Modification of cardiovascular response of adenosine A1 receptor agonist by cyclic AMP in the spinal cord of the rats

ELSEVIER

Neuroscience Letters 219 (1996) 195-198

NEUROSCI[HC[ lETTERS

Modification of cardiovascular response of adenosine A1 receptor agonist by cyclic AMP in the spinal cord of the rats Hyun Chul Koh a'*, In Chul Shin a, Se Jin Hwang b, Doo Jin Paik b aDepartment of Pharmacology, College of Medicine, Hanyang University, 17 Haengdang-Dong, Sungdong-Ku, Seoul 133-791, South Korea bDepartment of Anatomy, College of Medicine, Hanyang University, 17 Haengdang-Dong, SungdOng-Ku, Seoul 133-791. South Korea

Received 26 August 1996; revised version received 22 October 1996; accepted 22 October 1996

Abstract

This study was performed to investigate the influence of the spinal adenosine Am receptors on the central regulation of blood pressure (BP) and heart rate (HR), and to define whether its mechanism is mediated by cyclic AMP (cAMP) or cyclic GMP (cGMP). Intrathecal (i.t.) administration of drugs at the thoracic level were performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection (i.t.) of adenosine A I receptor agonist, Nr-cyclohexyladenosine (CHA; 1, 5 and 10 nmol) produced dose dependent decrease of BP and HR. Pretreatment with a cAMP analogue, 8-bromo-cAMP, attenuated the depressor and bradycardiac effects of CHA (10 nmol), but not with cGMP analogue, 8-bromo-cGMP. These results suggest that adenosine A~ receptor in the spinal cord plays an inhibitory role in the central cardiovascular regulation and that this depressor and bradycardiac actions are mediated by cAMP. Keywords: Adenosine Ai receptor; Spinal cord; Nr-Cyclohexyladenosine; Blood pressure; Heart rate; cAMP

Adenosine is coupled to adenylate cyclase via two types of receptor: A~ receptor that mediates an inhibition of adenylate cyclase and A 2 receptor that mediates a stimulation of the enzyme [9,11,14,19]. The A1 and A 2 receptors have distinct distribution in the central nervous system [6,17], and evidences for the existence of both receptors in the spinal cord of the rats were also demonstrated by Choca et al. [8]. The action of adenosine as a neurotransmitter or neuromodulator responsible for cardiovascular regulation has been suggested [1-4]. Its two receptors mediate different cardiovascular effects in the peripheral and central nervous system. In the peripheral autonomic nervous system, A1 receptor mediates negative inotropic effects on the heart, while A2 receptor mediates depressive effects on most of blood vessels including the coronary arteries. As for the central nervous system, the administration of adenosine or its agonist into the 3rd ventricle[16], 4th ventricle [1] and the nucleus tractus solitarius [4,15] all resulted in dose dependent decrease of the blood pressure (BP) and the heart rate (HR). * Corresponding author. Tel.: +82 2 2900651; fax: +82 2 2926686.

The autonomic preganglionic neurons in the spinal cord play an important role in central regulation of the cardiovascular system, and this is supported by experiments which show the alterations of cardiovascular responses after intrathecal (i.t.) administration of drugs. Recently, alterations in cardiovascular responses by i.t. administration of sodium nitroprusside [ 13] and glutamate [ 18] were reported. However, little is known about the cardiovascular regulatory effects of adenosine Al receptors in the spinal cord. This study was performed to examine the cardiovascular effects of adenosine Al receptor stimulation in the thoracic cord, and to define whether its mechanism is mediated by c A M P or cGMP. The experimental animals were categorized into three groups. The first group of the these groups was treated only with Nr-cyclohexyladenosine (CHA; 1, 5, 10 nmol), an adenosine Al receptor agonist. The second and third groups were individually treated with a c A M P analogue, 8 - b r o m o - c A M P (10 nmol) and a c G M P analogue, 8b r o m o - c G M P (10 nmol), respectively, 10 min before the injection of 10 nmol of CHA. CHA, 8-bromo-cGMP and 8 - b r o m o - c A M P were purchased from RBI Chemical Co. (USA). All drugs were dissolved in 0.9% NaC1 prior to

