Nitroxidergic nerve stimulation relaxes human uterine vein

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Journal of the Autonomic Nervous System 55 (1995) 189-192

Autonomic Nervous System

Nitroxidergic nerve stimulation relaxes human uterine vein Noboru Toda *, Toshio Kimura, Tomio Okamura Department of Pharmacology, Shiga University of Medical Sciences, Seta, Ohtsu 520-21, Japan Received 23 March 1995; revision received and accepted 18 May 1995

Abstract The predominant action of nitroglycerin, a nitric oxide (NO) donor, on veins over arterioles is well recognized. This study was carded out to determine whether endogenous NO derived from vasodilator nerve regulates the tone of human uterine venous strips. The isolated vein partially contracted with prostaglandin F2~ responded to nicotine with a contraction or a relaxation; the contraction was reversed to a relaxation by prazosin, and the relaxation was potentiated by the aradrenoceptor antagonist. In prazosin-treated strips, nicotine-induced relaxations were not affected by timolol, atropine and indomethacin but were abolished by oxyhemoglobin and NG-nitro-L-arginine (L-NA), a NO synthase inhibitor. The o-enantiomer was without effect. The inhibition by L-NA was reversed by L-arginine. The NO-induced relaxation was not influenced by L-NA but was abolished by oxyhemoglobin. It may be concluded that the human uterine vein is innervated by vasodilator nerves from which NO is liberated as a vasodilator neurotransmitter. Norepinephrine from adrenergic nerves contracts venous smooth muscle possibly via stimulation of ch-adrenoceptors. Keywords: Nitric oxide; Nicotine; Uterus; Human; Vasoeonstrictor/vasodilator innervation; Nitroxidergic nerve

1. Introduction The predominant action of nitroglycerin on veins over arterioles has been clinically recognized [5], which accounts for the efficacy of nitroglycerin in the management of heart failure and angina. The venoselectivity of other nitric oxide (NO) donors such as sodium nitroprusside, linsidomine and S-nitrosoglutathione has also been demonstrated in human blood vessels [10]. Since the discovery of the endogenous NO-generating system [12], a role of NO in the regulation of vascular tone was studied in human veins in vitro [4] and in vivo [26]; however, the studies were focused solely on the endothelial function. On the other hand, physiological roles of NO in nerve cells and fibers were suggested from immunohistochemical studies on localizations of NO synthase [3]. Our recent studies have provided evidence supporting the hypothesis that NO or its analog acts as a vasodilator neurotransmitter in cerebral [20-22] and peripheral arteries [23,24] of various species, including humans [18]. On the other band, little is known concerning vasodilator innervation in the vein, and the information on human veins is lacking.

* Corresponding author. Tel.: (81-775)48-2181; Fax: (81-775) 48-2183. 0165-1838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-1 8 3 8 ( 9 5 ) 0 0 0 4 6 - 1

The aims of the present study were, therefore, to determine vasodilator innervation in human uterine veins freshly excised from the uterus of myoma patients and to analyze the mechanism underlying the neurogenic vasodilatation, in special reference to NO synthesized in the nerve.

2. Materials and methods The human uterine vein accompanying the ascending branch of the uterine artery was isolated from the nonpregnant uterus excised by operation of myoma uteri. The patients (n = 12) were 37 to 52 years of age. The vein was cut into helical strips of approx. 20 mm, and the endothelium was removed by gently rubbing the intimal surface with cotton ball. The specimens were vertically fixed between hooks in a muscle bath containing the modified Ringer-Locke solution which was maintained at 37 :t: 0.3°C and aerated with 95% 0 2 / 5 % CO 2. The hooks anchoring the upper end of the strips were connected to the lever of a force-displacement transducer. The resting tension was adjusted to 0.7 g, which was optimal for inducing the maximal contraction. The composition of the solutions was as follows (in mM): NaCl 1 2 0 / K C I 5 . 4 / C a C l 2 2.2/MgCl 2 1.0/NaHCO 3 25.0/dextrose 5.6. The pH of

N. Toda et a l . / Journal of the Autonomic Nervous System 55 (1995) 189-192

190

H U M A N U T E R I N E VEIN - +10

Nicotine

1 0 -4 M

H U M A N U T E R I N E VEIN - -

Nicotine

10 - 4 M

0

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n=6

C

D-NA

L-NA

(7)

(4)

(7)

L-NA +D-Arg. (4)

L-NA + L-Arg. (7)

Fig. 1. Modifications by prazosin (PZ; 10 -3 M) of the contractile (left panel, n = 4) and relaxant responses (right, n = 6) to 10 -4 M nicotine in human uterine venous strips partially contracted with PGF2a. Contractions induced by 5 mM Ba 2+ were taken as 100% contraction, and relaxations by 10 -4 M papaverine were taken as 100% relaxation. The plus on the ordinate of the left panel represents contraction, and the minus represents relaxation. Significantly different from control, a p < 0.02 (unpaired t-test); b p < 0.05 (paired t-test), n, number of strips from separate individuals. Vertical bars represent SE.

