Changes in smooth muscle sensitivity to agonist drugs during development

Physiology& Behavior,Vol. 49, pp. 251-255. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

Changes in Smooth Muscle Sensitivity to Agonist Drugs During Development T H E O D O R E Y. L O T A N D F R A N C I S V. U D O H

The Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmaceutical Sciences University of Jos, P.M.B. 2084, Jos, Nigeria R e c e i v e d 12 June 1990

LOT, T. Y. AND F. V. UDOH. Changes in smooth muscle sensitivity to agonist drugs during development. PHYSIOL BEHAV 49(2) 251-255, 1991.--The receptors in the expansor secundariorum muscle of chicks were characterized pharmacologically and the changes in their response to nerve stimulation and agonist drugs determined during development. The muscle responded to noradrenergic nerve stimulation, noradrenaline and 5-hydroxytryptamine without any change in sensitivity during development. Expansor muscles from 15-day-old chicks were more sensitive to isoprenaline than muscles from older animals. The muscle from 15-dayold chicks responded to acetylcholine and histamine; the sensitivity to both drugs decreased progressively with increasing age of the chicks and disappeared by day 40 posthatching. The normal developmental decrease in response to acetylcholine and histamine were prevented by surgical denervation of the muscle; an intervention that also induced supersensivity to noradrenaline > isoprenaline > 5-hydroxytryptamine. The muscle responded to potassium chloride without any change in sensitivity during development or following surgical denervation. These f'mdings indicate that sympathetic nerves influence the responsiveness of the expansor secundariorum muscle to drugs, especially the developmental decrease in response to acetylcholine and histamine. Development

Surgical denervation

Agonist drugs

Chick smooth muscle

THE expansor secundariorum muscle (ESM), a smooth muscle in the wing of chicks, has been shown to be innervated by postganglionic noradrenergic nerves only (2, 6, 11). While earlier studies in 8-12-week-old chicks showed the absence of muscarinic receptors in the ESM (6), latter findings showed that the ESM possesses muscarinic receptors in chicks younger than 10 days posthatching which disappear by day 40 posthatching (1,8). Such developmental changes were not observed for noradrenaline. In 8-12-week-old chicks, the ESM does not respond to histamine (6,8). The possibility of a developmental change in response to histamine that might be similar to that of acetylcholine (ACh) has not yet been examined. Furthermore, although the muscle was found to be sensitive to various agonist drugs in 8-12-weekold chicks (6), it is not yet known whether there are any appreciable changes in the response of the ESM to such agonist drugs during the course of development. The aim of the present study was to examine the developmental changes in response of the ESM to various agonist drugs with a view to examining the influence of noradrenergic nerves on developmental changes in smooth muscle response to agonist drugs. Since chemical sympathectomy or surgical denervation prevent the developmental decrease in response of the ESM to ACh (1,7), the ESM was also surgically denervated to see if it can produce a similar effect for other agonist drugs.

used in the present study.

Denervation of the Expansor Secundariorum Muscle (ESM) Chicks used to study the effect of denervation on developmental changes in response of the ESM to agonist drugs were aged 30 days. While the chicks were under ether anaesthesia, the skin above the branch of the radial nerve supplying the ESM was incised and the nerve cut 1-2 cm from the muscle of the left wing with a pair of sterile scissors as described by Bennett et al. (I). The wound was sealed with an antiseptic spray dressing. The ESM from the right wing was left intact to serve as the control. The chicks were killed at the age of 50 days to study the effects of denervation.

Organ Bath Studies Chicks of different ages were killed by an overdose of ether and the ESM quickly dissected out. The ESM was freed of adherent tissue, except for the tendon and a piece of secondary feather at the base of the muscle. The feather was tied to a pin held in a perspex block and suspended in a 20-ml organ bath as described by Bennett et al. (1). The tendon was threaded 3 cm from the feather and tied to an isometric transducer connected to a Grass polygraph recorder (Model 7D). The organ bath contained 20 ml of freshly prepared physiological saline (sodium chloride, 118 mM; potassium chloride, 4.7 mM; magnesium sulphate, 2.5 mM; sodium dihydrogen ortho-

