A heat released substance from rat ileal muscle

Phurmac~ologic~alReseur-ch, Vol. 26. No. I, 1992

A HEAT

RELEASED

ZOHEIR

93

SUBSTANCE MUSCLE

A. DAMANHOURI

FROM

and OSAMA

RAT ILEAL

S. TAYEB

Department of Pharmacology, Faculty of Medicine, King Ahdulaziz Unil~ersit~, P.O. Box 9029, Jeddah 21413, Saudi Arabia Receilsed in,finalfbrm

13 September 1991

SUMMARY

Change in body temperature is reported to induce changes in muscle contraction by unconfirmed mechanisms. Therefore, the changes in isolated rat ileal muscle contractions at different surrounding temperatures were studied. The contraction was decreased by increase of the surrounding temperature from 37 “C to 40 “C, while it was increased by decrease of the temperature to 30 “C. Further, bathing the muscle in a conditioned medium prepared by incubation of the ileal muscle at 40 “C caused a decreasein contraction at a surrounding temperature of 37 “C. This indicates that increase of the temperature around muscles induces a release of the substance that causes a decrease in the contractile response. The effects of this heat-released substance were not blocked by propranolol, phentolamine and naloxone, or edrophonium, which indicates that these effects are not produced through an interaction with cholinesterase enzyme, a or p adrenergic receptors or opiate receptors. KFY WORDS: heat illnesses, muscle contractions, temperature effect, rat ileal.

INTRODUCTION

Heat-related illnesses are characterized by a variety of signs and symptoms which result from the deleterious effect of high body temperature, or possibly from compensatory mechanisms [l, 21. Among these effects, various degrees of disturbances of the nervous system are expected [2, 31. Such actions, which may be caused at least partially by a disturbance of the autonomic nervous system, include changes in muscular contractions. Several studies have reported changes in the contraction of vascular smooth muscles [2], tracheal smooth muscles, and skeletal muscles [ 1, 21. There are no reports about the changes in the contraction of ileal smooth muscles, but there are indications of such effects by the reported gastrointestinal disturbances in heat stroke cases [4, 51. Furthermore, drugs which have been shown to be effective in the management of heat stroke, such as Correspondence to: Osama S. Tayeb PhD.

lO43-6618/92/050093AX3/$03.0/0

0 1992 The Italian Pharmacological Soclet)

94

Pharmacological

Research,

Vol. 26. No. 1, 1992

dantrolene and naloxone [6,7] have a pronounced effect on smooth muscle contractions [8,9]. In view of the above, it is thought that studies on the effect of heat on isolated smooth muscle contractions may be of value in establishing the pathogenesis of heat-related illnesses, or some of the triggered compensatory mechanisms. Therefore, we have studied the contraction of ileal smooth muscle from the rat at various surrounding temperatures.

MATERIALS

AND METHODS

The effect of temperature on smooth muscle contraction was studied on isolated rat ileum preparation. The choice of this specific preparation was becausethe rat is one of the most widely used models for heat studies [lo, 1 I]. O ther models which involve the use of dogs, rabbit, and sheep are not as suitable and convenient for isolated preparation studies as the rat. Guinea pigs and mice were not reported in any studies on the effects of heat exposure. Male Wistar rats (200-250 g) were fasted 24 h before the time of the experiment. Then, they were killed by cervical dislocation, and dissected for the ileum. Short segments of the ileum, from the caecal end, were removed and put immediately in a petri dish containing oxygenated (95% 02/5% CO?) Krebs solution of the following composition-NaCl: 6.9 g/l; NaHC03: 2.1 g/l; glucose: 2 g/l; KH2P04: 0.16 g/l; KCl: 0.36 g/l; MgS04.7H20: 0.29 g/l; CaC1.2H20: 0.37 g/l. Isolated strips of muscles were trimmed, and one of these strips was transferred to a lo-ml muscle bath at 37 “C. The muscle was washed with Krebs solution and left for 30 min for equilibration. Muscle contractions were recorded by a Palmer Bioscience potentiometric recorder connected to the muscle through a Bioscience UFl isometric transducer and Al00 Bioscience coupler. Carbachol was added to the muscle bath in various concentrations in a graded manner. The muscle was washed after each addition of carbachol. This was continued until the dose that caused a maximum contraction of the muscle was reached. Then, in some experiments, bath temperature was changed to 30 “C or 40 “C and left for equilibration for 30 min, before the same series of carbachol doses was again tested. The bath temperature was brought back to 37 “C, and challenged with the same doses of carbachol after 30 min of equilibration. In some experiments, one of the following drugs was added to the bath before one minute of carbachol addition. These drugs are propranolol hydrochloride (Inderal, ICI, UK), phentolamine methylate (Ciba, Switzerland) and naloxone hydrochloride (Narcan, DuPont pharmaceuticals, USA). In some other experiments, a conditioned medium was prepared by incubation of a piece of the ileal muscle (similar in size to the muscle used in the muscle bath) in 10 ml Krebs, oxygenated by 95% OJ5% CO? at the desired temperature (40 “C or 30 “C) for 30 min. This conditioned medium was added to the bath in some experiments instead of normal Krebs solution, before we challenged the muscle with carbachol. A fresh conditioned medium was used for each dose of carbachol tested. In another set of experiments, carbachol responsewas tested in the same manner