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Fig. 1. Decrease of BP and HR induced by i.t. injection of CHA. (A) Representative tracings depicting the changes of BP and HR following microinjection of CHA ( 10 nmol, i.t.) (B) Dose dependent decrease of MAP by i.t. injection of CHA (1, 5, 10 nmol). (C) Dose dependent decrease of HR by i.t. injection of CHA (1, 5, 10 nmol). Results are expressed as mean _+SE. *P < 0.05, **P < 0.01, compared to baseline MAP and HR. administration. The drug administrations were performed in urethane-anesthetized (1.15 g/kg, i.p.), o-tubocurarineparalyzed (0.5 mg/kg, i.m.) and artificially ventilated (Ugo Basile, Italy), male S p r a g u e - D a w l e y rats ( 3 0 0 - 3 5 0 g). Rectal temperature was maintained at 37 _+ 0.5°C with a heating pad, and the rats were placed in stereotaxic instrument (Stoelting, USA) in the prone position. The posterior atlantooccipital membrane was exposed by an occipital incision. The atlantooccipital membrane was cut and a guide cannula (PE-10) was inserted i.t.; its tip was positioned at the thoracic cord level (6.5 cm from the lower margin of the occipital bone). Administration (i.t.) of drugs was made using the injector cannula (33-gauge stainless steel) through the guide cannula. All drugs were delivered at a rate of 5/xl/min with Hamilton syringe mounted on a micrometer. BP and HR were continuously monitored via a femoral arterial catheter (PE-50) connected to a pressure transducer (Spectramed, USA) and a polygraph (Grass, USA). Mean arterial pressure (MAP) was calculated as diastolic pressure + 1/3(systolic pressure - diastolic pressure). Data were expressed as mean _+ SEM of the maximal response following drug administration. Student's t-test for paired or unpaired data was used for statistical evaluation of the results. A representative experiment showing the effects of i.t. administration of C H A on BP and HR is illustrated in Fig. 1. Injection (i.t.) of C H A caused an decrease in M A P that reached maximum in 19.3 + 2.1 min after injection. The depressor response evoked by C H A was dose dependent (1, 5 and 10 nmol of C H A decreased the M A P by

2.4 + 2.0, 11.0 + 1.9 and 27.7 + 3.8 mmHg, respectively; n = 7 for I nmol, n = 5 for 5 and 10 nmol; Fig. 1). Dose dependent decrease of HR was also induced by CHA administration, maximum 15.7 + 3.2 min after injection. (1, 5 and 10 nmol of C H A decreased the HR by 10 +_ 7.1, 54.1 _+ 11.4 and 108 _+ 25.0 beats/rain, respectively; Fig. 1). Baseline M A P and HR for these rats were 85.7 _+ 6.5 m m H g and 334.5 _+ 9.3 beats/rain. Administration (i.t.) of an equivalent volume of normal saline did not affect the basal M A P and HR. Pretreatment with 8 - b r o m o - c A M P (10 nmol, i.t.) significantly attenuated the CHA-induced cardiovascular responses; decrease in M A P and HR were 2.1 +_ 1.9 m m H g and 14 + 19 beats/rain, respectively (n = 5; Fig. 2). However, i.t. administration of 8-bromo-cGMP (10 nmol, i.t.) prior to injection of 10 nmol of C H A did not alter the depressor nor bradycardiac response elicited by CHA; decrease in M A P and HR were 24.5 _+ 5.2 m m H g and 113 + 36.9 beats/min (n = 5; Fig. 3), respectively. Injection (i.t.) of 8 - b r o m o - c A M P (10 nmol) and 8b r o m o - c G M P (10 nmol) had no effects on basal M A P and HR. In present study, i.t. administration of C H A in anesthetized and artificially ventilated rats elicited dose-dependent decrease of M A P and HR. Many experimental evidences suggested that adenosine A1 receptors in the central nervous system plays a critical role in the mediation of cardiovascular responses [ 1,3,15]. Cardiovascular inhibitory action induced by adenosine or ade¢l~sffie A1 receptor agonists in the caudal and rostral nucleus tractus