Fig. 2. Modifications by nitro-arginine (L-NA and D-NA) and arginine (D-Axg. and L-Arg.) of the relaxant response to 10 -4 M nicotine in human uterine venous strips treated with 10 -3 M prazosin and partially contracted with PGF2a. Relaxations induced by 10 -4 M papaverine were taken as 100%. Significantly different from control (C), a p < 0.05; significantly different from the value with L-NA plus L-arginine, b p < 0.01 (Tukey's method). Numbers in parentheses indicate the number of strips from separate individuals. Vertical bars represent SE.

the solution was 7.35 to 7.43. Before the start of experiments, all strips were allowed to equilibrate for 60 to 90 rain in the bathing media, during which time the fluid was replaced every 10-15 min. Isometric mechanical responses were displayed on an ink-writing oscillograph. The contractile response to 5 mM Ba2+ was first obtained, and the venous strips were repeatedly washed with fresh media and equilibrated. The Ba 2+induced contraction was taken as a standard for the contraction caused by agonists. The strips were partially contracted with prostaglandin (PG) F2~ ((2-10)× 10 -7 M), the contraction being in a range between 30 and 43% of the contraction induced by 5 mMBa 2+. Nicotine and nitric oxide (NO) in single concentrations were successively applied, unless stated otherwise. After the end of each series of experiment, papaverine (10 -4 M) was applied to

attain the maximal relaxation, which was taken as a standard for relaxation. Venous strips had been treated for 15-20 min with blocking agents, before the effects of agonists were obtained. Results are expressed as mean values + SE. Statistical analyses were made using the Student's paired and unpaired t-tests and the Tukey's method after one-way analysis of variance. Drugs used were N~-nitro-L-arginine (LNA), Nt-nitro-o-arginine (D-NA) (Peptide Institute, Minoh, Japan), L-arginine, o-arginine, nicotine (base), hexamethonium bromide (Nacalai Tesque, Kyoto, Japan), prostaglandin (PG) F2~ (Upjohn, Tokyo, Japan), prazosin hydrochloride (Pfizer-Taito, Tokyo, Japan), indomethacin (Sigma, St. Louis, MO, USA), atropine sulfate (Tanabe, Osaka, Japan), timolol maleate (Banyu, Tokyo, Japan) and papaverine hydrochloride (Dalnippon, Osaka, Japan). Oxy-

HUMAN UTERINE V E I N - - N i c o t i n e

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Control

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L-NA + D-arg.

N NO PA -

NO

PA

NO N NO

PA

NO

PA

L-NA + L-arg.

PA N NO

PA

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10min

Fig. 3. Typical tracings of the response to nicotine (N; 10 -4 M) and NO (10 -6 M) in a human uterine venous strip before (lst tracing) and after treatment with 10 -3 M pmzosin (2nd to 6th tracings). Under treatment with the al-adrenoceptor antagonist, effects of NG-nitro-D-arginine (D-NA; 10 -6 M), Nt-nitro-L-arginine (L-NA; 10 -6 M), L-NA plus D-arginine (D-arg.; 3 × 10 -4 M) and L-NA plus L-arginine (L-arg., 3 × 10 -4 M) were determined. The strip was partially contracted with PGF2~ (3 × 10 -~ M). PA represents 10 -4 M papaverine to attain the maximal relaxation.

N. Toda et al. / Journal of the Autonomic Nervous System 55 (1995) 189-192 HUMAN UTERINE VEIN--NO

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Fig. 4. Modifications by NG-nitro-L-arginine (L-NA; l0 -6 M), L-NA plus L-arginine (L-Arg.; 3 X l0 -4 M) and oxyhemoglobin (OxyHb; 1.6 X l0 - s M) of the response to 10 -6 M NO in human uterine venous strips partially contracted with PGF2~. Relaxations induced by l0 -4 M papaverine were taken as 100%. Significantly different from control (C), a p < 0.001 (unpaired t-test). Numbers in parentheses indicate the number of strips from separate individuals. Vertical bars represent SE.

hemoglobin was prepared from hemoglobin (Sigma) by a method described by Martin et al. [11]. Responses to NO were obtained by adding NaNO 2 solution adjusted at pH 2 [8].

