The intercellular communication via nitric oxide and its regulation in coupling of cyclic GMP synthesis upon stimulation of muscarinic cholinergic receptors in rat superior cervical sympathetic ganglia

BRAIN RESEARCH ELSEVIER

Brain Research 650 (1994) 283-288

Research Report

The intercellular communication via nitric oxide and its regulation in coupling of cyclic GMP synthesis upon stimulation of muscarinic cholinergic receptors in rat superior cervical sympathetic ganglia Masato Ando *, Takatsugu Tatematsu, Shuichi Kunii, Yutaka Nagata Department of Physiology, Fujita Health University School of Medicine, Toyoake-shi, Aichi-ken 470-11, Japan

Accepted 22 March 1994

Abstract

Cyclic GMP (cGMP) production in rat superior cervical sympathetic ganglia (SCG) was markedly increased (ca. 7-9-fold) by the addition of either acetylcholine (ACh; 0.1 mM) or a muscarinic agonist, carbachol (Carb; 0.1 mM), in the presence of an inhibitor (3-isobutyl-l-methylxanthine) for cGMP hydrolytic enzyme during in vitro aerobic incubation at 37°C for 5 min. The ACh-induced accumulation of cGMP in SCG was effectively blocked ( - 7 3 % ) by the further addition of atropine (10/zM), a muscarinic antagonist, whereas a nicotinic blocker, hexamethonium (10/zM) partially antagonized ( - 4 1 % ) this ACh stimulation. The inhibitory effect of hexamethonium on ACh-evoked ganglionic cGMP production was effectively augmented ( - 8 3 % ) by addition of NG-monomethyl-L-arginine (L-NMMA, 50 /xM), a compound that inhibits nitric oxide (NO) synthesis from L-arginine. Comparable inhibition of cGMP formation was observed following application of L-NMMA to the SCG upon stimulation of Carb. In contrast, L-NMMA had no effect on the decreased level of ACh-evoked cGMP production caused by the muscarinic antagonist. The Carb-induced elevation of ganglionic cGMP synthesis was significantly reduced within 1 min of incubation in the medium containing hemoglobin (Hb; 20/zM), an agent that scavenges only the extracellular fraction of NO. Thereafter, the tissue cGMP formation attenuated to the control level by subsequent incubation for several minutes. Addition of protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 p.M) to the medium significantly decreased Carb-evoked cGMP synthesis ( - 6 1 % ) in SCG, whereas superoxide dismutase (SOD; 30 U/ml) only slightly suppressed the Carb stimulation. This finding supports the idea that PKC might play a role in dampening the muscarinic receptor-mediated increase in NO release within ganglionic tissue. In axotomized SCG one week prior to examination, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, no stimulatory effect of Carb-induced cGMP production via NO release was seen. These results provide evidence that a large fraction of NO generated upon stimulation of muscarinic receptors in sympathetic neuronal cells can possibly freely diffuse in extracellular space, and then be taken back into the same group of surrounding cells to stimulate cGMP production. Key words: Nitric oxide; Acetylcholine; Carbachol; Cyclic GMP; Hemoglobin; Protein kinase C; Rat superior cervical sympathetic ganglia; Denervation; Axotomy

I. Introduction

Electrical stimulation of preganglionic nerves increases the cyclic G M P (cGMP) synthesis in rat superior cervical sympathetic ganglia (SCG) [2,5]. Sheng et al. [20] have been indicated that this cGMP production is activated by nitric oxide (NO) derived from L-arginine

* Corresponding author. Fax: (81) (562) 93-2649. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00390-X

(L-Arg). Stimulation

of muscarinic cholinergic receptors also causes cGMP accumulation in ganglionic tissues of various animal species [7,25]. Current immunohistochemical analyses using an antibody to nitric oxide synthase (NOS) from bovine SCG have demonstrated that NOS localizes in postganglionic neuronal cell bodies of bovine SCG [19], suggesting that NO mediates ganglionic cGMP increase in response to muscarinic receptor activation. The NO molecule, which activates cytosolic guanylate cyclase (GC) to produce cGMP