METHOD Male chicks (Light Sussex and Rhode Island Red cross) were

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phosphate, l mM; sodium bicarbonate, 30 mM; glucose, 11.1 mM; calcium chloride, 2.5 mM) gassed with a mixture of 95% oxygen and 5% carbon dioxide. The bath was maintained at 37.0 +_ 1.0°C by means of a water jacket connected to a thermostatically controlled pump (Haake). Tissues were given 30 rain to equilibrate before experiments started. Parallel platinum wire electrodes were arranged on either side of the tissue and connected to a constant voltage square-wave stimulator (Grass $88 Stimulator). Electrical stimuli were delivered as pulses of 140 V and 0.2-ms duration for 10 s every 4 min. The frequency of stimulation was varied as desired. These parameters have been shown to stimulate selectively the noradrenergic nerves of the ESM (11). Drug solutions were freshly prepared in concentrations such that the addition of 0.2 ml gave the final bath concentration (g/ ml or M for potassium chloride). Ascorbic acid (1 x 10 4 M) was added to dilute solutions of noradrenaline (NA) or isoprenaline. The drug contact time was 1 min with an interval between successive doses of at least 5 min. Concentration-response curves were established by graded increases in the concentrations of agonist drugs, frequency-response curves by graded increases in frequency of stimulation. Antagonists were always applied 5 min before the agonist drugs, where they were used.

Drugs Drugs used were acetylcholine bromide (British Drug Houses), atropine sulphate (Sigma), Delysid or LSD25 (Sandoz), histamine acid phosphate (British Drug Houses), isoprenaline hydrochloride (British Drug Houses), mepyramine maleate (May & Bayker), noradrenaline bitartrate (Sigma), phentolamine mesylate (Ciba), propranolol hydrochloride (I.C.I.), and serotonin creatinine sulphate (Kotch-Light).

Tension (g)

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FIG. t. Mean contractions of the expansor secundariorum muscle (ESM) (n = 5 in each group) to graded increases in the frequency of nerve stimulation; vertical lines show s.e.m. ESM from 15-day-old chicks (X); ESM from 30-day-old chicks (A); ESM from 40-day-old chicks ([]); ESM from 50-day-old chicks ((3); ESM from 50-day-old chicks denervated 20 days previously (0). Abscissa scale: frequency of stimulation (Hz) on a log scale. (A) Ordinate scale: response as tension in grams. (B) Ordinate scale: response as a percentage of the maximum tension. *p<0.05 by Student's unpaired t-test compared to ESMs from 15-day-old chicks. +p<0.001 by Student's unpaired t-test compared to normal ESMs.

Statistical Analysis Regression lines with confidence limits were calculated for the linear portions of log concentration-response or log frequency-response curves. The significance of differences in slope was used as a measure of parallelism of the two lines. Log concentration or frequency limits at 50% of the maximum response were used in the analysis of the significance of concentration or frequency differences as described by Birmingham et al. (5). Maximum responses were compared by means of Student's unpaired t-test. RESULTS

Field Stimulation The expansor secundariorum muscle (ESM) taken from chicks of increasing ages all responded to field stimulation in a graded fashion. The responses were associated with age-related increases in the highest responses recorded which were significant (p<0.05) for ESMs from 50-day-old chicks compared to chicks aged 15 days (Fig. 1A). There was no significant difference (p>0.05) between the sensitivities of ESMs for the different chick ages (Fig. 1B). Surgical denervation abolished the response of the ESM to nerve stimulation since the denervated ESM was only weakly responsive to stimulation at higher frequencies (Fig. 1A).

ing more potent than isoprenaline (Fig. 2A, B). The contractions were associated with an increase in the maximum tension for NA and isoprenatine with increasing age of the chicks which were not always significant. The sensitivities of ESMs to NA from chicks of all the ages investigated did not differ (p>0.05) from each other (Fig. 2A). The sensitivities of ESMs to isoprenaline from chicks aged 30, 40 or 50 days were similar (p>0.05) but less (p<0.05) than the sensitivity of ESMs from 15-day-old chicks (Fig. 2B). Denervation increased the sensitivity of ESMs to NA compared to the contralateral control muscles (p<0.001) or ESMs from chicks aged 15, 30 and 40 days (p<0.001) (Fig. 2A). There was also an increase in sensitivity of denervated ESMs to isoprenaline compared to the control (p<0.001) which were similar to muscles from 30 and 40 days but less pronounced (p<0.001) when compared to ESMs from 15-day-old chicks (Fig. 2B). The increase in sensitivity following denervation was more marked for NA than isoprenaline (Fig. 2). The contractions of ESMs from chicks of all ages and of denervated ESMs evoked by NA were all abolished by 1 x 10-5 g/ml phentolamine but were unaffected by 1 x 10 -5 g/ml propranolol. The contractions of all normal ESMs or denervated ESMs evoked by isoprenaline were abolished by 1 x 10 - 5 g/ml propranolol but were unaffected by 1 x 10 -5 g/ml phentolamine.