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solitarius [2,18] were also reported. Our result is consistent with the opinion of above authors, but this is the first evidence of cardiovascular inhibitory action of adenosine AI receptor in spinal cord. Little is known about the autonomic preganglionic neurons which has adenosine receptors responsible for the cardiovascular regulation in the spinal cord. However, anatomical localization of nitric oxide synthetase in preganglionic autonomic neurons in intermediolateral cell column of thoracic and lumbar spinal cord was reported [5] and cardiovascular excitatory response elicited by i.t. administration of sodium nitroprusside, a nitric oxide donor, was demonstrated [13]. Presence of adenosine A l and A2 receptors in spinal cords were also demonstrated through the autoradiographic binding method [7,8]. However, those studies were primarily concerned with its localizations in substantia gelatinosa which has close association with pain modulation. Further experiments are required to identify morphologically the adenosine receptors responsible for cardiovascular regulation in the spinal cord. Administration (i.t.) of 8-bromo-cAMP significantly

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attenuated the cardiovascular depressor actions elicited by CHA, but 8-bromo-cGMP did not altered it. Adenosine receptors are linked to G-proteins known to be coupled negatively to the enzyme adenylate cyclase [10]. The actions of cAMP and cGMP are mediated by cAMP and cGMP dependent protein kinase, respectively. In recent study, Jiang et al. [12] reported that high concentration of cAMP could also activate cGMP dependent protein kinase as well as cAMP-dependent protein kinase, suggesting the close relationship between actions of cAMP and cGMP. But our result suggested that the cardiovascular inhibitory action of adenosine Aj receptor in spinal cord is mediated by cAMP dependent adenylate cyclase, independent from cGMP. In conclusion, our results show that adenosine A] receptor in the spinal cord plays an inhibitory role in central cardiovascular regulation and that this depressor and bradycardiac actions are mediated by cAMP. [1] Barraco, R.A., Campbell, W.R., Parizon, M., Shoener, E.P. and Shein, S.E., Cardiovascular effects of microinjections of adenosine analogs into the fourth ventricle of rats, Brain Res., 424 (1987) 1725.

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[2] Barraco, R.A., E1-Ridi, M.R., Ergene, E. and Phillis, J.W., Adenosine receptor subtypes in the brain stem mediate distinct cardiovascular response patterns, Brain Res., 26 (1991) 59-84. [3] Barraco, R.A., Janusz, C.A., Parizon, M., Posalek, P.M. and Roberls, P.A., Cardiovascular effects of microinjection of adenosine into the nucleus tractus solitarius, Brain Res. Bull., 20 (1988) 129-132. [4] Barraco, R.A., Janusz, C.A., Shoener, E.P. and Simpson, L.L., Cardiorespiratory function is altered by picomol injections of 5'N-ethylcarboxamidoadenosine into the nucleus tractus solitarius of rats, Brain Res., 507 (1990) 234-246. 151 Bredt, D.S., Glatt, C.E., Hwang, P.M., Fotuhl, M., Dawson, T.M. and Snyder, S.H., Nitric oxide synthetase protein and m-RNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase, Neuron, 7 (1991) 615624. [6] Bruns, R.F., Fergus, J.H., Badger, E.W., Bristol, J.A., Santay, L.A., Hartman, J.D., Hays, S.J. and Huang, C.C., Binding of the Ai selective adenosine antagonist 8-cyclopentyl-l,3-diproxylxanthine to rat brain membranes, Nannyn-Schmicdeberg's Arch. Pharmacol., 335 (1987) 59-63. [7] Choca, J.l., Green, R.D, and Proudfil, H.K., Adenosine Ai and Az receptors of the substantia gelatinosa are located predominantly on intrinsic neurons: an autoradiographic study, J. Phamacol. Exp. Ther., 247 (1988) 757 764. [81 Choca, J.l.. Proudfit, H.K. and Green, R.D., Identification of A i and A2 adenosine receptors in the rat spinal cord, J. Pharmacol. Exp. Ther.. 242 (1987) 905 910. 19] Daly, J.W., Butts-Lamb, P. and Padgett, W., Subclass of adenosine receptors in the central nervous system: interaction with caffeine and related methylxanthines, Cell. Mol. Biol., 3 (1983) 69-80. [101 Gerber, U. and Gfihwiler, B.H., GABA~ and adenosine receptors

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