Michiko Shinkai

Mechanism of action of nicotine in isolated urinary bladder of guinea-pig.

1 Nicotine produced a transient contraction of isolated strips of guinea‐pig urinary bladder. The response to nicotine was antagonized by the nicotinic receptor antagonist, hexamethonium but was insensitive to tetrodotoxin. 2 The nicotine‐induced contraction was potentiated by the cholinesterase inhibitor, physostigmine, and was reduced to 50% and 70% by the muscarinic cholinoceptor antagonist, atropine and the sympathetic neurone blocking drug, guanethidine, respectively. Chemical denervation with 6‐hydroxydopamine abolished the inhibitory effect of guanethidine. Simultaneous treatment with atropine and guanethidine did not abolish the response to nicotine, but the degree of inhibition was comparable to that obtained with atropine alone. 3 The nicotine‐induced contraction was insensitive to bunazosin and yohimbine (α1‐ and α2‐adrenoceptor antagonists, respectively), and exogenously applied noradrenaline did not cause a contraction even in the presence of blockade of noradrenaline uptake mechanisms with desipramine and normetanephrine and of β‐adrenoceptors with propranolol, suggesting a non‐adrenergic nature of the sympathomimetic effect of nicotine in this tissue. 4 The nicotine‐induced contraction in the presence of atropine was abolished after desensitization of P2‐purinoceptors with α, β‐methylene adenosine 5′‐triphosphate, a slowly degradable ATP analogue selective for P2‐purinoceptors. By this desensitization, the response to ATP, but not to histamine, was also abolished. 5 A cyclo‐oxygenase inhibitor flurbiprofen partially inhibited the nicotine‐induced contraction. The degree of the inhibition was more pronounced in the presence of atropine than in its absence. Flurbiprofen antagonized the response to exogenously applied ATP in an unsurmountable manner, but not that to carbachol. 6 The present results suggest that nicotine might induce a contraction through an interaction with nicotinic receptors located on the terminals of, possibly, (i) parasympathetic cholinergic, (ii) sympathetic non‐adrenergic and (iii) non‐sympathetic purinergic nerves in guinea‐pig detrusor preparations, and that a portion of the contraction due to the purine nucleotide released is possibly potentiated by intramural prostaglandin(s). Parasympathetic cholinergic output might be modulated by an unknown excitatory substance released by nicotine from sympathetic nerve. 7 Nicotine reveals a latent excitatory effect of the sympathetic hypogastric nerve which innervates guinea‐pig detrusor.

Tachykinin receptors of the NK2 type involved in the acetylcholine release by nicotine in guinea-pig bladder

1 The effects of guanethidine and tachykinins on nicotine‐ and electrical stimulation‐induced cholinoceptor responses were studied in isolated urinary bladder from the guinea‐pig. 2 Acetylcholine release and the contractile response stimulated by nicotine were partially reduced by a sympathetic nerve blocker, guanethidine. Neurokinin A (but not substance P methyl ester or senktide) enhanced both acetylcholine release and contraction by nicotine in the presence of guanethidine. 3 Frequency‐contraction curves (1 to 50 Hz) for electrical field stimulation (EFS) were partially reduced by atropine (1 μm), and after desensitization to α,β‐methylene adenosine 5′‐triphosphate, the atropine‐resistant contraction to EFS was completely abolished. Guanethidine, the tachykinin antagonist [d‐Arg1, d‐Pro2, Trp7,9, Leu11]‐substance P and application of neurokinin A or substance P did not change the contractile response to EFS. Preganglionic nerve stimulation (5 Hz and 20 Hz) also evoked a similar response to EFS and was not influenced at all by guanethidine or neurokinin A. 4 We conclude that the ability of nicotine to release acetylcholine is enhanced both by endogenous tachykinins (probably released from sympathetic nerves) and by exogenously applied tachykinins as a result of interaction with NK2 receptors in the urinary bladder.

Pre- and postjunctional actions of endothelin in the rat iris sphincter preparation.