except that the Krebs solution contained edrophonium chlorate (Tensilon, Roche. Switzerland). Carbachol-induced contractions were measured for each dose and the size of contraction was calculated as a percentage of the maximal contractions elicited by carbachol at 37 “C. Several groups of various exposures and treatment conditions were studied. Experiments of each group were repeated at least 10 times. Percentages of contractions induced by carbachol from different experiments under the same conditions were averaged. Results were plotted on a semi-log paper. Responses to similar doses of carbachol in different groups were compared to each other by Student’s r-test.

RESULTS

Effect of bath temperature Figure 1 shows the dose-response curves for carbachol-induced contractions of the isolated rat ileal muscle at different surrounding temperatures. Comparison of A

150 -

B

C

140 130. 120. 110

2

3

4

56

2 CARBACHOL

3

4

56

CONCENTRATION

2

3

4

56

Cg/ml)

Fig. 1. Log dose-responsecurves of the effect of carbachol on the contraction of the isolated rat ileum at the following different conditions: at 37 “C in presenceor absenceof CM40, and at 40 “C (a), at 37 “C in presenceor absenceof CM30 and at 30 “C (b), and at 30 “C in presenceor absence of CM40 and at 37 “C (c). Each point is the average of at least 10 repetitions. Lines above and below points represent SEM. The temperature figure next to each curve representsthe bath temperature at which experiments were performed. CM40 represents the use of a conditioned medium at 40 “C instead of normal Krebs around the muscle. CM30 representsthe use of a conditioned medium prepared at 30 “C instead of normal Krebs around the muscle. * indicates significant difference versus the responses in presence of normal Krebs. at 37 “C tP< 0.01).

Pharmacological

96

Research,

Vol. 26, No. I, 1992

these curves to the observed responsesat the normal natural body temperature of 37 “C, showed that the responses to carbachol at 40 “C were decreased significantly (P
I 2

CARBACHOL

/ 3

III 4

56

CONCENTRATION

lug/ml)

Fig. 2. Log dose-response curves of the effect of carbachol on the contraction of the isolated rat ileum in the presence of naloxone (Nal) at different surrounding temperatures. Each point is the average of at least IO repetition. Lines above and below points represent SEM. 0-O Response in presence of 8 rig/ml Nal at 37 “C. A-A Response in presence of 8 rig/ml Nal at 40 “C. O-O Response in presence of 4 rig/ml Nal at 37 “C. A-A Response in presence of 4 r&ml Nal at 40 “C. * indicates significant difference versus the responses of the corresponding Nal concentration at 37 “C (P
‘.>I

Phr~r.nrcrc~olo,~ic~ul Research. Vol. 26, No. 1, 1992

with the conditioned medium prepared at 30 “C around the muscle in the bath, at a surrounding temperature of 37 “C, did not cause any change in the responses to carbachol (Fig. 1b). Ejfect

of nuloxone

at different

bath temperatures

Figure 2 shows the responses to carbachol in hydrochloride at 37 “C and 40 “C. The responses at 40 “C than the responses observed at 37 “C (PcO.01). Further, difference between the two doses of naloxone used in this effect

oj’propranolol

at different

presence of naloxone were significantly lower there was no significant study.

