Relaxation of rat tail artery to electrical stimulation

Life Sciences, Vol. 33, pp. 303-309 Printed in the U.S.A.

Pergamon Press

RELAXATION OF RAT TAIL ARTERY TO ELECTRICAL STIMULATION Anthony B. Ebeigbe, Robin D. Gantzos and R. Clinton Webb Department of Physiology University of Michigan Ann Arbor, Michigan 48109 (Received in final form May 5, 1983) Summary Rat tail artery strips relax in response to electrical stimulation (0.1-8Hz, 9V, 1.Omsec) following contraction induced by norepinephrine (s.9xlO- 7M). The relaxation is not altered by treatment of the strips with atropine, propranolol, tetrodotoxin or indomethacin nor by chemical denervation with 6-hydroxydopamine. Incubation of strips in calcium-free solution reduced the contractile response to norepinephrine and blocked relaxation in response to 4Hz electrical stimulation. Histamine antagonists (HZ receptor subclass: cimetidine, metiamide) inhibited the relaxation to electrical stimulation in a dose~dependent manner. These results suggest that relaxation to electrical stimulation in rat tail artery is modulated by calcium and by the HZ subclass of histamine receptors. Many studies have described the occurrence of neurogenic relaxation in various isolated blood vessels in response to electrical stimulation (1-5). However, in the canine coronary artery, made to contract with various agonists, a non-neurogenic relaxation to electrical stimulation has been reported (6). The nature of the dilator substance is not clear, since the response persists in the presence of various antagonists of transmitter action. The present study was designed to characterize relaxation to electrical stimulation in rat tail arteries. Methods Helical strips (0.8 X 10.Omm) of tail arteries from adult male SpragueDawley rats (ZsO-300g) were prepared and suspended in physiological salt solution (PSS) in sOm1 tissue baths (see reference 7 for details). The composition of the PSS was (mM): NaCl, 130; KCl, 4.7; KHZP04, 1.18; MgS04-7HZO, 1.17; CaClz, 1.6; NaHC03, 14.9; dextrose, 5.5; and CaNaZ EDTA, 0.03. The PSS was maintained at 37°C, pH 7.4 and gassed with 95% 0Z/s% COZ. Each strip was connected to a force transducer (Grass FT.03) under a resting tension of 600mg, and allowed to equilibrate for 90min before the start of experiments. Electrical stimulation was effected by delivering supramaximal voltage (9V) across two platinum electrodes placed parallel to the strips, using a Grass model 6 stimulator at 1.Omsec pulse duration (7). Strips were made to contract in response to s.9xlO- 7M norepinephrine (NE). The effect of electrical stimulation on such NE-induced contractions was studied in the absence or presence of various pharmacological agents. In some experiments, tail artery strips were acutely denervated with 6hydroxydopamine (6-0HDA) according to the method of Aprig1iano and Hermsmeyer (8). Strips were incubated in unbuffered (bicarbonate-free), unaerated PSS with 1.Zx10-sM 6-0HDA for lOmin. The pH of this PSS was adjusted to 4.0 by 00Z4-320S/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

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adding 2.0xlO- SM glutathione. Following denervation, strips were allowed to recover for 2hr in normal PSS. Drugs used were: norepinephrine bitartrate (Levophed, Sterling Drug Inc.); atropine sulphate (Sigma Chemical Co.); propranolol (Inderal, Ayerst Laboratories Inc.); indomethacin (Sigma Chemical Co.); phentolamine mesylate (Regitine, Ciba); tetrodotoxin (Sigma Chemical Co.); cimetidine (Smith, Kline & French); metiamide (Smith, Kline & French); pyrilamine (Sigma Chemical Co.); histamine hydrochloride (Sigma Chemical Co.); 6-hydroxydopamine hydrobromide (Sigma Chemical Co.); prostaglandin F2~ (Sigma Chemical Co.); 4-methylhistamine and dimaprit (gifts from Dr. D.M. Cohen, Warner-Lambert, Parke-Davis Co.). Drugs were diluted in deionized-distilled water before use. Indomethacin was dissolved in ethanol. The bath concentration of ethanol did not exceed 0.1%; control tests contained the same concentration of ethanol, which had no observable effect on force generation. Values presented are means + standard error of the mean (SEM). Statistical assessment was made by Stude~t's t-test; significance level was P <0.05. Results Tail artery strips (n=20) studied contracted in response to electrical stimulation (4Hz, 9V, 1.Omsec) applied when the muscle was completely relaxed; force developed was 437±40mg. These cgntractions were absent in denervated strips, or following phentolamine (10- M) treatment. Following contraction induced by NE however, electrical stimulation produced relaxation (frequency range 0.1-8.0Hz; Figure 1). Tail artery strips contracted with prostaglandin F2~ (2.lxlO- sM, n=4) or serotonin (s.7xlO- 7M, n=12) showed comparable relaxa~ tion responses to electrical stimulation.