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284

f o r m a t i o n , is a h i g h l y r e a c t i v e a n d l i p o p h i l i c g a s w i t h v e r y s h o r t d e c a y a n d is c a p a b l e o f r a p i d d i f f u s i o n i n t o n e a r b y cells to m e d i a t e i n t r a - a n d / o r intercellular c o m m u n i c a t i o n [11,16]. T h i s p a t h w a y l e a d i n g to G C a c t i v a t i o n via N O r e l e a s e u p o n in v i t r o s t i m u l a t i o n o f m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s , p a r t i c u l a r l y in r a t S C G , by a n a g o n i s t s u c h as a c e t y l c h o l i n e ( A C h ) o r a m u s c a r i n i c a g o n i s t , c a r b a c h o l ( C a r b ) , h a s n o t still b e e n fully e l u c i d a t e d . T h e r a t S C G c o n t a i n s at l e a s t t h r e e c a t e g o r i e s o f cells, i.e. n e u r o n a l , glial, a n d v a s c u l a r . The postganglionic sympathetic neurons are well-pres e r v e d by d e n e r v a t i o n w h i c h , h o w e v e r , c a u s e s W a l l e rian degeneration of the preganglionic cholinergic nerve t e r m i n a l s a w e e k a f t e r t h e o p e r a t i o n . In c o n t r a s t , o n e week following axotomy, both retrograde degradation o f g a n g l i o n i c n e u r o n a l cell b o d i e s a n d r e a c t i v e p r o l i f e r a t i o n o f glial c e l l s c a n r e s u l t . T h e p r e s e n t s t u d i e s w e r e designed to examine the effects of muscarinic choliner-

2.3. hwuhation Isolated SCG were incubated in a bathing solution (BS) which consisted of a modified Krebs-Ringer buffered solution. The BS contained: 136 mM NaCI, 5.6 mM KCI, 2.2 mM CaCI:, 1.2 mM MgCI2, 1.2 mM NaH2PO 4, 16.2 mM NattCO 3, and 5.5 mM glucose at pH 7.4 [I,14]. After preincubation of the isolated SCG in the BS for 30 min, usually two ganglia were further incubated for 5 rain at 37°C in 1.0 ml BS with or without selective agents, at the concentrations indicated in the Tables and Figures, under continuous bubbling of 95% O ~ / 5 % C O 2 gas [2]. Throughout the incubation conditions an inhibitor of cGMP hydrolytic enzyme, a phosphodiesterase, 3-isobutyl-l-methylxanthine (IBMX; 0.5 mM) was added to the BS in order to prevent the degradation of the formed cGMP level [20].

2.4. Determination of c(;MP production

gic a g o n i s t o n N O - d e p e n d e n t c G M P p r o d u c t i o n in r a t SCG one week following denervation or axotomy. These r e s u l t s a r e to f o c u s o n p r o v i d i n g m o r e d i r e c t i n f o r m a tion concerning both the relative contribution of NO r e l e a s e d t h r o u g h i n t r a - a n d / o r i n t e r c e l l u l a r s p a c e to c G M P p r o d u c t i o n a n d m o d i f y i n g m e c h a n i s m s t h a t regu l a t e s u c h N O r e l e a s e in r a t g a n g l i o n i c t i s s u e .

At the end of the incubation period, the reaction was terminated by rapidly aspirating the medium and homogenizing the ganglia in 0.2 ml of ice-cold 5% trichloroacetic acid using a supersonic tissue homogenizer (Bioruptor) (Cosmobio Co., Tokyo, Japan) at 100 W for 5 min. Following centrifugation of the homogenate at 3,000 rpm for 10 min, the supernatant fraction was extracted with water-saturated ethylether to remove the acid. The ganglionic cGMP in the resulting extract was succinylated, and then quantified by radioimmunoassay method using [t25I]succinyl cGMP tyrosine methylester and its monoclonal antibody [9]. Protein content in the SCG samples was determined by the method of Bradford [3] with bovine serum albumin as the standard.

