The regulation of cyclic nucleotides in a sympathetic ganglion

Journal of the Autonomic Nervous System, 6 (1982) 65-72

65

Elsevier Biomedical Press

The regulation of cyclic nucleotides in a sympathetic ganglion Robert L. Volle, Linda F. Quenzer and Brian A. Patterson Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032 (U.S.A.) (Received January 5th, 1982) (Accepted March 1st, 1982)

Key words: 4-aminopyridine - cyclic AMP - cyclic G M P - cyclic nucleotides sympathetic ganglia

Abstract Preganglionic nerve terminal stimulation in rat superior cervical ganglia causes marked increases in the levels of cyclic nucleotides. Results are similar when preganglionic nerve stimulation is compared with elevated [K + ]0 or 4-aminopyridine. Although intact nerve terminals and Ca 2+ are required for the response to occur, pharmacological studies indicate that acetylcholine and adrenergic transmitters are not involved in the cyclic nucleotide response. It is suggested that cyclic nucleotide accumulation occurs in the nerve terminals or an unknown transmitter or substance participates in the postsynaptic accumulation of the cyclic nucleotides. Polypeptides tested thus far do not seem to be implicated. Interrelationships among phospholipid turnover, Ca z+-exchange and cyclic nucleotide accumulation in rat sympathetic ganglia are considered, but are difficult to establish.

Introduction The function of cyclic nucleotides in nervous tissue is not understood and their regulation by transmitters and by inorganic ions has not been defined. For adenyl cyclase, the view is held generally that the activation of subsynaptic receptors by transmitter substances is coupled to the enzyme system in such a way that the formation of adenosine 3',5'-monophosphate (cAMP) is accelerated. By a sequence of ill-defined cAMP-dependent steps, cAMP causes synaptic potentials to be formed, completing the process of transmission or, perhaps, synaptic modulation. For guanyl 0165-1838/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

66 cyclase, a similar view is held, i.e. transmitters lead to the accelerated formation of guanosine 3',5'-monophosphate (cGMP) and, by a cGMP-dependent process, the generation of synaptic potentials. This viewpoint may require modification. Since some sympathetic ganglia manifest long-lasting synaptic potentials, it was thought that transmitter-mediated activation of adenyl and guanyl cyclase participates in the genesis of the slow ganglion potentials [12,14,16], but, the evidence is inconclusive and unconvincing [3,4,14,21]. What is apparent, however, is that cAMP and c G M P are present in sympathetic ganglia, and their formation is accelerated during synaptic activity [4,11,12,16,21,26]. Transmitter release and synaptic activity evoked by preganglionic nerve stimulation, elevated [K + ]0 or the drug, 4-aminopyridine (4AP), are associated with a rise in ganglion levels of cAMP and cGMP that is caused by a mechanism requiring functionally intact nerve terminals and extracellular Ca 2+. Interestingly, the cyclic nucleotide responses to stimulation of the rat superior cervical ganglia do not appear to involve cholinergic or adrenergic steps. Two possibilities are considered; either the cyclic nucleotides accumulate in the nerve terminals or their formation in postsynaptic structures is regulated by an unknown transmitter or modulator substance [26].

Materials and Methods

Male Sprague-Dawley rats (200g) are used and the superior cervical ganglia removed, desheathed and incubated in Locke's solution (mM): NaC1, 136; KCI, 5.6; N a H C O 3, 20; NaH2PO4, 1.2; CaCI2, 2.2; MgCi 2, 1.2; glucose, 5.5 and theophylline, 5-10. The solution is aerated with 95% 02-5% CO 2 and the ganglia are equilibrated in this solution (pH 7.2) for 30 min at 37°C before incubation in drug-containing or test solutions. When receptor blocking agents are used, the ganglia are preincubated in solutions containing the drug alone for 20-40 min. The ganglia are homogenized within 30 s of their removal from the incubation solution in 6% trichloroacetic acid and cyclic nucleotide levels measured by radioimmunoassay. Ganglionic protein content is measured using the method of Lowry et al. [15]. Experimental details are described in previous papers [20,26].

