the experiments the brain was removed and the localization of the push-pull cannula verified.
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Fig. 1. Effect of superfusion of the posterior hypothalamus with muscarine on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with muscarine. Abscissa: time in min after beginning of the superfusion with muscarine. Circles: controls (n = 6), squares: 5 x I 0- 3M muscarine (n = 6), triangles: 1 x 10-3M muscarine (n = 6). Muscarine was superfused for 60 min (horizontal bar). For difference between controls and 5 x 10-3M muscarine and between controls and 1 x i0 -a M muscarine, P < 0.001 ; between 5 x 10 .3 M and 1 x 10-3M muscarine, P < 0.01. Mean values +_ S.E.M.
C. Substances Used
responses were taken as 100 ~o and subsequently the hypothalamus was superfused with various concentrations of muscarine for 60 rain. Muscarine gradually decreased the pressor responses; however, 5 x 10 -3 M muscarine seemed to be less effective than I x 10 -3 M. The inhibitory effect of muscarine persisted during the subsequent superfusion with muscarine-free CSF.
N-methylatropine bromide (Merck, Darmstadt), oxotremorine (EGA Chemie, Steinheim), arecoline hydrochloride (Ferak, Berlin), hcxamethonium chloride (Fluka, Buchs), N-benzyl-3-pyrrolidyl acetate methobromide (A. H. Robins, Richmond), (+_)-muscarine chloride (Ciba-Geigy, Basel).
B. Effect of Centrally Applied MethyIatropine on the Enhancement of Pressor Responses Caused by Muscarine
B. Statistical Analysis The statistical significance was calculated by analysis of variance (Winne, 1965). In some experiments, Student's t-test was additionally used.
RESULTS
A. Effects of Superfusion of the Hypothalamus with Muscarine on the Pressor Responses to Hypothalamic Stimulation The posterior hypothalamus was superfused with CSF through the double barrelled push-pull cannula. Electrical stimulation every 10 min elicited pressor responses of constant height for at least 12 stimulation periods (Fig. 1). In other animals the constant pressor
In order to examine whether the inhibition of the pressor responses caused by muscarine was due to the muscarinic properties of this agent, the hypothalamus was superfused with 5 x 10-4M methylatropine for 60 min and thereafter with CSF which contained this concentration of methylatropine and 1 x 10-3M muscarine for another 60 min. Central application of methylatropine abolished the inhibitory effect of muscarine on the pressor responses. 5 x 10-4M methylatropine did not affect the rise of blood pressure caused by hypothalamic stimulation (Fig. 2).
Effects of Centrally Applied Muscarinic Drugs on Pressor Responses
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Fig. 2. Effect of superfusion of the posterior hypothalarnus with methylatropine on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with methylatropine. Abscissa: time in rain after beginning of the superfnsion with methylatropine. Circles: controls (n = 6), squares: 5 • 10-* M methylatropine (n ~ 6). Methylatropine was superfused for 60 min (horizontal bar). Difference between controls and methylatropine n.s. Mean values • S.E.M.
C. Effect of Hypothalamic Superfusion with AHR 602 on the Pressor Responses Superfusion of the hypothalamus for 60 min with I x 10 -2 M AHR 602 which acts on muscarinic receptors (Franko et al., 1963; Jones et al., 1963; Jaramillo and Volle, 1967) did not affect the rise of blood pressure elicited by electrical stimulation of the hypothalamus, while superfusion with 5 x 10 -2 M greatly reduced the pressor responses (Fig. 3). The impairment of the pressor responses was persistent and became even more pronounced during the subsequent superfusion with drug-free CSF. Hypothalamic superfusion with 5 x 10-* methylatropine for 60 rain prior to and together with AHR 602 abolished the inhibitory effect of the latter agent on the pressor responses (Fig. 4). In fact, after superfusion with methylatropine, AHR 602 seemed to temporarily enhance the pressor responses. Although calculation of statistical significance of the curves as a whole by analysis of variance did not reveal significant difference between control animals and animals treated with the drugs, calculation by Student's t-test showed that the pressor response at 20 min was enhanced when the hypothalamus was superfused with methylatropine and AHR 602. The "resting" arterial blood pressure was not affected either by muscarine and AHR 602 or by methylatropine.
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Ordinate: rise of blood pressure as per cent of that before superfusion with A H R 602. Abscissa : time in rain after beginning of the superfusion with AHR602. Circles: controls (n = 5), squares: I x 10 -z M A H R 602 (n = 5), triangles: 5 x 10 -2 M AHR (n = 5). A H R 602 was superfused for 60 rain (horizontal bar). For differences between controls and 5 • 10- 2M A H R 602 and between I x 10- 2M A H R 602 and 5 x 10-2M AHR 602, P < 0.001. Difference between controls and i x 10-2M A H R 602 n.s. Mean values _+ S.E.M.
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Fig. 4. Effect of superfusion of the posterior hypothalamus with methylatropine and A H R 602 in the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with A H R 602. Abscissa: time in rain after beginning of the superfusion with A H R 602. Circles : controls (n = 5), squares: after a presuperfusion with 5 x 10-4M methylatropine for 60 rain the hypothalamus was superfused with 5 x 10-4 M methylatropine and 5 x 10-SM A H R 602 for 60 min (n = 5). Difference between controls (superfusion with CSF) and superfusion with methylatropine and A H R 602 n.s. * P < 0.05 (Student's t-test). Mean values • S.E.M.