3. Results

In human uterine venous strips (10 from different individuals) partially contracted with PGF2,,, the addition of nicotine in a submaximal concentration of 10 -4 M [22] produced a slight, transient contraction in 4 and a relaxation in the remaining 6. The contraction was reversed to a relaxation by treatment with 10 -5 M prazosin, and the relaxation was potentiated (Fig. 1). The relaxant response of prazosin-treated strips was abolished by treatment with 10 -5 M hexamethonium (n = 10). Therefore, the mechanism of action of nicotine was analyzed in the strips treated with the c~l-adrenoceptor antagonist in the remainder of this study. The nicotine-induced relaxation was not influenced by Table I Modification by antagonists of the relaxation induced by 10 -4 M nicotine in human uterine venous strips treated with 10 -2 M praz~sin Treatment

Indomethacin, 10 -6 M Atropine, 10 -7 M Timolol, 10 -7 M Oxyhemoglobin, 1.6)< 10 -5 M

Nicotine-induced relaxation a n

Control

Experimental

5 5 5 5

34.1-t-5.4 32.64-6.5 36.64-5.9 36.3+5.6

32.5+4.9 36.94-5.7 37.1+7.9 0.8+ 1.2 b

n, Number of strips from separate individuals. a Relaxation relative to that caused by 10 -4 M papaverine. b Significantly different from control, P < 0.001 (unpaired t-test).

191

treatment with D-NA (10 -6 M) but was abolished or reversed to a slight contraction by L-NA (10 -6 M) (Fig. 2). The depressed relaxation was restored by 3 × 10 -4 M L-arginine but not by D-arginine. The typical tracings are demonstrated in Fig. 3. In the venous strips, similar magnitude of relaxation was induced by the addition of 10 -6 M NO; the response was not influenced by L-NA and Larginine but was abolished by treatment with oxyhemoglobin (1.6 × 10 -5 M) (Fig. 4). Relaxations induced by nicotine were not influenced by indomethacin ( 1 0 - 6 M ) , a t r o p i n e ( 1 0 - 7 M ) and timolol (10 -7 M) but were abolished by oxyhemoglobin (1.6 × 10 -s M). The results are summarized in Table 1.

4. Discussion

Nicotine stimulates nerve terminals, increases the influx of Ca 2+ and liberates neurotransmitters in vasculature [14,16]. Responses to non-adrenergic, non-cholinergic nerve stimulation by nicotine and electrical pulses in canine cerebral arteries are similarly affected by pharmacological antagonists, such as NO synthase inhibitors, hemoglobin, methylene blue, non-selective Ca2+-channel inhibitors, calmodulin, etc., except for the fact that the response to nicotine is blocked by hexamethonium but not by tetrodotoxin, and the opposite is true in the case of response to electrical nerve stimulation [15,17,20-22,25]. Therefore, nicotine would be a useful tool to chemically stimulate nerve terminals. Human uterine veins partially contracted with PGF2~ responded to nicotine with contractions in 4 out of 10 strips and with relaxations in the remaining 6. The responses were abolished by hexamethonium. The contraction was reversed to a relaxation by treatment with prazosin, whereas the relaxation was potentiated, suggesting that aradrenoceptors are stimulated by norepinephrine released from adrenergic nerves in response to nicotine. In guinea pig [7,13] and human uterine arteries [19], norepinephrine-induced contractions are mediated predominantly by al-adrenoceptors. Relaxations seen in prazosintreated strips were not influenced by timolol, atropine and indomethacin, indicating that /3-adrenoceptors, muscarinic receptors and PG-related mechanisms are not involved. The nicotine-induced venous relaxation was not influenced by D-NA but was abolished by L-NA, and the inhibition was reversed by the addition of L-arginine but not by D-arginine. Oxyhemoglobin, a NO scavenger [11], also abolished the response. Relaxations caused by exogenously applied NO were not affected by L-NA but were abolished by oxyhemoglobin. Similar findings were also obtained in guinea pig [10] and human uterine arteries [19]. Histochemical study has demonstrated perivascular nerves containing NADPH-diaphorase in human uterine veins [28]. According to Dawson et al. [6], NO synthase and NADPH diaphorase are identical in neurons. These findings may

192

N. Toda et al./ Journal of the Autonomic Nervous System 55 (1995) 189-192

indicate that human uterine veins are innervated by nonadrenergic, non-cholinergic vasodilator nerves from which NO is liberated as a neurotransmitter. Non-cholinergic vasodilator innervation is postulated in guinea-pig uterine arteries, although the mechanism is not clarified [2]. We have reported that human uterine arteries are also innervated by the NO-mediated vasodilator nerve [19]. The relaxation elicited by nicotine is similar to that seen in the veins. Despite a less innervation in human veins that is observed by histochemical study with NADPH diaphorase [28], functioning of the nerve does not appreciably differ in the arteries and veins. In summary, the tone of human uterine veins is regulated by adrenergic vasoconstrictor and nitroxidergic [23] vasodilator nerves. Recently, attention is directed to the relationship between the NO-mediated response or constitutive NO synthase in uterine arteries and pregnancy or female sex hormones [1,9,27]. Nitroxidergic nerve in the vein, as well as artery may play an important role in the control of blood flow in pregnant and non-pregnant uteri.

[10]

[11]

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