5-Hydroxytryptamine (5-HT) and LSD25 Adrenergic Agonists and Antagonists Noradrenaline (NA) and isoprenaline contracted the ESM taken from chicks of increasing ages in a dose-related fashion, NA be-

The responsiveness of ESMs to 5-HT were comparable to those for NA (Fig. 2). The decrease in sensitivity of the ESM to 5-HT with increasing age of the chicks was not significant (p>0.05)

SMOOTH MUSCLE SENSITIVITY CHANGES

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Nomdrenoline (- Log. g;ml)

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HG. 2. Mean response of expansor secundariorum muscles (ESMs) (n=6 in each group) from chicks aged 15 days (X), 30 days (~), 40 days (D), 50 days ((3) or 50 days with the ESM denervated 20 days previously (0) to graded increases in concentration of (A) noradrenaline, (B) isoprenaline and (C) 5-hydroxytryptamine. Vertical lines show s.e.m. Ordinate scale: response as a percentage of the maximum tension.

(Fig. 2C). However, there was an increase in the maximum tension developed to 5-HT with increasing age of the chicks which was not always significant. Denervation increased the sensitivity of ESMs to 5-HT compared to normal muscles (0.01>p>0.001) from 40- or 50day-old chicks but not muscles from 15- or 30-day-old chicks (p>0.05) (Fig. 2C). The increase in sensitivity of ESMs to 5-HT was less than those for isoprenaline and NA (Fig. 2). Responses of normal or denervated ESMs to 5-HT were abolished by 1 × 10 - 6 g/ml LSD25.

by increases in the maximum tension developed that were not all significant (Fig. 3A). This was not associated with a significant change (p>0.05) in the sensitivity of the ESM to KC1. Denervation depressed the maximum tension (p<0,01) developed to KC1 (Fig. 3A) without affecting the sensivity of the ESM to KC1.

Acetylcholine (ACh) and Atropine ACh contracted the ESM from 15-day-old chicks in a doserelated fashion with the highest responses being comparable to those of NA. This had decreased by 30 days posthatching. Above 30 days of age, the responsiveness of the ESM to ACh rapidly declined (Fig. 3C). Denervation prevented the decline in response of ESMs to ACh

Potassium Chloride (KCI) Potassium chloride contracted the ESM taken from chicks of increasing ages in a dose-related fashion which was accompanied

A

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FIG, 3. Mean response of expansor secundariorum muscles (ESMs) (n=6 in each group) from chicks aged 15 days (X), 30 days (A), 40 days (D), 50 days (O) or 50 days with the ESM denervated 20 days previously (0) to graded increases in concentration of (A) potassium chloride, (B) acetylcholine and (C) histamine. Vertical lines show s.e.m. Ordinate scale: response as tension in grams; note the expanded scale in (C).*p<0.05 by Student's unpaired t-test compared to ESMs from 15-day-old chicks.

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with age since such muscles were as responsive to ACh as muscles from 15-day-old chicks (Fig. 3B). All responses of normal or denervated ESMs to ACh were abolished by 1 × 10- 6 g/ml atropine.

Histamine and Mepyramine Histamine was the weakest agonist investigated since the responses of ESMs from 15-day-old chicks were the smallest (Fig. 3C). This was followed by a marked decline in responsiveness seen with muscles from older chicks, the ESMs from chicks above 30 days being unresponsive to histamine. Denervation maintained the response of the ESM to histamine which was associated with an increase (p<0.001) in the maximum tension developed compared to muscles from 15-day-old chicks (Fig. 3C). The response of normal or denervated ESMs to histamine were always less (p<0.05) than the corresponding ones for ACh (Fig. 3B, C). All responses of normal or denervated ESMs were abolished by 1 × 10 -6 g/ml mepyramine. DISCUSSION