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D. Effects of Hypothalamic Superfusion with Oxotremorine on the "Resting" Arterial Blood Pressure and on the Pressor Responses to Hypothalamic Stimulation; Impairment of the Effect of Oxotremorine by Methylatropine Superfusion with 1 x 10-=M or 5 x 1 0 - 2 M oxotremorine for 60 rain did not appreciably affect the pressor responses to hypothalamic stimulation (Fig. 5). However, the evaluation of the height of the pressor responses was very difficult and inaccurate, because hypothalamic superfusion with this agent caused a dose-dependent fall of the "resting" arterial blood pressure (Fig. 6). The depression of the blood pressure might be due to leakage Of oxotremorine from the hypothalamic area and action of the agent on muscarinic receptors of peripheral tissues. Indeed, an intravenous injection of 0.5 mg/kg methylatropine which does not readily penetrate the blood-brain barrier abolished the effect of the hypothalamic application of oxotremorine on the "resting" blood pressure (Fig. 6). Under these conditions, i.e. after inhibition of the muscarinic receptors of peripheral tissues, superfusion of the hypothalamus with 5 x 10-ZM oxotremorine caused a Very pronounced reduction of the pressor response to hypothalamic stimulation (Fig. 7). The effect of oxotremorine on the pressor responses was of hypothalamic origin, since it was
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Fig. 6. Effect of superfusion of the posterior hypothalamus with oxotremorine on the "resting" arterial blood pressure. Ordinate: arterial blood pressure as per cent of that before superfusion with oxotremorine. Abscissa: time in rain after beginning of the superfusion with oxotremorine. Oxotremorine was superfused for 60 rain (horizontal bar), Open circles: controls (n = 5)~ open triangles: ] x 10-2M oxotremorine (n = 5), solid circles: 5 • 10-2M oxotremorine (n = 5), solid triangles: 5 • oxotremorine after intravenous injection of methylatropine (0.5 mg/kg). For differences between controls and 5 x 10-2M oxotremorine and between 5 x l 0 - = M oxotremorine and 5 • oxotremorine after intravenous injection of methylatropine, P < 0.001. Differences between controls and 1 x 10-2M oxotremorine and between controls and 5 x I 0 - 2 M oxotremorine after intravenous injection of methylatropine n.s. Mean arterial blood pressure before beginning of the superfusion (mm Hg); controls: 100 • 2, 1 x 10-ZM oxotremorine: 80 __ 4, 5x 1 0 - a M oxotremorine: 87 + 4, 5 x 10-=M oxotremorine after intravenous injection ofmethylatropine: 98 + 4. All values are means + S.E.M.
completely inhibited when the hypothalamus was additionally superfused with 5 x 10-*M methylatropine for 60 rain prior to and during superfusion with oxotremorine.
E. Effect of Hypothalamic Superfusion with Arecoline on the Pressor Responses," Influence of Methylatropine and Hexamethonium on the Effect of Arecoline Hypothalamic superfusion with arecoline (1 x 10-2 M) was ineffective on the pressor responses to hypothalamic stimulation, while a higher concentration (5 x 10 -2 M) increased them (Fig. 8). Since the enhancement of the pressor responses might be due to an action on nicotinic receptors, the hypothalamus was superfused with 1 x 10 -2 M hexamethonium for
Effects of Centrally Applied Muscarinic Drugs on Pressor Responses
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Fig. 7. Effect of superfusion of the posterior hypothalamus with oxotremorine on the pressor responses to hypothalamic stimulation after intravenous injection of methylatropine. Ordinate: rise of blood pressure as per cent of that before superfusion with oxotremorine, Abscissa: time in min after beginning of the superfusion with oxotremorinc. Oxotremorine was superfused for 60 rain (horizontal bar). Circles: controls (n = 6), squares: 5 x 10-2M oxotremorine after intravenous injection of methylatropine (0.5 rag/ kg). For difference between controls and oxotremorine, P < 0.001. Mean values + S.E.M.
60 rain prior to arecoline; subsequently, the hypothalamus was superfused with the same concentration of hexamethonium and 5x 1 0 - 2 M arecoline. This concentration of hexamethonium does not influence the pressor responses to hypothalamic stimulation (Philippu et al., 1974a) but reversed the effect of arecoline (Fig. 9); thus, after superfusion with hexamethonium arecoline caused a slight but significant decrease of the pressor responses to hypothalamic stimulation. On the other hand, superfusion of the hypothalamus with 5x l O - 4 M methylatropine for 60 rain prior to and during superfusion with 1 x 10-2M arecoline led to a significant enhancement of the pressor responses (Fig. 9), although this concentration of arecoline failed to influence the pressor responses in the absence of methylatropine (cf. Fig. 8).
DISCUSSION The intravenous injection of physostigmine elicits a rise of the arterial blood pressure which is due to an action of the drug on the central nervous system (Varagi6, 1955; Varagid and Vojvodid, 1962). Similarly, the intraventricular injection of muscarinic agents such as oxotremorine and arecoline causes pressor responses (Phan and Gy6rgy, 1973). On the other hand, Brezenoff (:1972) observed that the responses of the
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Fig. 8. Effect of superfusion of the posterior hypothalamus with arecoline on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with arecoline. Abscissa: time in min after beginning of the superfusion with arecoline. Circles : controls (n = 6), triangles : l x 10-2M arecoline (n = 6), squares: 5 x 10 2M arecoline (n = 6). Arecoline was superfused for 60 rain (horizontal bar). For difference between controls and 5 • 10-2M arecoline, P < 0.001. Difference between controls and i x 10-2M arecoline n.s. Mean values • S.E.M.
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Fig. 9. Effect of superfusion of the posterior hypothalamus with arecoline and hexamethonium or arecoline and methylatropine on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before snperfusion with arecoline. Abscissa: time in rain after beginning of the superfusion with arecoline. Circles: controls (n = 6), solid squares: after a presuperfusion with I x 10-2M hexamethonium for 60 rain the hypothalamus was superfused with 1 x I 0 - 2 M hexamethonium and 5 x 10 - 2 M arecoline (n = 6), open squares : after presuperfusion with 5 • 10-4M methylatropine for 60 rain the hypothalamus was superfused with 5 x 10-4M methylatropine and 1 x 10-2M arecoline (n = 6). For differences between controls and superfusion with arecoline and hexamethonium or between controls and superfusion with arecoline and methylatropine P < 0.01. Mean values _+ S.E.M.