Results and Discussion

Isolated rat ganglia respond to preganglionic stimulation (0.5-30 Hz) with a frequency-dependent increase in ganglion levels of both cAMP and c G M P [4,16,26]. When ganglia are stimulated for 60s at 10 Hz, c G M P levels increase from 0.1-0.3 pmol. m g - l wet weight to about 1.0-1.5 pmol. m g - 1 (Table I). For cAMP, preganglionic nerve stimulation at 10 Hz, increases levels from about 3-5 pmol. mg 1 to 8 - 9 pmol- m g - I (Table II). Similar results have been obtained by others [2,4]. The c G M P and cAMP responses to preganglionic stimulation require Ca 2÷ and are resistant to block by atropine or hexamethonium (C 6, Tables I and II; ref. 25). When 4AP (5 X 10 -4 M) or tetraethylammonium TEA (3 X 10 - 3 M) are included in the

67 TABLE I c G M P RESPONSE T O P R E G A N G L I O N I C N E R V E T E R M I N A L S T I M U L A T I O N cGMP pmol. m g -

Locke's solution Low [Ca 2+ ]0 d Atropine (10 - 5 M) C 6 (5 × 10 - 4 M) Denervation e

b c d e

1

N.S. a

[K + ]o b

10 Hz-60 s - l

45 raM

1.3-+0.4 0.1-+0.1 1.1 -+0.2 1.3--+0.3 -

2.7-+0.2 0.1-+0.05 2.3-+0.1 3.0-+0.2 0.1 -+0.07

(30) ¢ (6) (12) (12)

(32) (8) (5) (4) (12)

Preganglionic nerve stimulation. Replacement of 45 m M NaC1 by 45 m M KC1. Mean-+S.E. N u m b e r of ganglia. Ca2+ replaced by 5 m M MgCI 2. Preganglionic nerve sectioned for at least 2 days.

bathing (Table

solution, II). 4 A P

the cyclic nucleotide and TEA

Like preganglionic ganglion

cGMP

Ganglia

denervated

promote

response

transmitter

nerve stimulation, or more

stimulation

is e n h a n c e d g a n g l i a [5,24].

e l e v a t e d [ K ÷ ]0 c a u s e s a m a r k e d

that requires intact nerve terminals for 48h

to nerve

release in mammalian

do not

and extracellular

respond

to elevated

increase in

C a 2+ ( T a b l e I). [K+]0

with

an

T A B L E II C Y C L I C N U C L E O T I D E C O N T E N T OF I S O L A T E D R A T S U P E R I O R C E R V I C A L G A N G L I A T R E A T E D W I T H 4 - A M I N O P Y R I D I N E (4AP; 5 × l0 - 4 M) cGMP

Locke's solution Denervation Low [Ca 2+ ]0 Atropine (10 -5 M) C 6 (5 × 10 - 4 M) Phentolamine (10 -5 M) Practolol (10 - 4 M) 10 H z . 6 0 s - i 30 H z . 6 0 s - I

cAMP

Control

4AP pmol. r a g - 1

Control

4AP

0.3--+0,01 a 0.3 -+ 0.07 0.2-+0.03 0.4-+0.4 1.3-+0.4 c -

1.1 --+0.2 0.2 -+ 0.3 0.3-+0.1 1.8-+0.2 1.1 -+0.3 3.0_+0.8 c -

5.1 -+0.5 4.5 -+ 0.3 3.4-+0.1 3.8-+0.3 ?b 4.0+-0.1 4.0-+0.1 9.4-+0.2 c 13.1_+0.7 c

9.1 -+0.6 3.6 --+0.2 3.2-+0.2 16.1 -+0.8 ? 8.4-+0.2 8.5 -+0.2 16.4-+0.2 ~ 24.2-0.9 c

a Mean_+S.E. At least 3 ganglia were used for each test. b Indeterminate because C 6 inceases c A M P levels in some ganglia. c Not blocked by atropine (10 -5 M).