The expansor secundariorum muscle (ESM), a smooth muscle in the wing of chicks, has been shown to be wholly innervated by postganglionic noradrenergic nerves (2, 6, 11). The parameters used in the present study have been reported to selectively stimulate the noradrenergic nerves causing contraction of the ESM through activation of et-adrenoceptors (11). Results from the present study showed that the sensitivity of the ESM to noradrenergic nerve stimulation did not change during the course of development of the chicks up to the age of 50 days posthatching. The ESM has been shown to have a-adrenergic and muscarinic receptors (1, 8, 11) which were confirmed in this study using specific antagonists. The presence of other receptor types was also identified in this study and includes serotonergic receptors, [3-adrenergic receptors and histamine (Ht) receptors which all contracted the muscle when activated in that decreasing order of potency. It is now generally accepted that potassium chloride (KC1) causes depolarization of excitable tissue, resulting in direct muscle contraction. This was found to be the case and it is of interest that the sensitivity of the ESM to such direct stimulation did not change during the course of development. Thus, changes in response of the ESM to agonist drugs described subsequently are unlikely to be due to a direct muscle effect but could be attributed to other mechanisms like changes in receptor numbers and sensitivity. The sensitivity of the ESM to noradrenaline (NA) did not change during the course of development up to the age of 50 days posthatching. This is consistent with the findings for noradrenergic nerve stimulation whose effect is also mediated by NA released by the nerves. A similar observation for NA has previously been reported (7,8). These findings would tend to suggest that the et-adrenergic receptors of the ESM do not appear to change with age. The ESM was found to be less sensitive to isoprenaline than NA as previously reported by other workers (6,8). Another difference between the actions of the two adrenergic agonists was the finding that there was a marked decline in isoprenaline sensitivity between the age of 15 and 30 days posthatching. The reason for this decline could not be determined in this study but might be due to an easier access of isoprenaline to the 13-adrenoceptors in the younger chicks. A more likely explanation could be a decline in the number of 13-adrenoceptors in the muscle between 15 and 30 days posthatching. With 5-hydroxytryptamine (5-HT), there was a gradual de-

cline in sensitivity with increasing age of the chicks. This pattern thus differs from that seen with noradrenergic nerve stimulation, KC1, NA or isoprenaline. The reason for the change in sensitivity to 5-HT was not determined but could also be due to a change in its number of receptors since its effect was found to be mediated by activation of serotonergic receptors. Another possibility is an increase in the uptake mechanism of the older nerves as reported for 6-hydroxy-dopamine (4). In spite of such a pattern of change, the responsiveness of the ESM to 5-HT was comparable to that of NA as previously reported (6). The noradrenergic innervation of the ESM has been reported to influence its responsiveness to drugs (1, 7, 9). Based on such reports, the developmental decrease in responsiveness of the ESM to acetylcholine (ACh) is now well documented and has been ascribed to a decrease in the number of postsynaptic muscarinic receptors (1). Results from the present study are consistent with such previous findings and further confirm the influence of noradrenergic nerves on muscarinic receptors of smooth muscle. It has been reported that ESMs from 8-12-week-old chicks were unresponsive to histamine (6,8). In those studies, the effects of histamine on ESMs from younger chicks were not investigated, neither did the workers examine developmental changes in response of ESMs to histamine. Results from the present study showed that histamine contracted the ESM from 15-day-old chicks through activation of histamine (Hi) receptors; this effect declined thereafter and disappeared by 40 days posthatching. In this regard, the effect of histamine on the ESM resembles that of ACh although our findings indicate that histamine-induced contractions might disappear earlier than those induced by ACh. This finding raises the interesting possibility that noradrenergic nerves might influence histamine receptors as reported for muscarinic receptors ( 1). It has been shown that chemical sympathectomy or surgical denervation prevents the developmental decrease in response of the ESM to ACh (1,7). Results from the present study are consistent with this since surgically denervated ESMs responded to ACh. Such findings have been ascribed to the prevention of the decline in number of postsynaptic muscarinic receptors following denervation (1). In this study, it was found that surgical denervation prevented the developmental decrease in response to histamine, also possibly due to the prevention in the decline of postsynaptic histamine receptor population. In addition, there was a marked increase in the maximum response of denervated ESMs to histamine, possibly indicating proliferation of postsynaptic histamine receptors as an additional mechanism. Proliferation of postsynaptic receptors following sympathectomy has also been reported for the rat submaxillary gland (12). Denervation increased the sensitivity of ESMs to various agonist drugs in the order NA > Isoprenaline > 5-HT. This change in sensitivity was likely to be receptor mediated since the effects were abolished by their specific antagonists and denervation did not produce an appreciable change in sensitivity to KC1. Based on this contention, the likely explanation would be the loss of uptake sites producing supersensitivity to catecholamines (3, 10, 13, 14). The greater sensitivity seen with NA would tend to suggest that it is more influenced by loss of neuronal uptake sites. It is of interest that supersensitivity with 5-HT was only significant when compared with responses from 40- or 50-day-old chicks, indicating a smaller role of neuronal uptake for 5-HT. Another interesting finding from this study was the observation that the maximum tension developed by ESMs to nerve stimulation, NA, isoprenaline, 5-HT and KC1 all increased with increasing age of the chicks. It is thus likely that a similar mechanism may account for this phenomenon. Such a likely explanation could be an increase in the muscle size with increasing age of the chicks. In contrast, the maximum tension developed to