6 blood pressure to intrahypothalamic application of cholinergic drugs is greatly dependent on the area of the hypothalamus involved; thus, the posterior and the ventromedial nuclei of the hypothalamus seem to mediate a rise of the blood pressure, while the dorsomedial and the premammillary nuclei mediate hypotensive responses. Furthermore, the hypotensive response following the intrahypothalamic injection of muscarine, methacholine or oxotremorine was prevented by the central application of atropine (Brezenoff and Wirecki, 1970). Another approach to the effects of centrally applied drugs on the blood pressure and to the localization of their sites of action is to apply drugs centrally and to examine their effects on the pressor responses elicited by electrical stimulation of the hypothalamus. By using a double-barrelled push-pull cannula it was recently found that superfusion of the posterior hypothalamus with DMPP or carbachol increased the rise of blood pressure caused by hypothalamic stimulation, the enhancement of the pressor responses being abolished by hexamethonium (Philippu et al., 1974 a). From the agents with muscarinic properties used in this study, only oxotremorine led to a fall of the "resting" arterial blood pressure. This was due to leakage of the drug into the blood probably because of its highly lipophilic character (Herz et al_, 1966). Indeed, intravenous administration of methylatropine which does not readily penetrate the blood-brain barrier, completely inhibited the effect of oxotremorine on the "resting" arterial blood pressure. Under these conditions, superfusion of the posterior hypothalamus with oxotremorine elicited a very pronounced reduction of the pressor responses caused by dectrical stimulation of the hypothalamus. The depression of the pressor responses was probably due to an action of oxotremorine on muscarinic receptors of the posterior hypothalamus, since it was abolished when the hypothalamus was superfused with methylatropine prior to and during superfusion with oxotremorine. Thus, it seemed that the impairment of the pressor responses by oxotremorine was due to the muscarinic properties of the agent. In this connection it should be noted that the concentration of methylatropine (5 x 10 -4 M) used in the present experiments did not affect the pressor responses to hypothalamic stimulation; it merely inhibited the reduction of the pressor responses caused by oxotremorine. A similar impairment of the pressor responses to hypothalamic stimulation was observed when the posterior hypothalamus was superfused with A H R 602. However, superfusion of the hypothalamus with methylatropine prior to and during superfusion with A H R 602 did not merely abolish the reduction of the pressor responses by this agent; in fact, under
A. Philippu and N. Bohuschke these conditions A H R 602 elicited a temporary enhancement of the pressor responses. Hence, A H R 602 seemed to possess two actions on the pressor responses. After inhibition of the depressant one, the enhancing action became visible; the second one might be due to an action of A H R 602 on nicotinic receptors, since nicotinic agents increase the rise of blood pressure caused by hypothalamic stimulation (Philippu et al., 1974a). On the other hand, Fozard and Muscholl (1972) have shown that A H R 6 0 2 facilitates the release of noradrenaline from the heart caused by electrical stimulation of the sympathetic nerves. This effect of A H R 602 is atropine-resistant (Fozard and Muscholl, 1972) and might be of significance for the enhancement of the pressor response. Although muscarine also inhibited the pressor responses, the effect of a higher concentration (5x 10 .3 M) of the drug was less than that of a lower one (1 x 10 -3 M). Since muscarine possesses both muscarinic and nicotinic properties (Ambache et al., 1956; Konzett and Waser, 1956), it might be assumed that the failure of the dose-response relationship was due to a nicotine-like action when higher concentrations of muscarine were used. Indeed, the inhibition of the pressor responses by the lower concentration of muscarine was blocked when the hypothalamus was superfused with methylatropine. Unfortunately, the amount of muscarine was insufficient for a more extensive investigation. The enhancement by arecoline (5x 10 -2 M) of the pressor responses to hypothalamic stimulation was not surprising, since also this agent possesses nicotinic and muscarinic properties (Feldberg and Vartiainen, 1934; Euler and Domeij, 1945). If the increase of the pressor responses were indeed due to the nicotinic properties of this agent, its enhancing action should be impaired by hexamethonium. In fact, superfusion of the hypothalamus with hexamethonium reversed the action of arecoline, thus leading to a significant reduction of the pressor responses; probably, block of the nicotinic receptors by hexamethonium unmasked the action of arecoline on the muscarinic ones. On the other hand, superfusion of the hypothalamus with a lower concentration of arecoline (1 x 10 -2 M) did not influence the pressor responses. However, superfusion of the hypothalamus with methylatropine prior to an during superfusion with the low concentration of arecoline greatly increased the pressor responses to hypothalamic stimulation. Thus, the enhancing action of arecoline seemed to be due to its nicotinic character, because it was abolished by hexamethonium; superfusion with methylatropine blocked the muscarinic receptors, thus enhancing the nicotinic component of the areco-
Effects of Centrally Applied Muscarinic Drugs on Pressor Responses
line action. It is noteworthy that superfusion of the hypothalamus with hexamethonium does not affect either the pressor responses to hypothalamic stimulation or the "resting" arterial blood pressure, while it completely inhibits the increase of pressor responses caused by hypothalamic application of DMPP (Philippu et al., 1974a). These results indicate that superfusion of the posterior hypothalamus with drugs possessing only muscarinic properties, such as oxotremorine, depresses the pressor responses to hypothalamic stimulation. Drugs possessing both muscarinic and nicotinic properties, such as muscarine, arecoline and AHR 602, may either enhance or impair the pressor responses; their overall action seems to depend on the relative potencies on the two types of receptor. Acknowledgements. The authors wish to thank Dr. H. Brunner, Ciba-Geigy, Basel, for muscarine chloride and Dr. N. Chremos, A. H. Robins, Richmond, for AHR 602. They also thank Miss Inge Smolarek for her skilful technical assistance.