68 increase in c G M P levels. Neither atropine nor C6 has any effect on the c G M P response of intact ganglia to elevated [K+]0, a result similar to that reported by Roch and Kalix [22] showing that these drugs have no effect on the accumulation of cAMP by bovine sympathetic ganglia treated with elevated [K ÷ ]0. Both 4AP and TEA increase the c G M P response to nerve stimulation, but only 4AP increases the resting level of the nucleotide. The c G M P content of rat ganglia (Table II) is increased about 4-fold and the cAMP content (Table I1) about 2-fold by 4AP (5 × 10 4 M). 4AP has no effect on the cyclic nucleotide content of denervated ganglia or on intact ganglia bathed in solutions lacking Ca 2+ . In addition, the cyclic nucleotide response to 4AP is unaffected by atropine. The relationship between nicotinic receptor blockade and the cAMP response to 4AP was difficult to study because C 6 increases cAMP content in some, but not all, ganglia. However, it is clear that C 6 has no effect on the c G M P response to 4AP (Table II). Because there is some suggestion that ganglionic cAMP levels are regulated by an adrenergic transmitter [16], tests were made of the cAMP response to 4AP in ganglia pretreated with a- and /3-adrenergic receptor blocking drugs (Table II). Neither phentolamine nor practolol, in concentrations that prevent the cAMP accumulation caused by isoproterenol [21,25], alter the cAMP accumulation caused by 4AP. Thus, synaptic activation by preganglionic stimulation, elevated [K + ]0 and 4AP, causes cyclic nucleotide content to increase in the face of muscarinic and nicotinic receptor blockade by atropine and C6, respectively, and adrenergic receptor blockade by phentolamine and practolol. This makes it unlikely that the activation of adenyl and guanyl cyclase involves cholinergic or adrenergic transmitters in the rat ganglion. Of the catecholamines (Table III), isoproterenol, a fl-receptor agonist, is the most potent activator of ganglionic adenyl cyclase [2,21], whereas dopamine, implicated by some [14,16] as a transmitter in mammalian ganglia, is without effect on cAMP levels in rat ganglia [2]. Bethanechol, a muscarinic receptor agonist, has no effect on ganglion cAMP, evidence against a disynaptic sequence of cholinergic-adrenergic activation of adenyl cyclase in rat ganglia [21]. In some experiments [26], but not others [4] bethanechol causes an increase in ganglion cGMP. Ganglion depolarization during nicotinic receptor activation by dimethylphenylpiperazinium (DMPP) affects neither cAMP nor cGMP. Therefore, like their antagonist counterparts, cholinergic and adrenergic receptor agonists have actions that are not consistent with the notion that cholinergic or adrenergic transmission in ganglia activates adenyl or guanyl cyclase. It was reasonable, therefore, to test polypeptides postulated as transmitters or modulators in autonomic ganglia [8,10,18]. Although all possibilities have not been examined, these have little effect on c G M P content (Table III). Since the finding of noncholinergic and nonadrenergic accumulation of cAMP in rat ganglia is recent [25], not all polypeptides and modulators have been tested for their effects on cAMP accumulation. The result with VIP is encouraging. Even though the search for transmitters that activate adenyl and guanyl cyclase has been unsuccessful, the observation that denervated ganglia respond to sodium azide with a marked increase in c G M P content [20] and to isoproterenol with a

69 TABLE Ill CYCLIC N U C L E O T I D E C O N T E N T OF ISOLATED RAT S U P E R I O R CERVICAL G A N G L I A T R E A T E D W I T H PUTATIVE T R A N S M I T T E R SUBSTANCES A N D AGONISTS Agonist

cGMP

cAMP

% of Control Isoproterenol Dopamine Bethanechol DMPP b Sub-P c LHRF d Met-5-Enk e VIPf

(10 -6 M) (10 -3 M) (10 -4 M) (10-4M) (10 4M) (10-4 M) (5)< 10 -5 M) ( 5 × 10 - 6 M )