2. M a t e r i a l s

3. R e s u l t s

and methods

2. l. Chemicals NC~-monomethyI-L-arginine (L-NMMA) was purchased from Calbiochem Co. (La Jolla, CA, USA). [125I]Succinyl cGMP tyrosine methylester (1,500-2,0011 cpm/fmol) and its monoclonal antibody were obtained from Yamasa Shoyu Co. (Choshi, Japan). All other chemicals used were of analytical grade from Nagoya-Katayama Chemicals Co. (Nagoya, Japan).

3.1. Effects o f cholinergic antagonists a n d N O S inhibitor on A C h - i n d u c e d ganglionic c G M P production W h e n S C G w e r e i n c u b a t e d in v i t r o f o r 5 m i n in t h e BS containing ACh at varying concentrations (1-1,000 txM), an accelerated profile of ganglionic cGMP prod u c t i o n w a s o b s e r v e d a t a c o n c e n t r a t i o n as low as 10 /xM, a n d t h e s t i m u l a t i v e e f f e c t o f A C h w a s d o s e - d e -

2.2. Surgical operations (denercation and axotomy) of the SCG All experiments were carried out using SCG excised from adult rats (Wistar strain) of either sex weighing 180-200 g. Transection of prc- (deuervation) or post- (axotomy) ganglionic nerves was performed under anesthesia with intraperitoneal (Ip) injection of pentobarbiturate (40 mg/kg b.wt.) [l]. In brief, a ganglion was denervated by cutting the preganglionic nerve trunk about 5 mm away from the ganglion body and folding back the cut end of the nerve. Axotomy was done by cutting both internal and external carotid nerve trunks about 2 3 mm from the contralateral ganglion body. Special care was taken not to touch the ganglion body, the preganglionic nerve trunk, or blood vessels, to avoid hemorrhage. These surgical operations were performed under aseptic conditions and procedures. Success of an operation (denervation and axotomy) was confirmed by observing the subsequent narrowing of the palpebral fissure and constriction of the pupils (Homer's syndrome) when the rat recovered from anesthesia. At one week following the operation, rats were again anesthetized by Ip injection of urethane (1.5 g/kg b.wt.). The SCG were dissected out of both sides of the neck, and the surrounding connective tissue capsules were carefully removed with fine-tipped forceps in a chilled physiological saline solution under stereotactic microscopic examination.

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Fig. 1. Dose-dependent profiles of the cGMP production by ACh or carbachol in intact SCG during aerobic incubation in the presence of 3-isobutyl-l-methylxanthine (IBMX; 0.5 mM) for 5 min. Each point represents the mean of at least 4 experiments; the vertical bar shows S.E.M.

M. Ando et al. ~Brain Research 650 (1994) 283-288 Table 1 Effects of cholinergic antagonists a n d / o r L-NMMA on ACh-induced c G M P production in intact SCG Conditions

No addition + acetylocholine (ACh, 0.1 mM) + ACh + atropine (10/~M) + ACh + hexamethonium (10/zM) + L-NMMA (50/~M) + ACh + atropine + L-NMMA + ACh + hexamethonium + L-NMMA + A C h + I.-NMMA

cGMP production (pmol/ mg protein)

% change

12.6_+3.4 (6) 89.3 +_6.6 (6) a 23.9_+3.6 (5) a 52.4+3.9 (5) " 14.5_+2.7(5) 19.9 + 3.5 (5) 15.0_+3.1 (5)

100 709 190 416 115 158 119

100 27 59 16 22 17

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Excised SCG were incubated for 5 min at 37°C in the BS containing IBMX (0.5 mM) and eserine (0.01 mM) under the indicated conditions, and then assayed for cGMP accumulation. Each value expressed as the m e a n + S . E . M . , with the n u m b e r of experiments in parentheses. a p < 0.001 significantly different from the value at no addition control. L-NMMA, NG-monomethyl-L-arginine.