REFERENCES Ambache, N., Perry, W. L. M., Robertson, P. A.: The effect of muscarine on perfused superior cervical ganglia of cats. Brit. J. PharmacoI. 11, 442-448 (1956) Brezenoff, H. E.: Cardiovascular responses to intrahypothalamic injections of carbachol and certain cholinesterase inhibitors. Neuropharmacology 11,637-644 (1972) Brezenoff, H. E., Wirecki, T. S. : The pharmacological specificity of muscarinic receptors in the posterior hypothalamus. Life Sci. 9, 99-109 (1970) v. Euler, U. S., Domeij, B.: Nicotine-like actions of arecoline. Acta pharmacol. (Kbh.) 1, 263-269 (1945) Feldberg, W., Vartiainen, A. : Further observations on the physiology and pharmacology of sympathetic ganglion. J. Physiol. (Lond.) 83, 103-128 (1935) Fozard, J. R , Muscholl, E. : Effects of several muscarinic agonists on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart. Brit, J. Pharmacol. 45, 616-629 (1972) Franko, B. V., Ward, J. W., Alphin, R. S. : Pharmacologic studies of N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602),
7 a ganglion stimulating agent. J. Pharmacol. exp. Ther. 139, 2 5 - 30 (1963) Herz, A., Holzh/iuser, H., Teschemacher, H. : Zentrale und periphere Wirkungen yon Cholinomimetica und ihre Abh/ingigkeit v o n d e r Lipoidl6slichkeit. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 253, 280-297 (1966) Jaramillo, J., Volle, R. L. : Ganglion blockade by muscarine, oxotremorine and AHR-602. J. Pharmacol. exp. Ther. 158, 80-88 (1967) Jones, A., Gomez Alonso de la Sierra, B., Trendelenburg, U. : The pressor response of the spinal cat to different groups of ganglionstimulating agents. J. Pharmacol. exp. Tber. 139, 312--320 (1963) Konzett, H., Waser, P. G. : Zur ganglionS.ren Wirkung von Muscarin. Helv. physiol, pharmacol. Acta 14, 202-206 0956) Merlis, J. K. : The effect of changes in the calcium content ofcerebrospinal fluid on spinal reflex activity in the dog. Amer. J. Physiol. 13, 6 7 - 72 (1940) Phan, D. V., Gy6rgy, L.: The pressor effect of oxotremorine and arecoline in rats. Acta physiol. Acad. Sci. hung. 43, 7 1 - 8 4 (1973) Philippu, A.: Central regulation of the arterial blood pressure. Clin. Sci. molec. Med. 48, 191s- 194s (1975) Philippu, A., Demmeler, R., Roensberg, G.: Effects of centrally applied drugs on pressor responses to hypothaIamic stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol. 282, 389-400 (1974a) Philippu, A., Przuntek, H., Roensberg, W.: Superfusion of the hypothalamus with gamma-aminobutyric acid : effect on release of noradrenaline and blood pressure. Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 103--118 (1973) Philippu, A., Reich, N., Bohuschke, N. : Influence of cholinergic drugs on the pressor responses to hypothalamic stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol. 285, R 62 (1974b) Reinoso-Suarez, F.: Topographischer Hirnatlas der Katze. Darmstadt: Merck AG 1961 Varagic, V. : The action of eserine on the blood pressure of the cat. Brit. J. Pharmacol. 10, 349-353 (1955) Varagic, V., Vojvodic, N. : Effect of guanethidine, hemicholinium and mebutamate on the hypertensive response to eserine and catecholamines. Brit. J. Pharmacol. 19, 451-457 (1962) Winne, D.: Zur Auswertung von Versuchungsergebnissen: die Priifung, ob Kurven sich in ihrem Verlauf unterscheiden. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 250, 383396 (1965)
Received June 25/Accepted September 29, 1975
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 292, i - 7 (1976)
Pharmacology
9 by Springer-Verlag 1976
Hypothalamic Superfusion with Muscarinic Drugs Their Effects on Pressor Responses to Hypothalamic Stimulation* A. Philippu and N. Bohuschke ]{nstitut ffir Pharmakologie und Toxikologie der Universitfit Wtirzburg, Versbacher Landstrasse 9, D-8700 Wfirzburg, Federal Republic of Germany
Summary. The posterior hypothalamus of cats anaesthetized with pentobarbital sodium was superfused with artificial cerebrospinal fluid through a push-pull cannula and electrically stimulated with the noninsulated tip of the cannula. The effects of muscarinic drugs on the pressor response to stimulation of the hypothalamus were investigated. Superfusion with muscarine, oxotremorine or N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602) decreased the pressot responses to hypothalamic stimulation. Superfusion with methylatropine did not influence the pressor responses to hypothalamic stimulation; however, superfusion with methylatropine 60 min prior to and during superfusion with the muscarinic drugs abolished the inhibitory effects of muscarine and oxotremorine and temporarily reversed that of A H R 602 on the pressor responses. Superfusion of the posterior hypothalamus with arecoline enhanced the rise of blood pressure elicited by hypothalamic stimulation. When the hypothalamus was superfused with hexamethonium 60 min prior to and during superfusion with arecoline, arecoline reduced the pressor responses to electrical stimulation of the hypothalamus. Superfusion with methylatropine prior to and together with an ineffective concentration of arecoline increased the rise of blood pressure elicited by hypothalamic stimulation. From the drugs studied here only oxotremorine caused a fall of the "resting" arterial blood pressure; it was abolished by the intravenous injection of methylatropine. From these results it was concluded that superfusion of the posterior hypothalamus with muscarinic drugs impairs the pressor responses ro hypothalamic Send offprint requests to." A. Philippu at the above address. * This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.
stimulation. Drugs possessing both nicotinic and muscarinic properties either enhance or diminish the pressor responses according to their relative potencies on the two types of receptor.