0

20

:gc::: ~

40

~

~ ~

Control

60

80

100

0.1

0.5 Frequency

2

8

(Hz)

FIG 1 Frequency-dependent relaxation of s.9xlO- 7M NE contracted rat tail artery strips to electrical stimulation in the absence (e) and presence (0) of 4.0x 10-sM cimetidine. Strips were exposed to cimetidine 10min prior to and during the frequency-response tests. Values are the means + SEM for 7 experiments.

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305

The effect of various neurotransmitter antagoni sts were s t udi ed by exposing strips to each anta gon ist 10min prior to NE stimulation, and during subsequent frequenc y-response tests; propranolol, ouabain, atropine, pyrilamine, tetrodotoxin, indomethacin and 6-0HDA treatment did not significantly affect r elaxation to electrical stimulation (Table 1). The concentrations of various antagonists used preven t ed the responses to t heir respect ive a gonists. Propranolol, metiamide, cim etidine, ouabain, tetrodotoxin, indomethacin and atropine had no observable eff ec t on ba s el i ne tension or responsiveness to NE. Pyrilamine did not alter baseline tension, but depre ssed contractile respons es to NE by a bou t 35%; the reduced con t rac t i l e response was unrelated to the magnitude .of relaxation i n r esponse to electrical stimulation. In all experiment s, cimetidine, 4.0xlO- 5M (n=8) did not affect the magnitude of NE-induced contraction , but significantly (P < 0.05) shifted the frequenc y-re sponse curve t o the r i ght (Fig. 1). Pretreatment of strips with 4.0x 10-5M cimetidine blocked relaxations to 4Hz electrical stimulation (Fig. 2). In other experiments (n =6) , the effect of varying concentrations of cimetidine was studied on relaxation induced by 4Hz electrical s t i mul a t i on . The ED50 for c i me t id i ne --i nduc e d inhibition was 5.0(+0.2)xlO- 6M (Fig. 2). The blockade by cimetidine could be partially overcome by increasing the f requency of stimulat ion t o 16 or 32Hz. Me t iamide (4.lxlO- 5M, n=4), anot her h i s t ami ne H2-receptor antagonist, also blocked relaxations to electrical s timulat i on (Table 1). The possib ility that relaxations to electrical s t i mula t i on may be calciumdependent was also studied. Str ips (n=6) were ex posed to Ca-fr e e PSS (no added calcium) f or 10min, f ollowed by 5.9xlO- 7M NE s t imul a tion. The contraction produced (14l+l2mg) was no t significantl y affected by 41l~ electrical s t i mul a t i on (Fi g. 3). - In 6 experiments, 5.9xlO- 9M NE s t imula t i on produced comparable contractions (155+24mg) in normal Ca-PSS as 5.9xlO- 7M NE stimulation in Ca-free PSS; these contractions we r e compl etely relaxed by 4Hz electrical stimulation (Fi g. 3) . TABLE 1 Ef fect of va rious e xpe rimental conditions on t he relaxation of rat tail artery strips to electrical s timula tion (9V, 4Hz, l .Omsec) f ol l owi ng 5 .9xlO- 7M NE s timulation. Expe r i ment a l condition

% Relaxation

Control

95.3 + 2.1 (20)

Propranolol (lO-6M)

93.5 + 4.6 (4)

Ouabain (lO-3M)

96 .3 + 3.1 (5)

Tetrodotoxin (10-6M)

94.8 + 2.9 (4)

Indomethacin (1.4xlO- 5M)

93 . 7 + 3.4 (6)

± 0.3

6-hydroxydopamine treatment

96.8

Pyrilamin e (3 .5xlO- 6M)

94.3 + 2 .5 (6)

At r opi ne (10-6M)

96.3 + 3.1 (5)

Metiamide (4.lxlO- 5M) Cimetidine (4.0xlO- 5M)

8.1 5.0

(2)

± 4.3*(4) ± 1.8*(8)

Values a re me ans + SEM. The a s t e r i s ks indicate significant difference from control. Numbe r of rats in parentheses. Strips were exposed to each experiment al condition lOmin prior to, and during frequenc y response tests.

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0

20

c::

.~

~ 40

...~

~ 60

~

80 100

3..

10-7

10- 6

3..

10-5

[ Cimetidine] FIG 2

3a

10-5

10- 6

(M)