pendent in manner except at concentrations of 500/zM and more (Fig. 1). This increase in ganglionic cGMP level was maximal (ca. 7-fold) at the concentration of 100 /xM ACh (Fig. 1). As shown in Table 1, the ACh-induced elevation of cGMP synthesis in SCG was effectively blocked ( - 7 3 % ) by the addition of a muscarinic cholinergic antagonist, atropine (10 /zM). Application of hexamethonium (10 tzM), a nicotinic cholinergic blocker, to the BS caused a partial inhibition ( - 4 1 % ) of the ACh stimulation of ganglionic cGMP formation. The hexamethonium-induced blockade effect on ganglionic cGMP production in response to ACh was augmented ( - 8 3 % ) by the further addition of NG-monomethyl-L-arginine (L-NMMA, 50/zM), a compound that selectively inhibits NO synthesis from L-Arg substrate [16]. However, L-NMMA had no further effect on decreased levels of ACh-evoked cGMP production caused by atropine. The L-NMMA effect on ACh-evoked cGMP synthesis was similar extent that shown by atropine inhibition. 3.2. Effect of Carb on cGMP synthesis via NO release in SCG To confirm the possible involvement of specific muscarinic cholinergic receptors in the process of cholinergic stimulation-evoked cGMP production via NO release in the SCG tissue, we conducted additional examination to know the effect of a muscarinic cholinergic agonist, Carb, on such stimulation-induced changes. Application of Carb to the medium at 100 #M, a concentration that generates a peak stimulative effect on ganglion cGMP synthesis (Fig. 1), also showed a prominent, approximately 9-fold elevation of the cyclic

Fig. 2. Time course of carbachol-induced cGMP production with or without hemoglobin (Hb) in intact SCG. Each point represents the mean of at least 5 experiments; the vertical bar shows S.E.M.

nucleotide level in SCG, which was of greater magnitude than the ACh response. This Carb-induced cGMP formation was almost nullified ( - 81%) by the addition of L-NMMA.

3.3. Effect of Hb on Carb-evoked cGMP production via NO release in SCG Under conditions where Carb stimulated the cGMP accumulation in SCG, hemoglobin (Hb; 20 p~M), an agent that scavenges only extracellular NO gas [15], abolished the increase as rapidly as 1 rain following its addition to the BS (Fig 2), and the level of ganglionic cGMP declined to near control value ( - 8 5 % ) during subsequent 5 min longer incubation (Fig. 2 and Table 2). This evidence suggests that there is diffusion of an activator for GC, from inside responsive donor cells to extracellular space surrounding the cells. And the likely candidate for an influential diffusable activator may be Table 2 Effects of modulators of nitric oxide synthase on carbachol-induced cGMP production in intact SCG Conditions

cGMP production ( p m o l / m g protein)

% change

No addition + carbachol (Carb; 0.1 mM) + Carb + L-NMMA (50/~M) + Carb + hemoglobin (20 ~zM) + Carb + SOD (30 U / m l ) + C a r b + T P A (1 # M ) + Carb + T F P ( 1 0 / x g / m l )

12.6 _+3.4 (6) 109.8_+7.1 (6) 21.2+6.2(5) 16.3 + 4.0 (5) 98.5+7.0(5) 42.4_+6.4(5) 115.1 +8.7 (5)

100 871 168 129 782 337 913

~ b a a ~

100 19 15 90 39 105

Excised SCG were incubated for 5 min at 37°C in the BS containing IBMX (0.5 raM) under the indicated conditions, and then assayed for c G M P accumulation. Each value expressed as the mean + S.E.M., with the number of experiments in parentheses. L-NMMA, NG-monomethyl-L-arginine; SOD, superoxide dismutase; TPA, 12-O-tetradecanoylphorbol 13-acetate; TFP, trifluoperazine. a p < 0.001, b p < 0.05 significantly different from the value at no addition control.