Key words: Hypothalamus - Electrical stimulation Pressor responses - Muscarinic drugs - Methylatropine - Hexamethonium.
INTRODUCTION Recently, the posterior hypothalamus of cats was superfused with cholinergic agents through a double barrelled push-pull cannula and electrically stimulated with the cannula tip. It was shown that both 1,]dimethyl-4-phenylpiperazinium (DMPP) and carbachol caused a dose-dependent enhancement of the pressor responses to the electrical stimulation of the hypothalamus; furthermore, their effects were abolished when the hypothalamus was superfused with hexamethonium (Philippu et al., 1974a). Hence, it was concluded that the enhancement of the pressor responses was due to the nicotinic character of these agents. In the present paper the posterior hypothalamus was superfused with drugs acting predominantly on muscarinic receptors and their effects on the rise of blood pressure elicited by hypothalamic stimulation were studied. Preliminary results of this study were presented at the Meeting of the German Pharmacological Society in Graz, September 1974 (Philippu et al., 1974 b) and the Third Symposium of the International Society of Hypertension, Milan, September 1974 (Philippu, 1975).
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METHODS
A. Superfusion and Stimulation of the Hypothalamus with a Push-Pull Cannula Cats of either sex and weighing 1.6-2.2 kg were anaesthetized with pentobarbital sodium (36 mg/kg i.p.). Tracheotomy was performed and the head fixed in a stereotaxic instrument (la Pr6cision Cinematographique). The mean arterial blood pressure was monitored from the femoral artery with a Statham transducer connected to a Schwarzer recorder. For the intravenous application of drugs a catheter was inserted into the femoral vein. A push-pull eannula with its stilettes (Philippu et aI., 1973) was stereotaxically inserted into the posterior hypothalamus according to the stereotaxic atlas of Reinoso-Suarez (1961) (anterior 10 mm, lateral: 2 ram, vertical: 7 mm above zero point). The outer surface of the double barrelled cannula was insulated. The stilettes were removed and the posterior hypothalamus was superfused through the cannula with artificial cerebrospinal fluid (CSF) (Merlis, 1940). The CSF was saturated with a mixture of 95 ~o Oz and 5 ~o CO2; the pH of the saturated fluid was 7.2. Hypothalamic superfusion was carried out at the rate of 0.075 ml/min and the hypothalamus stimulated with the non-insulated tip of the cannula. The indifferent electrode was attached at the ipsilateral ear. Stimulation was carried out every 10 rain with 8 - 1 0 V, 40 Hz, 6 msec with square waves (H. Sachs Stimulator I). A stimulation period of 1 0 - 3 0 sec elicited a rise of the arterial blood pressure of 45 - 50 mm Hg. Optimal voltage and stimulation period were adjusted at the beginning of, and maintained throughout, the experiment. Superfusion with drugs was carried out only when the pressor responses to at least three stimulation periods were identical. The drugs were dissolved in CSF and snperfused for 60 min. In most experiments, the hypothalamus was subsequently superfused with drug-free CSF for another 60 min. At the end of the experiments the brain was removed and the localization of the push-pull cannula verified.
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Fig. 1. Effect of superfusion of the posterior hypothalamus with muscarine on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with muscarine. Abscissa: time in min after beginning of the superfusion with muscarine. Circles: controls (n = 6), squares: 5 x I 0- 3M muscarine (n = 6), triangles: 1 x 10-3M muscarine (n = 6). Muscarine was superfused for 60 min (horizontal bar). For difference between controls and 5 x 10-3M muscarine and between controls and 1 x i0 -a M muscarine, P < 0.001 ; between 5 x 10 .3 M and 1 x 10-3M muscarine, P < 0.01. Mean values +_ S.E.M.
C. Substances Used
responses were taken as 100 ~o and subsequently the hypothalamus was superfused with various concentrations of muscarine for 60 rain. Muscarine gradually decreased the pressor responses; however, 5 x 10 -3 M muscarine seemed to be less effective than I x 10 -3 M. The inhibitory effect of muscarine persisted during the subsequent superfusion with muscarine-free CSF.
N-methylatropine bromide (Merck, Darmstadt), oxotremorine (EGA Chemie, Steinheim), arecoline hydrochloride (Ferak, Berlin), hcxamethonium chloride (Fluka, Buchs), N-benzyl-3-pyrrolidyl acetate methobromide (A. H. Robins, Richmond), (+_)-muscarine chloride (Ciba-Geigy, Basel).
B. Effect of Centrally Applied MethyIatropine on the Enhancement of Pressor Responses Caused by Muscarine
B. Statistical Analysis The statistical significance was calculated by analysis of variance (Winne, 1965). In some experiments, Student's t-test was additionally used.
RESULTS
A. Effects of Superfusion of the Hypothalamus with Muscarine on the Pressor Responses to Hypothalamic Stimulation The posterior hypothalamus was superfused with CSF through the double barrelled push-pull cannula. Electrical stimulation every 10 min elicited pressor responses of constant height for at least 12 stimulation periods (Fig. 1). In other animals the constant pressor
In order to examine whether the inhibition of the pressor responses caused by muscarine was due to the muscarinic properties of this agent, the hypothalamus was superfused with 5 x 10-4M methylatropine for 60 min and thereafter with CSF which contained this concentration of methylatropine and 1 x 10-3M muscarine for another 60 min. Central application of methylatropine abolished the inhibitory effect of muscarine on the pressor responses. 5 x 10-4M methylatropine did not affect the rise of blood pressure caused by hypothalamic stimulation (Fig. 2).
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Fig. 2. Effect of superfusion of the posterior hypothalarnus with methylatropine on the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with methylatropine. Abscissa: time in rain after beginning of the superfnsion with methylatropine. Circles: controls (n = 6), squares: 5 • 10-* M methylatropine (n ~ 6). Methylatropine was superfused for 60 min (horizontal bar). Difference between controls and methylatropine n.s. Mean values • S.E.M.