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ACh and histamine decreased with age. This has been attributed to the decline in the number of postsynaptic muscarinic receptors for ACh (1, 7, 8) with a similar possibility for histamine as seen in this study.

The present study has therefore shown that noradrenergic nerves influence the responsiveness of smooth muscle to agonist drugs through their receptors particularly as found with muscarinic receptors and histamine (H~) receptors.

REFERENCES 1. Bennett, T.; Lot, T. Y.; Strange, P. G. The effects of noradrenergic denervation on muscarinic receptors of smooth muscle. Br. J. Pharmacol. 76:177-183; 1982. 2. Bennett, T.; Malmfors, T. The adrenergic nervous system of the domestic fowl (Gallus domesticus L.). Z. Zellforsch. 106:22-50; 1970. 3. Bennett, T.; Malmfors, T. Regeneration of the noradrenergic innervation of the cardiovascular system of the chick following treatment with 6-hydroxy-dopamine. J. Physiol. 242:517-532; 1974. 4. Bennett, T.; Malmfors, T.; Cobb, J. L. S. Fluorescence histochemical study of the degeneration and regeneration of the noradrenergic nerves in the chick following treatment with 6-hydroxydopamine. Z. Zellforsch. 142:103-130; 1973. 5. Birmingham, A. T.; Paterson, G.; Wojcicki, J. Comparison of the sensitivities of innervated and denervated rat vasa deferentia to agonist drugs. Br. J. Pharmacol. 30:748-754; 1970. 6. Buckley, G. A.; Wheater, L. D. The isolated expansor secundariorum--A smooth muscle preparation from the wing of the domestic fowl. J. Pharm. Pharmacol. 20(Suppl.): 114S-121S; 1968. 7. Kuromi, H.; Hagihara, Y. Influence of sympathetic nerves on development of responsiveness of the chick smooth muscle to drugs. Eur. J. Pharmacol. 36:55-59; 1976.

8. Kuromi, A.; Hasegawa, S. Changes in acetylcholine and noradrenaline sensitivity of chick smooth muscle wholly innervated by sympathetic nerve during development. Eur. J. Pharmacol. 33:41-45; 1975. 9. Lot, T. Y. The consequences of loss followed by recovery of noradrenergic nerve function on muscarinic receptors in the chick expansor secundariorum muscle. Br. J. Pharmacol. 90:633--639; 1987. 10. Lot, T. Y.; Bennett, T. Comparison of the effects of chronic chloroquine treatment and denervation on noradrenergic mechanisms. Med. Biol. 60:25-33; 1982. 11. Lot, T. Y.; Bennett, T. Comparison of the effects of chloroquine, quinacrine and quinidine on autonomic neuro-effector mechanisms. Med. Biol. 60:307-317; 1982. 12. Pimoule, C.; Briley, M.; Arbilla, S.; Langer, S. Z. Chronic sympathetic denervation increases muscarinic cholinoceptor binding in the rat submaxillary gland. Naunyn Schmiedebergs Arch. Pharmacol. 312:15-18; 1980. 13. Trendelenburg, U. Supersensitivity and subsensitivity to sympathomimetic amines. Pharmacol. Rev. 15:225-276; 1963. 14. Trendelenburg, U. Mechanisms of supersensitivity and subsensitivity to sympathomimetic amines. Pharmacol. Rev. 18:629-640; 1966.