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the NO molecule. So far, we have found both muscarinic receptor-mediated cGMP production and release of NO are functionally operating in the process of cholinergic synaptic transmission in rat ganglionic tissue. 3.4. Effects of SOD, TPA or TFP on Carb-induced cGMP synthesis in SCG We examined the modulative effects of superoxide dismutase (SOD), 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), and trifluoperazine (TFP), an antagonist of calmodulin, on stimulation of NOS coupled with increases in ganglionic cGMP formation. As indicated in Table 2, SOD (30 U / m l ) only slightly suppressed the effect of Carb in ganglionic cGMP production. However, TPA (1 /~M) decreased cGMP formation in SCG upon stimulation of Carb by 39%. Almost no effect of TFP (10 > g / m l ) on Carb-evoked increases in cGMP accumulation was seen. 3.5. Effect of denervation or axotomy on Carb-induced increases in cGMP production via NO release in SCG In denervated ganglia (Fig. 3), addition of Carb to the medium had a stimulatory effect on tissue cGMP

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Fig. 3. Effect of L-NMMA (NG-monomethyl-L-arginine) or hemoglobin on carbachol-induced cGMP production in intact, denervated and axotomized SCG during aerobic incubation for 5 rain. The value of cGMP synthesis at no addition in each intact, denervated and axotomized SCG is as follows: 12.6_+3.4, 4.9_+1.8 and 5.8_+2.3 p m o l / m g protein (as the mean of at least 5 experiments _+S.E.M.), respectively. Horizontal bar shows S.E.M. ** P < 0.001 significantly different from the value at each no addition, as determined by the Student's t-test.

formation (ca. 3.7-Mid), although thc magnitude of thc response was less than one half of that shown in intact SCG. Either L-NMMA or Hb effectivcly blocked thc effect of Carb on cGMP synthesis in dcnervated ganglionic tissue. In contrast, a significant diminution (ca. 1.6-fold) of the effect by Carb on cGMP synthesis was detected in axotomized SCG. Further addition of either L-NMMA or Hb to the medium had no effect on Carb-induced cGMP level (Fig. 3).

4. Discussion

Nitric oxide (NO), which is known to be generated from endothelial cells, causes vasorelaxation through activation of soluble guanylate cyclase (GC) in adjacent vascular smooth muscle cells [17,18]. The released NO molecule subsequently increases cGMP production [13], and thus has been implicated in cell to cell communication as a putative neurotransmitter [23]. In recent findings of Sheng et al. [19]., the bovine SCG contains a constitutive NO synthase (NOS), that is Ca24/cal modulin-regulated and requires N A D P H and tetrahydrobiopterin as cofactors. >-NMMA, a potent inhibitor of NOS, blocks the increase in cGMP level caused by ACh stimulation, and also inhibits the partially purified NOS from the SCG. As with bovine SCG, the present results support that a fraction of the muscarinic receptor-mediated increase in cGMP synthesis in rat SCG is likely to be responsible for the action via released NO molecules, which can be generated by the catalysis of NOS from its intrinsic substrate, L-Arg, as shown in previous reports [10,20]. The present observations also agree with previous reports [10] that co-addition of the nicotinic blocker, hexamethonium, and L-NMMA effectively blocked the ACh-induced increase in ganglionic cGMP production (Fig. 1 and Table 1). INMMA also completely abolished the elevation of cGMP synthesis caused by stimulation of the muscarinic agonist, Carb (Fig. 1 and Table 2). Thus, it virtually confirms that NO molecules are participating in the muscarinic cholinergic signal transmission process. The NO is a small lipophilic molecule and is easily diffusable through cell membrane. The NO released from inside donor cells or in extracellular space possibily mediates intra- a n d / o r intercellular communication coupled with cGMP accumulation [11]. Hemoglobin is an useful tool to scavenge only extracellular NO gas since the heme protein is incapable of diffusing across cell membrane into cytoplasm [15,24]. In the present study, we found very rapid and effective attenuation (within 1 min) of the increase in ganglionic cGMP level induced by Carb when Hb was added to the incubation medium, i.e. the extracellular space of the ganglionic tissue (Fig. 2 and Table 2). It is evident