temperutures

Figure 3 shows the responses to carbachol in presence of propranolol hydrochloride at 37 “C and 40 “C. The responses at 40 “C were significantly lowe] than the responses observed at 37 “C (PcO.01). Further, there was no significant difference between the two doses of propranolol used in this study. Ejfect

of phentolamine

at diflerent

temperatures

Figure 4 shows the responses to carbachol in presence of phentolamine at 37 “C and 40 “C. The responses at 40 “C were significantly lower than the responses observed at 37 “C (PcO.05). Further, there was no significant difference between the two doses of phentolamine used in this study. Effect

of edrophonium

Figure 5 shows the responses to carbachol in presence of edrophonium

at 37 “C

loogo-

*

80-

?

g

70-

f, g

GO-

l l

J+

i.t 50SQ 40-

l !S l

*

~

302010-

3

2

CARBACHOL

4

56

CONCENTRATION

@g/ml)

Fig. 3. Log dose-response curves of the effect of carbachol on the contraction of the isolated rat ileum in the presence of propranolol (Propr.) at different surrounding temperatures. Each point is the average of at least IO repetitions. Lines above and below points represent SEM.

O-O

Response in presence of IO rig/ml Propr. at 37 “C. A---A Response in presence of

IO rig/ml Propr. at 40 “C. 0-O Response in presence of 30 rig/ml Propr. at 37 “C. &-A Response in presence of 30 rig/ml Propr. at 40 “C. * indicates significant difference versus the responses of the corresponding Propr. concentration at 37 “C (P
98

Pharmac,ological

Research.

Vol. 26, No. I. 1992

(normal Krebs and 40 “C conditioned medium), and at 40 “C. The responses at 40 “C, or at 37 “C in the presence of 40 “C conditioned medium were lower than the responses observed at 37 “C in normal Krebs (PcO.05). Further, there was no

20 IO 1 I

I

2

3

CARBACHOL

III 4

56

CONCENTRATION

@g/ml)

Fig. 4. Log dose-response curves of the effect of carbachol on the contraction of the isolated rat ileum in the presence of phentolamine (Phent) at different surrounding temperatures. Each point is the average of at least 10 repetition. Lines above and below points represent SEM. 0-O Response in presence of 10 rig/ml Phent. at 37 “C. A-A Response in presence of 10 rig/ml Phent. at 40 ‘C. O-O Response in presence of 30 rig/ml Phent. at 37 “C. A-A Response in presence of 30 rig/ml Phent. at 40 “C. * indicates significant difference versus the responses of the corresponding dose at 37 “C (PcO.05).

37% + Edro.

loo-

CM40 at 37%+ Edro.

90-

40°C+Edro. si

6t-

5 $

70-

’ 8

605040 I

I

I,,

2

3

456

CARBACHOL CONCENTRATION @g/ml)

Fig. 5. Log dose-response curve of the effect of carbachol on the contraction of the isolated rat ileum in presence of edrophonium (Edro.) at different conditions. Each point is the average of at least 10 repetition. Lines above and below points represent SEM. The temperature figure next to each curve represents the bath temperature at which experiments were performed. CM40 represents the use of a conditioned medium prepared at 40 “C instead of normal Krebs around the muscle. * indicates significant difference versus the responses at 37 “C (P ~0.05).

Pharmucolo,qicul Research, Vol. 26, No. I, 1992

99

significant difference between the responses at 40 “C or at 37 “C in the presence of 40 “C conditioned medium.

