Effect of tachykinins on the acetylcholine output stimulated by nicotine from guinea-pig bladder

Regulatory Pepndes, 46 (1993) 399-401

399

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Effect of tachykinins on the acetylcholine output stimulated by nicotine from guinea-pig bladder Michiko ShmkaJ and Issei T a k a y a n a g ! Department of Chemical Pharmacology, Toho Umverslty School of Pharmaceutlcal Sciences. Chlba (Japan) Key words" N K 2 tachykinm receptor; Sympathetic

nerve; [D-mrg 1, D-Pro 2, D-Trp 7"9, Leu 11]-SP,

Radloimmunoassay; Guanethldine

Summary Effects of tachykinins on contractile responses and acetylcholine release evoked by nicotine were determined by isotonic transducer and radioimmunoassay. A sympathetic nerve blocker guanethidine and tachykinin antagomst, [o-Arg l, D-Pro 2, D-Trp 7'9, Leu ix ]-substance P (rpwwL- SP) partially inhibited the acetylcholine release evoked by nicotine to much the same degree. In preparations treated with rpwwL-SP to block the tachykinin receptors, guanethidine had no efect on the response to nicotine and vice versa, suggesting an exclusive contributlon of the sympathetic nerve commumcations to the action of tachykinir~s. A concentraction response curve to neurokmm A but not SP methyl ester or Tyr°-neurokinin B was shifted to the left after cholmeseterase inhibition with methanesulphonyl fluoride (MSF) and was partially inhibited by atropine. In the presence of guanethidine, an acetylcholine output by nicotine was facihtated in the presence of neurokinin A (10 nM) but not SP methyl ester (10 nM) or senktlde (10 nM). These results indicate that acetylcholine release evoked by nicotine is increased by the coordinated action of sympathetic nerves and tachykmms. Tachykinm receptor subtype involved in acetylcholine release is N K 2.

We have shown that nicotine produced a transient, tetrodotoxan-resistant contraction of isolated detrusor strips of guinea-pig, and suggested that the drug Correspondence to. M. Shmkm,Departmentof Chemical Pharmacology,Toho UmversltySchoolof PharmaceuticalSciences,2-2-1, Mlyama, Funabashl, Cluba 274, Japan

might interact with the presynaptic nicotinic receptors located on (1) parasympathetic cholinerglc, (2) sympathetic non-adrenergic and (3) non-sympathetic punnergic nerves to induce a release of two excitatory final transmatters, acetylcholine and a purine nucleotide [1,2]. In this study, we determined the role of tachykinms and sympathetic nerves on

400 TABLE I Effect of drugs on acetylcholine release and on the contractile resonses induced by nicotine in guinea-pig bladder treated with methanesulphonyl fluoride (MSF) Treatment

Acetylcholine release (%)

Contraction ( % )

Nicotine 0.1 m M + hexamethonium, 10 # M + tetrodotoxin, 30/aM + guanethidine, 3 # M + rpwwL-SP, 10/aM Nicotine 0.1 m M in the presence of rpwwL-SP, 10 # M + guanethidine, 3 # M Nicotine 0.1 mM in the presence of guanethidine, 3 # M + rpwwL-SP, 10/aM

100 15.6 ± 3.5* 112.9 ± 17.9 31.8 ± 9.7* 27.6 ± 5.8*

100 26.1 97.3 81.3 77.6

± 4.6* ± 7.7 _+2.7* ± 3.4*

99.5±20.3

101.5±3.4

94.6±8.8

112.7±6.3

* Significant difference from 100% at P<0.05. rpwwL-SP: [l)-Arg ~, D-Pro 2, D-Trp 7'9, Leu tl ]-substance P. Each value is presented as a mean + S.E. of 4-6 experiments.

nicotine-induced contraction and acetylcholine release simultaneously. Female Hartley strain guinea-pigs were stunned by a blow on the head and exsanguinated from the fem-

oral artery. The urinary bladder was rapidly removed and a longitudinal strip (2 m m x 20 m m ) of the detrusor muscle was prepared. Each strip was suspended vertically under a resting load of 1 g. The

ACh release (%) . . . . O

DO O

O

Nicotine + GUA

+ GUA SPOMe

i~ ?