C. Effect of Hypothalamic Superfusion with AHR 602 on the Pressor Responses Superfusion of the hypothalamus for 60 min with I x 10 -2 M AHR 602 which acts on muscarinic receptors (Franko et al., 1963; Jones et al., 1963; Jaramillo and Volle, 1967) did not affect the rise of blood pressure elicited by electrical stimulation of the hypothalamus, while superfusion with 5 x 10 -2 M greatly reduced the pressor responses (Fig. 3). The impairment of the pressor responses was persistent and became even more pronounced during the subsequent superfusion with drug-free CSF. Hypothalamic superfusion with 5 x 10-* methylatropine for 60 rain prior to and together with AHR 602 abolished the inhibitory effect of the latter agent on the pressor responses (Fig. 4). In fact, after superfusion with methylatropine, AHR 602 seemed to temporarily enhance the pressor responses. Although calculation of statistical significance of the curves as a whole by analysis of variance did not reveal significant difference between control animals and animals treated with the drugs, calculation by Student's t-test showed that the pressor response at 20 min was enhanced when the hypothalamus was superfused with methylatropine and AHR 602. The "resting" arterial blood pressure was not affected either by muscarine and AHR 602 or by methylatropine.
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Ordinate: rise of blood pressure as per cent of that before superfusion with A H R 602. Abscissa : time in rain after beginning of the superfusion with AHR602. Circles: controls (n = 5), squares: I x 10 -z M A H R 602 (n = 5), triangles: 5 x 10 -2 M AHR (n = 5). A H R 602 was superfused for 60 rain (horizontal bar). For differences between controls and 5 • 10- 2M A H R 602 and between I x 10- 2M A H R 602 and 5 x 10-2M AHR 602, P < 0.001. Difference between controls and i x 10-2M A H R 602 n.s. Mean values _+ S.E.M.
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Fig. 4. Effect of superfusion of the posterior hypothalamus with methylatropine and A H R 602 in the pressor responses to hypothalamic stimulation. Ordinate: rise of blood pressure as per cent of that before superfusion with A H R 602. Abscissa: time in rain after beginning of the superfusion with A H R 602. Circles : controls (n = 5), squares: after a presuperfusion with 5 x 10-4M methylatropine for 60 rain the hypothalamus was superfused with 5 x 10-4 M methylatropine and 5 x 10-SM A H R 602 for 60 min (n = 5). Difference between controls (superfusion with CSF) and superfusion with methylatropine and A H R 602 n.s. * P < 0.05 (Student's t-test). Mean values • S.E.M.
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D. Effects of Hypothalamic Superfusion with Oxotremorine on the "Resting" Arterial Blood Pressure and on the Pressor Responses to Hypothalamic Stimulation; Impairment of the Effect of Oxotremorine by Methylatropine Superfusion with 1 x 10-=M or 5 x 1 0 - 2 M oxotremorine for 60 rain did not appreciably affect the pressor responses to hypothalamic stimulation (Fig. 5). However, the evaluation of the height of the pressor responses was very difficult and inaccurate, because hypothalamic superfusion with this agent caused a dose-dependent fall of the "resting" arterial blood pressure (Fig. 6). The depression of the blood pressure might be due to leakage Of oxotremorine from the hypothalamic area and action of the agent on muscarinic receptors of peripheral tissues. Indeed, an intravenous injection of 0.5 mg/kg methylatropine which does not readily penetrate the blood-brain barrier abolished the effect of the hypothalamic application of oxotremorine on the "resting" blood pressure (Fig. 6). Under these conditions, i.e. after inhibition of the muscarinic receptors of peripheral tissues, superfusion of the hypothalamus with 5 x 10-ZM oxotremorine caused a Very pronounced reduction of the pressor response to hypothalamic stimulation (Fig. 7). The effect of oxotremorine on the pressor responses was of hypothalamic origin, since it was
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Fig. 6. Effect of superfusion of the posterior hypothalamus with oxotremorine on the "resting" arterial blood pressure. Ordinate: arterial blood pressure as per cent of that before superfusion with oxotremorine. Abscissa: time in rain after beginning of the superfusion with oxotremorine. Oxotremorine was superfused for 60 rain (horizontal bar), Open circles: controls (n = 5)~ open triangles: ] x 10-2M oxotremorine (n = 5), solid circles: 5 • 10-2M oxotremorine (n = 5), solid triangles: 5 • oxotremorine after intravenous injection of methylatropine (0.5 mg/kg). For differences between controls and 5 x 10-2M oxotremorine and between 5 x l 0 - = M oxotremorine and 5 • oxotremorine after intravenous injection of methylatropine, P < 0.001. Differences between controls and 1 x 10-2M oxotremorine and between controls and 5 x I 0 - 2 M oxotremorine after intravenous injection of methylatropine n.s. Mean arterial blood pressure before beginning of the superfusion (mm Hg); controls: 100 • 2, 1 x 10-ZM oxotremorine: 80 __ 4, 5x 1 0 - a M oxotremorine: 87 + 4, 5 x 10-=M oxotremorine after intravenous injection ofmethylatropine: 98 + 4. All values are means + S.E.M.
completely inhibited when the hypothalamus was additionally superfused with 5 x 10-*M methylatropine for 60 rain prior to and during superfusion with oxotremorine.