1 0 0+- 7 100± 6 2 5 0+- 10 110+20 135-+30 76+18 100 +- 5 190±25

(8) a (8) (6) (6) (3) (3) (10) (3)

950 + 100 (5) 116 + 8 (5) 112 + 7 (8) 95 + 7(3)

800-+75 (3)

a b c d

Mean+S.E. Number of ganglia in parenthesis. Dimethylphenylpiperazinium. Substance P. Leutinizing hormone-releasing factor. Methionine-enkephalin. f Vasoactive polypeptide.

marked increase in cAMP content [21] demonstrates that ganglion or glial cells contain the enzyme systems required for their synthesis. Accordingly, the idea that adenyl and guanyl cyclase can be activated by a transmitter or some other substance acting on or within postsynaptic structures retains some appeal. Because guanyl cyclase activation by transmitters requires Ca 2÷ , but has not been demonstrated in cell-free systems, the idea has been advanced that Ca 2+ mediates the activation of the enzyme [23]. According to this scheme, cell depolarization increases Ca2+-conductance, elevates [Ca2+]i and causes the activation of guanyl cyclase. That this may not be so in rat sympathetic ganglia is suggested by the findings: (a) that elevated [K+]0 does not increase cGMP content of denervated ganglia [20] but does increase 45Ca-uptake [1]; and (b) that DMPP does not increase cGMP content in intact rat ganglia but does increase 45Ca-uptake [26]. Both elevated [K+]o and DMPP are known to depolarize ganglion cells. Thus, under some conditions, when ganglia cells are depolarized and Ca2+-exchange accelerated, cGMP accumulation does not occur. For this reason, depolarization and elevation of [Ca2+]i per se are inadequate stimuli in ganglia for the activation of guanyl cyclase, suggesting that additional factors may be required. Michell [17] suggests that muscarinic receptors, phospholipid metabolism, Ca 2+exchange and guanyl cyclase activity are interrelated. For example, one model holds that muscarinic receptor activation causes the turnover of phosphatidylinositol (PI), causing an increase in Ca2+-conductance and leading to the activation of guanyl cyclase. Isolated rat superior cervical ganglia may be suitable for a study of these interrelationships, if they exist, because each of these parameters is altered during preganglionic stimulation (Table IV). Preganglionic stimulation increases cGMP

70 TABLE IV ALTERATION OF cGMP LEVELS, 45Ca2+-UPTAKE AND PHOSPHATIDYLINOSITOL (PI) TURNOVER IN RAT SYMPATHETIC GANGLIA Response

N.S.

[K+ ]o

DMPP

BCh

cGMP levels PI turnover 45Ca2+-uptake

t a 1"c Ta

I, a,b

No change q"~ ,f a

Tc ~d No change ¢

T Ta

" Not blocked by atropine [4,20,26], b Does not occur in denervated ganglia [20]. Blocked by atropine (refs. 19, 26; see refs. 9 and 13). a McN-343 was used as a muscarinic receptor agonist [19]. (Ref. 26).