M. Ando et al. / Brain Research 650 (1994) 283-288

that a large fraction of NO generated upon activation of muscarinic receptors in rat SCG by Carb does easily diffuse into extracellular space and then can be captured by Hb existing there at a faster rate than its reuptake by its donor cells, although a small portion of NO might remain intracellulary. In order to shed more light into the cellular localization of such NO-mediated communication processes, we have compared the effects of L-NMMA and Hb on the Carb-evoked cGMP formation in the SCG preparations following denervation or axotomy. In axotomized ganglia one week prior to examination, where postganglionic sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress [1], a significantly diminished effect of Carb stimulation (ca. 1.6-fold) on ganglionic cGMP synthesis was shown compared with that caused in either intact ganglion, or in denervated SCG in which preganglionic nerve terminals were degenerated but either postganglionic neurons or glial components were preserved [1] (Fig. 3). Application of either LNMMA or Hb to the medium caused no modification in the cGMP formation exerted by Carb in axotomized ganglia, while these agents effectively diminished the Carb-induced cGMP production in intact or denervated SCG (Fig. 3). Recently, Kiedrowski et al. [12] demonstrated that cerebellar astrocytes do not appear to possess NOS enzyme, but do contain GC, which can be activated by an NO-like factor generated in cerebellar granule cells in response to NMDA stimulation. Conversely, NOS-like activity seems to be induced in microglia, astroglia, and a related glioma cell line [21]. Until now, NOS action of glial cell component in SCG is not confirmed. The present study might underscore that not glial but neuronal cells in the SCG might be under obligation to exert both NO release and cGMP formation upon stimulation of muscarinic receptors by Carb. Thus, it appears that the NO-mediated communication process occurs almost exclusively in neuronal cells, then NO is again taken back into its donor neuronal ceils to increase cGMP formation by activation of soluble GC. Superoxide dismutase (SOD), which catalyses the dismutation of superoxide ions into oxygen and hydrogen peroxide [8], only slightly suppressed the Carb-induced cGMP production (Table 2). SOD is known to exist widely in the biological kingdom, and to exert a significant protective role for toxic effect of peroxidation [8]. Thus, modulation of the generated NO upon stimulation of Carb is not likely to be mediated by SOD enzymes. It is well-accepted that protein kinase C (PKC) plays an important role in the regulation of stimulus-response coupling in muscarinic receptors [6]. In mouse neuroblastoma clone N1E-115 cells, Hu et al. [10] showed that PKC exerted the effect of dampening the muscarinic receptor-mediated increase in NO release. They also demonstrated that activation of PKC

287

clearly suppresses muscarinic receptor-mediated cGMP production, and inhibits phosphoinositide hydrolysis [10]. The present findings indicate that TPA, an activator of PKC [10], significantly decreased ganglionic cGMP synthesis in response to Carb. However, TFP, an antagonist of calmodulin [22], has no effect on the Carb stimulation (Table 2). These results strongly suggest that the mechanism of action of PKC in attenuating the NO release in coupling of cGMP formation observed here occurs at the level of phosphorylation of NOS, which results in inhibition of this enzyme activity [4]. In conclusion, stimulation of muscarinic receptors in rat sympathetic neuronal cells increases the release of NO from inside the cell into extracellular space. A large fraction of released NO, then, could simply and rapidly diffuse back into the same group of cells, where it accelerates the generation of cGMP. These processes mimic the 'paracrine' and 'autocrine' schemes proposed for the intercellular communication process by NO action in other tissue [11]. In addition, we have provided possible evidence that muscarinic receptormediated release of NO in rat ganglionic tissue might be effectively regulated by the PKC enzyme.

Acknowledgements This work was supported in part by the Grant-in-Aid for Scientific Research (05680685) from the Ministry of Education, Science and Culture, Japan.

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