E. Effect of Hypothalamic Superfusion with Arecoline on the Pressor Responses," Influence of Methylatropine and Hexamethonium on the Effect of Arecoline Hypothalamic superfusion with arecoline (1 x 10-2 M) was ineffective on the pressor responses to hypothalamic stimulation, while a higher concentration (5 x 10 -2 M) increased them (Fig. 8). Since the enhancement of the pressor responses might be due to an action on nicotinic receptors, the hypothalamus was superfused with 1 x 10 -2 M hexamethonium for
Effects of Centrally Applied Muscarinic Drugs on Pressor Responses
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Fig. 7. Effect of superfusion of the posterior hypothalamus with oxotremorine on the pressor responses to hypothalamic stimulation after intravenous injection of methylatropine. Ordinate: rise of blood pressure as per cent of that before superfusion with oxotremorine, Abscissa: time in min after beginning of the superfusion with oxotremorinc. Oxotremorine was superfused for 60 rain (horizontal bar). Circles: controls (n = 6), squares: 5 x 10-2M oxotremorine after intravenous injection of methylatropine (0.5 rag/ kg). For difference between controls and oxotremorine, P < 0.001. Mean values + S.E.M.
60 rain prior to arecoline; subsequently, the hypothalamus was superfused with the same concentration of hexamethonium and 5x 1 0 - 2 M arecoline. This concentration of hexamethonium does not influence the pressor responses to hypothalamic stimulation (Philippu et al., 1974a) but reversed the effect of arecoline (Fig. 9); thus, after superfusion with hexamethonium arecoline caused a slight but significant decrease of the pressor responses to hypothalamic stimulation. On the other hand, superfusion of the hypothalamus with 5x l O - 4 M methylatropine for 60 rain prior to and during superfusion with 1 x 10-2M arecoline led to a significant enhancement of the pressor responses (Fig. 9), although this concentration of arecoline failed to influence the pressor responses in the absence of methylatropine (cf. Fig. 8).
DISCUSSION The intravenous injection of physostigmine elicits a rise of the arterial blood pressure which is due to an action of the drug on the central nervous system (Varagi6, 1955; Varagid and Vojvodid, 1962). Similarly, the intraventricular injection of muscarinic agents such as oxotremorine and arecoline causes pressor responses (Phan and Gy6rgy, 1973). On the other hand, Brezenoff (:1972) observed that the responses of the
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6 blood pressure to intrahypothalamic application of cholinergic drugs is greatly dependent on the area of the hypothalamus involved; thus, the posterior and the ventromedial nuclei of the hypothalamus seem to mediate a rise of the blood pressure, while the dorsomedial and the premammillary nuclei mediate hypotensive responses. Furthermore, the hypotensive response following the intrahypothalamic injection of muscarine, methacholine or oxotremorine was prevented by the central application of atropine (Brezenoff and Wirecki, 1970). Another approach to the effects of centrally applied drugs on the blood pressure and to the localization of their sites of action is to apply drugs centrally and to examine their effects on the pressor responses elicited by electrical stimulation of the hypothalamus. By using a double-barrelled push-pull cannula it was recently found that superfusion of the posterior hypothalamus with DMPP or carbachol increased the rise of blood pressure caused by hypothalamic stimulation, the enhancement of the pressor responses being abolished by hexamethonium (Philippu et al., 1974 a). From the agents with muscarinic properties used in this study, only oxotremorine led to a fall of the "resting" arterial blood pressure. This was due to leakage of the drug into the blood probably because of its highly lipophilic character (Herz et al_, 1966). Indeed, intravenous administration of methylatropine which does not readily penetrate the blood-brain barrier, completely inhibited the effect of oxotremorine on the "resting" arterial blood pressure. Under these conditions, superfusion of the posterior hypothalamus with oxotremorine elicited a very pronounced reduction of the pressor responses caused by dectrical stimulation of the hypothalamus. The depression of the pressor responses was probably due to an action of oxotremorine on muscarinic receptors of the posterior hypothalamus, since it was abolished when the hypothalamus was superfused with methylatropine prior to and during superfusion with oxotremorine. Thus, it seemed that the impairment of the pressor responses by oxotremorine was due to the muscarinic properties of the agent. In this connection it should be noted that the concentration of methylatropine (5 x 10 -4 M) used in the present experiments did not affect the pressor responses to hypothalamic stimulation; it merely inhibited the reduction of the pressor responses caused by oxotremorine. A similar impairment of the pressor responses to hypothalamic stimulation was observed when the posterior hypothalamus was superfused with A H R 602. However, superfusion of the hypothalamus with methylatropine prior to and during superfusion with A H R 602 did not merely abolish the reduction of the pressor responses by this agent; in fact, under
A. Philippu and N. Bohuschke these conditions A H R 602 elicited a temporary enhancement of the pressor responses. Hence, A H R 602 seemed to possess two actions on the pressor responses. After inhibition of the depressant one, the enhancing action became visible; the second one might be due to an action of A H R 602 on nicotinic receptors, since nicotinic agents increase the rise of blood pressure caused by hypothalamic stimulation (Philippu et al., 1974a). On the other hand, Fozard and Muscholl (1972) have shown that A H R 6 0 2 facilitates the release of noradrenaline from the heart caused by electrical stimulation of the sympathetic nerves. This effect of A H R 602 is atropine-resistant (Fozard and Muscholl, 1972) and might be of significance for the enhancement of the pressor response. Although muscarine also inhibited the pressor responses, the effect of a higher concentration (5x 10 .3 M) of the drug was less than that of a lower one (1 x 10 -3 