DISCUSSION The observed changes in the response of rat ileal muscle to carbachol at different bath temperatures are of some interest. High surrounding temperature decreases this response, while low surrounding temperature causes an increase. Although the observed change in response at 30 “C confirms previous findings in other muscle preparations [ 121, there are no reports of a definite effect of high temperature. There are two possibilities for the mechanism of these actions of high or low surrounding temperatures. It is possible that these actions were caused through a direct effect on the contractile element of the muscle, or they could be caused indirectly through some sort of a mediator. The observations that the conditioned medium prepared from muscles incubated at 30 “C did not cause any change in the response if tested at 37 “C, indicate that the effect of low surrounding temperature is a direct effect on the muscle. On the other hand, the conditioned medium prepared at 40 “C caused a decrease in the muscle response to carbachol at 37 “C. This indicates that upon incubation of the muscle at 40 ‘C, substances are released and that they cause a decrease in the response of the muscle which released it and other muscles which did not release it. Further, the observation that the response of muscles at 30 “C was decreased if bathed in 40 “C conditioned medium, confirms the existence of a heat-released substance or substances (HRS) in the medium prepared from incubation of muscles at 40 “C. As the HRS caused a decrease in the response of ileal muscle to carbachol. we have tried to identify it by using antagonists to the inhibitory agents for ileal smooth muscle contraction. Among these agents, opiates and adrenergic stimulants are the most widely known to have such an action [13, 141. None of the antagonists to these agents (naloxone, propranolol and phentolamine) blocked the effect of HRS. This indicates that HRS does not exert its actions through opiate receptors nor through a or p adrenergic receptors. Another possibility for the mechanism of HRS actions is that HRS could be a cholinesterase enzyme or an activator of some type for such an enzyme. Therefore. we have tested the effect of high surrounding temperature, and the effect of conditioned medium prepared at 40 “C, on the contractions of the ileal muscle in the presence of a cholinesterase inhibitor. The effect of HRS persisted even in the presence of edrophonium. This indicates that HRS is not a cholinestrase or an aclivator of such an enzyme. In conclusion, high surrounding temperature causes the release of the substance HRS which causes a decrease in the response of ileal muscle to carbachol. The effects of HRS are not blocked by antagonists to the known agents that cause a decrease in muscle contractions. Investigations are continuing in our laboratory to isolate and characterize HRS.

100

Pharmacological Research,Vol. 26. No. I, 1992

REFERENCES I. Anderson RJ, Reed G, Knochel J. Heat Stroke. Advanced Int Med 1983; 28: I 15-40. 2. Bova CM. Heat Illness. In: Greenberg MI, Roberts FA, eds. Emergency medicine. Philadelphia: Davies Publishing Co, 1982: 197-2 17. 3. Bark N. Heat stroke in psychiatric patients. Two cases and a review. .I C/in Psychiatry 1982; 43: 377-80. 4. Barry ME, King BA. Heat stroke. S African Med J 1962; 36: 455-61. 5. Carson J, Webb JF. Heat illness in England. J Roy Army Med Corps 1973; 119: 2-83. 6. Tayeb OS, Marzouki ZM. Effect of dantrolene preteatment on heat stroke in sheep. Pharmacol Res 1990; 22: 565-73. 7. Tayeb OS, Marzouki ZM. Effect of naloxone pretreatment on heat stroke in sheep. J Wilderness Med 1992 In press. 8. Conte-Camerino D, Lograno MD, Siro-Brigiani G, Negna G. Dantrolene sodium: Stimulatory and depressant effects on contractility of guinea-pig uterus in vitro. Eur J Pharmacol 1983; 92: 291-4. 9. Martin WR. Naloxone. Ann Intern Med 1976; 85: 765-8. 10. Hubbard RW, Boweres WD, Mathew WT. Rat model of acute heat stroke mortality. J Appl Physiol Respirat Environ Exercise Physiol 1977; 42: 809-16. I 1. Kielblock AJ, Strydom NB, Burger FJ, Pretorious PJ, Manjoo M. Cardiovascular origins of heat stroke pathophysiology. An anesthetized rat model. Aviat Space Env Med 1982; 53: 171-8. 12. Bratton DL, Tanaka DT, Grunstein MM. Effects of temperature on cholinergic contractility of rabbit airway smooth muscle. JAppI Physiol 1987; 53: 1933-41. 13. Jaffe JH, Martin WR. Optimal analgesics and antagonists. In: Gilman AG, Goodman LS, Gilman A, eds. The Pharmacological basis qf therapeutics, 6th edn. New York: Macmillan Publishing Co, 1980: 494-534. 14. Mayer SE. Neurohumoral transmission and the autonomic nervous system. In: Gilman AG, Goodman LS, Gilman A, eds. The pharmacological basis of therapeutics, 6th edn. New York: Macmillan Publishing Co, 1980: 56-90.