Inhibition by cimetidine of relaxatio~electrical stimulation in 6 rat tail artery strips. Strips were contracted with 5.9xlO- 7M NE and then relaxed by 4Hz electrical stimulation. Gimetidine was applied lOmin prior to and during electrical stimulation. Values are means + SEM. Relaxations to exogenous histamine were studied by addition of 5.4xlO- 4M histamine during 5.9xlO- 7M NE contraction. Strips (n=8) were expo~ed to 3.0x lO-6M pyrilamine (to prevent action on histamine HI-receptors) lOmin prior to and during histamine relaxation tests. The same dose of pyrilamine had no effect on relaxation to electrical stimulation (Table 1). The maximum relaxation to histamine was 44 + 5% of control NE responses, and was completely blocked with 4.0xlO- 5M cimetidine. In the absence of pyrilamine, dimaprit (4.0xlO- 4M) and 4-methyl histamine (4.0xlO- 4M) caused relaxation of strips (n=4) contracted with 5.9xlO- 7M NE; the magnitudes of the responses were: 1) dimaprit = 42 + 4%; and 2) 4-methyl histamine = 52 + 4%. Gimetidine (4 .0x 10-5M) blocked relaxations in response to both of these selective histamine H2-receptor agonists. In 7 other experiments, 4Hz electrical stimulation did not affect the contractile response to 40 or 130mM K+ (Fig. 4, high K+ PSS was prepared by equimolar replacement of NaGl, and contained lO-6M phentolamine). Discussion The present study reports that electrical stimulation of NE-contracted rat tail artery strips causes relaxation comparable to that observed in canine coronary arteries (6). Prior treatment of strips with adrenergic and cholinergic blocking drugs, as well as tetrodotoxin and 6-hydroxydopamine, did not modify the pattern of relaxation to electrical stimulation. Thus, the relaxation cannot be due to neurogenic mechanisms. A role for vasodilator prostaglandins is also unlikely, since relaxation persists in the presence of indomethacin. The relaxation is not attributable to stimulation of the electrogenic sodium pump since ouabain did not modify it. In the present experiments, the observation that relaxations to electrical stimulation are prevented by cimetidine and metiamide (antagonists of histamine

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307

_l ._. _ ._l~_ 60°1

mo

R

I 5.91110. 7 M Norepinephrine

oj.

---

---------1

Electrical stimulation - 4 Hz Calcium-free

R

I 5.91110'7 M Norepinephrine

2min

Electrical stimulation -4Hz

12°1 mo

___r:__u----_.--.-_.~:":'::_=r.._

.

.._

I 5.9IdO' 9 M Norepinephrine

R

--..f ~t _

Electrical stimulation -4Hz

FIG 3 Relaxation to electrical stimulation (4Hz, 9V, 1.Omsec) in rat tail artery strips. Strips were contracted with 5 .9xlO- 7M NE in normal (top panel) or Ca-free (middle panel) PSS; and 5.9xlO- 9M NE in normal PSS (bottom panel). The forc e developed in the middle and bottom panels i s similar, yet no relaxation occurred in Ca-free PSS. Tissue was rinsed at "R".

2 min ---.....

1

60 0 mil

______J __ •

I

40 mM KCI

_

Electrical stimulation- 4 Hz

FIG 4 Failure of electrical stimulation (4Hz, 9V, 1.Omsec) to relax 40mM ~-induced contraction in a rat tai l artery strip. Tissue was rinsed at "R".

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at HZ-receptor sites) suggests an involvement of Hz-receptors - a subclass of histamine receptors (9,10). In more recent experiments (data not shown), we have also observed that relaxation in response to electri~al stimulation in canine coronary arteries is sensitive to Hz-receptor antagonists. The cimetidine-induced inhibition of relaxation to electrical stimulation in rat tail arteries was dose-dependent (Fig. Z). The results, thus; suggest that electrical stimulation may cause the release of histamine, which then stimulates Hz-receptors, leading to relaxation. Evidence for the presence of mast cell (11) as well as non-mast cell histamine (lZ) has been reported for various vascular tissues. However, it is also possible that histamine may merely be playing a modulatory role for some unidentified transmitter. This possibility is supported by the observation that the maximal relaxations produced by exogenously applied histamine, as well as dimaprit or 4-methy1 histamine (selective HZ agonists), are not as great as those produced by electrical stimulation. The calcium-dependence of relaxation to electrical stimulation is suggestive of a role for calcium in the secretory process of the dilator substance. The lack of relaxation to electrical stimulation in Ca-free treated strips appears to be unrelated to the magnitude of NE contraction in Ca-free medium, since a comparable contraction produced by a lower dose of NE (Fig. 3, bottom panel) is completely relaxed by electrical stimulation. The failure of electrical stimulation to relax high ~ contractions is contrary to observations on coronary arteries (6); the difference may be due to the lower concentration of ~ (10mM) in their study, as against 40 and 130 mM in the present study which produce greater depolarization. In conclusion, this paper reports a calcium-dependent, Hz-receptor-antagonist-sensitive relaxation of the rat tail artery to electrical stimulation, due to release of an endogenous dilator substance. Acknowledgements This study was supported by grants from the National Institutes of Health (HL-18575 and HL-Z70Z0). Dr. Ebeigbe is a Visiting Associate Research Scientist from the Department of Physiology, University of Benin, Nigeria, and a recipient of a Fulbright Award for an African Senior Research Scholar. R.D. Gantzos is a recipient of a Michigan Heart Association Student Fellowship. Dr. Webb is a recipient of a Research Career Development Award from the National Institutes of Health (HL-008l3). 1. Z. 3. 4. 5. 6. 7. 8. 9.

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10. 11. 12.

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N.CHAND and P. EYRE. Agents and Actions 5,277-295 (1975). C.J. GARLAND and W.R. KEATINGE. J. Physio1. 327,363-376 (1982). T.M. EL ACKAD and M.J. BRODY. Blood Vessels 12,181-191 (1975).

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