Effects of endothelins (ETs) were studied in the rat iris sphincter preparation. Three peptides (ET1, ET-2 and ET-3) caused contractile responses, and the rank order of agonist potency was: ET-1 = ET-2 > ET-3. The concentration-response curve to ET-1 was shifted to the right by the ETA receptor antagonist cyclo [d-Asp-l-Pro-d-Val-l-Leu-d-Trp] (BQ-123: 10−7 M), the pA2 value of which was 7.41 ± 0.09 (n = 4).ET-1 and ET-3, at the concentration of 10−9 M, potentiated cholinergic contractions evoked by electrical field stimulation (5 and 20 Hz) without affecting the postjunctional sensitivity to carbachol. This potentiating effect was not influenced by BQ-123 (10−6 M). The ET-evoked percentage increase in the stimulation-induced contraction observed at 5 Hz was significantly greater than that at 20 Hz. A release of immunoreactive ET was detected when the preparation was stimulated at 20 Hz (1.81 ± 0.36 pg/sphincter n = 6). ET release evoked by 20 Hz stimulation was completely abolished by tetrodotoxin (10−7 M).In conclusion, ET interacts with two different receptor types, ETA and non-ETA receptors (probably ETB) which exist post- and presynaptically at cholinergic neuroeffector junctions of the rat iris preparation. Stimulation of ETA receptor results in a direct muscle contraction and non-ETA receptor activation facilitates the acetylcholine output from cholinergic nerve endings. It is suggested that ET released from a tetrodotoxin-sensitive site is involved in the modulation of acetylcholine release in the rat iris sphincter preparation.

Facilitatory effects of tachykinins and guanethidine on the acetylcholine output stimulated by nicotine from guinea-pig bladder

1 Contractile responses and acetylcholine release evoked by nicotine in guinea‐pig detrusor strips were determined by isotonic transducer and radioimmunoassay, respectively. Nicotine stimulated acetylcholine release and a contractile response in guinea‐pig detrusor strips treated with the cholinesterase inhibitor, methanesulphonyl fluoride (MSF). Both actions evoked by nicotine were antagonized by the nicotinic receptor antagonist, hexamethonium but were insensitive to tetrodotoxin. 2 A sympathetic nerve blocker, guanethidine and a tachykinin antagonist, [d‐Arg1, d‐Pro2, d‐Trp7,9, Leu11]‐substance P (rpwwL‐SP) partially inhibited the acetylcholine release evoked by nicotine to much the same degree. The inhibitory effects of guanethidine and rpwwL‐SP on acetylcholine release were significantly greater than corresponding effects on the contraction evoked by nicotine. 3 In preparations treated with rpwwL‐SP to block the tachykinin receptors, guanethidine had no effect on the response to nicotine. Conversely, after treatment with guanethidine to block release of a mediator from sympathetic nerve endings, nicotine‐induced responses were not affected by rpwwL‐SP. 4 Nicotine‐induced contraction was reduced to 30% by the muscarinic cholinoceptor antagonist, atropine and completely abolished after desensitization of P2‐purinoceptors with α,β‐methylene ATP in the presence of atropine. 5 A concentration‐contractile response curve to neurokinin A (NKA) was shited to the left after cholinesterase inhibition with MSF. Atropine abolished the facilitatory effect of MSF and partially inhibited contractions induced by NKA at 100 nm to 1 μm. The contractile responses to substance P methyl ester (SPOMe) and Tyro‐neurokinin B (Tyro‐NKB) were not influenced by MSF or atropine. 6 After desensitization of NR1 tachykinin receptors with SPOMe or preincubation with senktide, the cholinergic component of the nicotine‐induced contraction was the same as the control value (100%). 7 Our findings give further support to our previous results: nicotine stimulates acetylcholine release in a tetrodotoxin‐resistant manner in guinea‐pig bladder and acetylcholine release evoked by nicotine is increased by the coordinated action of sympathetic nerves and tachykinin(s). It is suggested that the tachykinin receptor subtype involved in acetylcholine release is NK2.

Mechanism of action of nicotine in isolated iris sphincter preparations of rabbit

1 Nicotine produced a transient contraction of rabbit isolated iris sphincter muscle, a parasympathetic ganglion‐free tissue. The response to nicotine was antagonized by hexamethonium, but was insensitive to tetrodotoxin (TTX). While single treatments with atropine, capsaicin or [d‐Arg1, d‐Pro2, d‐Trp7,9, Leu11]‐substance P (rpwwL‐SP) partially blocked the response, combined treatment abolished it. 2 Chronic treatment of animals with nicotine added to the drinking water (about 12 mg kg−1 per day) had no effect on the responsiveness to nicotine or the pharmacological properties of nicotine‐induced contraction. 3 These results suggest that acetylcholine and tachykinin(s) released via sodium channel‐independent mechanisms from nerve terminals of parasympathetic and primary sensory nerves, respectively, are involved in the nicotine‐induced contractile response.