+ GUA NKA + GUA senktide

i~ ~

~

~

,

~

~

105.8

I - --,

27.7

Fig. 1. Effect of tachykinin receptor agonists on acetylcholine release evoked by nicotine from guinea-pig bladder. GUA, guanethidine 3 /aM; NKA, neurokinin A 1 nM; SPOMe, substance P methyl ester 10 nM; senktide, senktide 10 nM. Nicotine induced aeetylcholine release was reduced by guanethidine to block modulator output from sympathetic nerves. Inhibitory effects of guanethidine were reversed by neurokinin A but not SPOMe or senktide. Each value is presented as a mean + S.E. of four experiments.

401

response to drugs was recorded isotonically. Acetylcholine was determined by radioimmunoassay according to the method of Kawashima et al. [3]. The effects of some drugs on contractile response and acetylcholine release induced by nicotine are summerized in Table I. A sympathetic nerve blocker guanetbidine and tachykinin antagonist, rpwwL-SP partially inhibited the acetylcholine release evoked by nicotine to much the same degree. In preparations treated with rpwwL-SP, guanethidine had no effect on the response to nicotine and vice versa (Table I). We confirmed the previous finding that the final effect of the sympathetic nerve and tachykinins is an increased acetylcholine release evoked by nicotine [1]. But it cannot be denied that tachykinins from some unknown site stimulated a release of mediator (other than noradrenaline [ 1]) from sympathetic nerve endings. In this case, the excitatory effect of tachykinins (increasing acetylcholine output) was apparently abolished by a sympathetic nerve blocker. As shown in Fig. 1, even in the presence of guanethidine which blocked the modulator release from sympathetic nerves, neurokinin A enhanced the acetylcholine output stimulated by nicotine. We may therefore, reasonably conclude that sympathetic nerve is related to the action of nicotine and is the site from which tachykinins are released. Neither SP methyl ester nor senktide which were selective for NK~ and NK 3 receptor subtypes, respectively, had any effect on the cholinergic responses in guinea-pig bladder (Fig. 1). N K 2 but not NK~ or N K 3 tachykinin receptor subtypes might therefore be

involved in the increased acetylcholine release evoked by nicotine. Consistent with this result, a concentration response curve to neurokinin A was shifted to the left after cholinesterase inhibition with MSF. Atropine abolished the facilitatory effect of M S F and partially inhibited contractions induced by neurokinin A at 100 nM to 1 #M. The pD 2 values for neurokinin A before and after treatment with M S F were 6.65 + 0.01 (n = 6) and 7.21 + 0.03 (n = 6). In contrast the pD 2 values for Tyr°-neurokinin B (water soluble analogue of neurokinin B) and SP methyl ester were not influenced by M S F (6.48 + 0.13 to 6.65 + 0.14 n = 6 and 7.43 + 0.14 to 7.40 + 0.12 n = 6, respectively). These results indicate that acctylcholinc release evoked by nicotine is increased by the coordinated action of sympathetic nerves and tachykinins. Tachykinin receptor subtype involved in acetylcholine release is NK2.

References i Hisayama, T.,,Shinkai, M., Takayanagi, I. and Toyoda, T., Mechanism of action of nicotine in isolated urinary bladder of guinea-pig, Br. J. Pharmacol., 95 (1988) 465-472. 2 Shinkai, M., Takayanagi, I. and Kato, T.,'Contrasting effects of tachykinins and guanethidine on the acetylcholinc output stimulated by nicotine from guinea-pig bladder, Br. J. Pharmacol., 103 (1991) 1191-1195. 3 Kawashima, K., Fujimoto, K., Suzuki, T. and Oohata, H., Direct determination of acetylcholine release by radioimmunoassay and presence of M I muscarinic receptors in guinea-pig ileum, J. Pharmacol. Exp. Ther., 244 (1988) 1036-1039.