levels, accelerates PI turnover [ 13,19], and increases 45Ca 2 + -uptake [ 1]. However, the neurally-induced increases in c G M P levels and 45Ca2+-uptake are unaffected by muscarinic receptor-blocking drugs, whereas the turnover of PI is prevented by atropine [19]. As noted before, in denervated ganglia, elevated [K]0 increased 45CaZ+-uptake but does not cause c G M P accumulation and c G M P accumulation in intact ganglia is unaffected by atropine. Similar divergence is noted with cholinergic agonists. D M P P increases PI turnover by an atropine-sensitive process [19] but causes 45CaZ+-uptake by a C6-sensitive process and has no effect on c G M P levels [26]. Muscarinic agonists increase ganglionic c G M P (refs. 12, 26, but see ref. 4) and increase PI turnover [19] but do not alter 45CaZ+-uptake [26]. In any event, interrelationships a m o n g these responses to preganglionic nerve stimulation are difficult to identify and further, are difficult to relate to cholinergic, muscarinic transmission. It m a y be that the use of an heterogeneous tissue (nerve endings, ganglion cells, glial cells, etc.) and c o m p a r t m e n t a l i z a t i o n of systems within ganglion cells are confounding factors. That c G M P accumulation, Ca 2+-uptake and phospholipid turnover reflect events in the nerve terminals is a possibility that deserves attention. Although the presence in nerve terminals of the cyclic nucleotides is difficult to establish, some evidence exists demonstrating that nerve terminals are a site of cyclic nucleotide formation. c G M P release from adrenergic nerves by elevated [K + ]0 has been d e m o n s t r a t e d [27] and a catecholamine-sensitive adenyl cyclase has been located in the presynaptic terminals of chick l u m b a r sympathetic ganglia [7]. The finding that 4AP, a drug with p r o f o u n d effects on transmitter release and on c G M P (and c A M P ) accumulation, enhances exocytosis suggests presynaptic interactions a m o n g these events. Since p h o s p h a t i d a t e (PA) turnover occurs in preganglionic nerve terminals during stimulation [9] and PA causes transmitter release [6], connections m a y exist a m o n g PA, Ca2+-influx and cyclic nucleotide m e t a b o l i s m in the nerve terminals. It is easy to imagine that the turnover of plasma and vesicle m e m b r a n e constituents could result in the formation of products capable of activating either of the two cyclase systems

71

and that changes in cyclic nucleotide metabolism reflect rather than cause transmitter release. In summary, evidence is lacking for a regulatory role by transmitter substance in the enhanced formation of cyclic nucleotides during synaptic transmission in rat superior cervical ganglia. It is possible that cAMP and cGMP accumulation occurs primarily in nerve terminals in response to exocytosis and the turnover of vesicle and plasma membrane constituents.

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72 19 Pickard, M.R., Hawthorne, J.N., Hayaski, E. and Yamada, S., Effects of surugatoxin and other nicotinic and muscarinic antagonists on phosphatidylinositol metabolism in active sympathetic ganglia, Biochem. Pharmacol., 26 (1977) 448-450. 20 Quenzer, L.F., Patterson, B.A. and Voile, R.L., K+-induced accumulation of guanosine 3',5'-monophosphate in sympathetic ganglia, J. Neurochem., 34 (1980) 1782-1784. 21 Quenzer, L., Yahn, D., Alkadhi, K. and Voile, R.L., Transmission blockade and stimulation of ganglionic adenylate cyclase by catecholamines, J. Pharmacol. exp. Ther., 208 (1979) 31-36. 22 Roch, P. and Kalix, P., Adenosine 3',5'-monophosphate in bovine superior cervical ganglion: effect of higher extracellular potassium, Biochem. Pharmacol., 24 (1975) 1293-1296. 23 Schultz, G., Hardman, J.G., Schultz, K., Baird, C.E. and Sutherland, E.W., The importance of calcium ions for the regulation of guanosine 3',5'-monophosphate levels, Proc. nat. Acad. Sci. (U.S.A.), 70 (1973) 3889-3893. 24 Shimizu, T. and Dun, N.J., A study of 4-aminopyridine on sympathetic ganglion cells, Fed. Proc., 40 (1981) 257. 25 Voile, R.L. and Patterson, B.A., Noncholinergic, nonadrenergic increase of adenosine 3',5'-monophosphate (cAMP) levels in rat superior cervical ganglia during preganglionic nerve stimulation, Fed. Proc., 41 (1982) 1000. 26 Voile, R.L., Quenzer, L.F., Patterson, B.A., Alkadhi, K.A. and Henderson, E.G., Cyclic guanosine 3',5'-monophosphate accumulation and 45Ca-uptake by rat superior cervical ganglia during preganglionic stimulation, J. Pharmacol. exp. Ther., 219 (1981) 338-343. 27 Zatz, M. and Weinstock, M., Electric field stimulation releases norepinephrine and cyclic GMP from rat pineal gland, Life Sci., 22 (1978) 767-772.