Pharmacological action of nicotine in the isolated urinary bladder from rabbit: Special reference to the chronic nicotine treatment

1. A mode of action of nicotine and a change of the responsiveness to nicotine following chronic nicotine treatment in the urinary bladder of rabbit were investigated. 2. Nicotine induced only a contraction in the urinary bladder of rabbit, and the response to nicotine was reduced by hexamethonium, atropine and capsaicin. These findings suggest that the contractile response to nicotine was mediated through an action on the nicotinic receptors and partially due to the release of acetylcholine and tachykinins. 3. Tetrodotoxin did not inhibit the contractile response to nicotine in the rabbit detrusor muscle, suggesting that the nicotine-induced response may be produced mainly through a sodium action potential-independent process. 4. Nicotine-induced contraction was reduced following the chronic nicotine treatment without a change of its pharmacological properties. These findings suggest that chronic nicotine treatment might cause a decrease of the amounts of nicotinic receptors and also receptors for mediators released by nicotine.

Contractile responses of rat aorta to phenylephrine and serotonin, and aging.

1. The potency (pD2 value) of phenylephrine increased with age from 3 to 10 weeks, but decreased thereafter from 10 to 80 weeks, while the affinity (pKA value) of phenylephrine to alpha 1-adrenoceptors did not alter with aging. 2. The potency (pD2 value) of serotonin did not alter with aging. 3. There is no significant difference between slopes of regression lines between a cytosolic free Ca2+ level ([Ca2+]i) and tension in the presence of phenylephrine in aorta strips from 10- and 60-week-old rats, suggesting that the sensitivity of contractile system to Ca2+ did not alter with aging. 4. Effect of ryanodine on the transient increase of [Ca2+]i and the followed sustained contraction induced by phenylephrine or serotonin in Ca2+ free solution did not alter with aging.

Inhibitory effects of d-nicotine on the responses evoked by 1-isomer in trachea and bronchus isolated from guinea-pig and rabbit

1. The effects of d-nicotine on the responses induced by 1-isomer were studied in tracheae and bronchi isolated from guinea-pigs and rabbits. In guinea-pigs trachea 1-nicotine produced a biphasic response consisting of initial contraction and following relaxation. In other airway preparations 1-nicotine produced only contraction. 2. d-Nicotine did not produce any responses except for the case of guinea-pig trachea. d-isomer produced only relaxation and relative potency was approximately 0.44 in guinea-pig trachea. 3. Pretreatment with d-nicotine (30-300 microM) reduced concentration response curves for 1-isomer in a non-competitive manner in all preparations used in this study. 4. 1-nicotine at the concentration of 3 microns, which did not produce any response itself, reduced the concentration response curve of 1-nicotine in guinea-pig trachea. 5. Inhibition by d-nicotine or 1-nicotine (3 microM) of the concentration-response curve of 1-nicotine may be due to desensitization of nicotine receptors.

Effect of ageing on response to nicotine in rabbit bronchial preparation

1. Effect of ageing on the response to nicotine was tested in the bronchial muscle preparations from 5, 13, 100 and 125 week-old rabbits. The pD2 value (potency) of nicotine significantly increased in the preparation from the 125 week-old rabbits. No age-related change was found in the pD2 value of carbamylcholine or pA2 value of atropine. 2. No age-related change in characteristics of nicotine receptors. Choline acetyltransferase activity, the amount of acetylcholine released by nicotine and acetylcholineesterase activity decreased in the preparations from the 125 week-old rabbits. 3. Decrease in the pD2 value of nicotine in the preparation from the older rabbit is due to a decline in choline acetyltransferase activity followed by a reduction in the acetylcholine released, and not to a change in characteristics of nicotine receptors. 4. These results also suggest that enzymes may be influenced more easily with age than drug receptors.

Effect of ω-conotoxin GVIA on tetrodotoxin-insensitive acetylcholine release by nicotine in guinea-pig bladder

1. Contractile responses and acetylcholine release evoked by nicotine and electrical field stimulation (EFS) were determined by isotonic transducer and radioimmunoassay, respectively. 2. Nicotine-induced contraction was reduced to 30% by nicotinic receptor antagonist, hexamethonium but was insensitive to tetrodotoxin. EFS-induced contraction was abolished by tetrodotoxin but was insensitive to hexamethonium. Replacement of external Na by choline completely abolished the contractile responses evoked by nicotine and EFS. 3. Both contractions evoked by nicotine and EFS were inhibited by omega-conotoxin GVIA, and inhibitory effects of the toxin were greater in low Ca concentrations. 4. In the condition that external Na or Ca is omitted from physiological solution, acetylcholine release evoked by nicotine was not observed. Nicotine-induced acetylcholine release was partially inhibited by omega-conotoxin but was insensitive to tetrodotoxin. 5. In conclusion, nicotine interacts with nicotinic receptors located on nerve terminals and produces transmitter release which depends on external Na through tetrodotoxin-insensitive mechanisms. It is suggested that voltage-dependent omega-conotoxin sensitive Ca channels are partially involved in the nicotine-induced transmitter release.