M). Since muscarine possesses both muscarinic and nicotinic properties (Ambache et al., 1956; Konzett and Waser, 1956), it might be assumed that the failure of the dose-response relationship was due to a nicotine-like action when higher concentrations of muscarine were used. Indeed, the inhibition of the pressor responses by the lower concentration of muscarine was blocked when the hypothalamus was superfused with methylatropine. Unfortunately, the amount of muscarine was insufficient for a more extensive investigation. The enhancement by arecoline (5x 10 -2 M) of the pressor responses to hypothalamic stimulation was not surprising, since also this agent possesses nicotinic and muscarinic properties (Feldberg and Vartiainen, 1934; Euler and Domeij, 1945). If the increase of the pressor responses were indeed due to the nicotinic properties of this agent, its enhancing action should be impaired by hexamethonium. In fact, superfusion of the hypothalamus with hexamethonium reversed the action of arecoline, thus leading to a significant reduction of the pressor responses; probably, block of the nicotinic receptors by hexamethonium unmasked the action of arecoline on the muscarinic ones. On the other hand, superfusion of the hypothalamus with a lower concentration of arecoline (1 x 10 -2 M) did not influence the pressor responses. However, superfusion of the hypothalamus with methylatropine prior to an during superfusion with the low concentration of arecoline greatly increased the pressor responses to hypothalamic stimulation. Thus, the enhancing action of arecoline seemed to be due to its nicotinic character, because it was abolished by hexamethonium; superfusion with methylatropine blocked the muscarinic receptors, thus enhancing the nicotinic component of the areco-
Effects of Centrally Applied Muscarinic Drugs on Pressor Responses
line action. It is noteworthy that superfusion of the hypothalamus with hexamethonium does not affect either the pressor responses to hypothalamic stimulation or the "resting" arterial blood pressure, while it completely inhibits the increase of pressor responses caused by hypothalamic application of DMPP (Philippu et al., 1974a). These results indicate that superfusion of the posterior hypothalamus with drugs possessing only muscarinic properties, such as oxotremorine, depresses the pressor responses to hypothalamic stimulation. Drugs possessing both muscarinic and nicotinic properties, such as muscarine, arecoline and AHR 602, may either enhance or impair the pressor responses; their overall action seems to depend on the relative potencies on the two types of receptor. Acknowledgements. The authors wish to thank Dr. H. Brunner, Ciba-Geigy, Basel, for muscarine chloride and Dr. N. Chremos, A. H. Robins, Richmond, for AHR 602. They also thank Miss Inge Smolarek for her skilful technical assistance.
REFERENCES Ambache, N., Perry, W. L. M., Robertson, P. A.: The effect of muscarine on perfused superior cervical ganglia of cats. Brit. J. PharmacoI. 11, 442-448 (1956) Brezenoff, H. E.: Cardiovascular responses to intrahypothalamic injections of carbachol and certain cholinesterase inhibitors. Neuropharmacology 11,637-644 (1972) Brezenoff, H. E., Wirecki, T. S. : The pharmacological specificity of muscarinic receptors in the posterior hypothalamus. Life Sci. 9, 99-109 (1970) v. Euler, U. S., Domeij, B.: Nicotine-like actions of arecoline. Acta pharmacol. (Kbh.) 1, 263-269 (1945) Feldberg, W., Vartiainen, A. : Further observations on the physiology and pharmacology of sympathetic ganglion. J. Physiol. (Lond.) 83, 103-128 (1935) Fozard, J. R , Muscholl, E. : Effects of several muscarinic agonists on cardiac performance and the release of noradrenaline from sympathetic nerves of the perfused rabbit heart. Brit, J. Pharmacol. 45, 616-629 (1972) Franko, B. V., Ward, J. W., Alphin, R. S. : Pharmacologic studies of N-benzyl-3-pyrrolidyl acetate methobromide (AHR 602),
7 a ganglion stimulating agent. J. Pharmacol. exp. Ther. 139, 2 5 - 30 (1963) Herz, A., Holzh/iuser, H., Teschemacher, H. : Zentrale und periphere Wirkungen yon Cholinomimetica und ihre Abh/ingigkeit v o n d e r Lipoidl6slichkeit. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 253, 280-297 (1966) Jaramillo, J., Volle, R. L. : Ganglion blockade by muscarine, oxotremorine and AHR-602. J. Pharmacol. exp. Ther. 158, 80-88 (1967) Jones, A., Gomez Alonso de la Sierra, B., Trendelenburg, U. : The pressor response of the spinal cat to different groups of ganglionstimulating agents. J. Pharmacol. exp. Tber. 139, 312--320 (1963) Konzett, H., Waser, P. G. : Zur ganglionS.ren Wirkung von Muscarin. Helv. physiol, pharmacol. Acta 14, 202-206 0956) Merlis, J. K. : The effect of changes in the calcium content ofcerebrospinal fluid on spinal reflex activity in the dog. Amer. J. Physiol. 13, 6 7 - 72 (1940) Phan, D. V., Gy6rgy, L.: The pressor effect of oxotremorine and arecoline in rats. Acta physiol. Acad. Sci. hung. 43, 7 1 - 8 4 (1973) Philippu, A.: Central regulation of the arterial blood pressure. Clin. Sci. molec. Med. 48, 191s- 194s (1975) Philippu, A., Demmeler, R., Roensberg, G.: Effects of centrally applied drugs on pressor responses to hypothaIamic stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol. 282, 389-400 (1974a) Philippu, A., Przuntek, H., Roensberg, W.: Superfusion of the hypothalamus with gamma-aminobutyric acid : effect on release of noradrenaline and blood pressure. Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 103--118 (1973) Philippu, A., Reich, N., Bohuschke, N. : Influence of cholinergic drugs on the pressor responses to hypothalamic stimulation. Naunyn-Schmiedeberg's Arch. Pharmacol. 285, R 62 (1974b) Reinoso-Suarez, F.: Topographischer Hirnatlas der Katze. Darmstadt: Merck AG 1961 Varagic, V. : The action of eserine on the blood pressure of the cat. Brit. J. Pharmacol. 10, 349-353 (1955) Varagic, V., Vojvodic, N. : Effect of guanethidine, hemicholinium and mebutamate on the hypertensive response to eserine and catecholamines. Brit. J. Pharmacol. 19, 451-457 (1962) Winne, D.: Zur Auswertung von Versuchungsergebnissen: die Priifung, ob Kurven sich in ihrem Verlauf unterscheiden. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 250, 383396 (1965)
Received June 25/Accepted September 29, 1975
