Contractile effect of prolactin on guinea pig isolated ileum

European Journal of Pharmacology, 72 (1981) 11--16

11

Elsevier/North-Holland Biomedical Press

CONTRACTILE EFFECT OF PROLACTIN ON GUINEA PIG ISOLATED ILEUM N. PADMANABHA PILLAI, SUBRAMANIAN RAMASWAMY, VENKATASUBRAMANIAN GOPALAKRISHNAN * and MANINDRA N. GHOSH

Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry-605006, India Received 23 January 1981, accepted 4 March 1981

N.P. PILLAI, S. RAMASWAMY, V. GOPALAKRISHNAN and M.N. GHOSH, Contractile effect of prolactin

on guinea pig isolated ileum, European J. Pharmacol. 72 (1981) 11--16. Prolaction (PRL) at high concentrations contracted the guinea pig isolated ileum. The maximum response elicited by PRL was 44% of that of histamine-induced responses. There was no significant difference in potency between PRL preparations obtained from two different sources. PRL responses were nullified by denaturation or proteolytic digestion of the hormone. The contractile response was antagonised by atropine and potentiated by neostigmine, b u t unaffected by the prostaglandin antagonist SC-19220. The PA2 values of atropine against PRL and ACh were similar. Preincubation with morphine, which inhibits ACh release, produced slight inhibition of PRL-evoked contractions. Even high concentration of PRL failed to produce any response in neostigminetreated frog rectus muscle preparations. This suggests that PRL may produce contractions through a cholinergic mechanism involving muscarinic receptors. Enhanced gut motility reported earlier for hyperprolactinemic states may be attributed to this cholinomimetic effect of PRL on the intestinal tract. Prolactin

Guinea pig ileum

Contraction

1. Introduction

Several investigations have been carried out to study the role of prolactin (PRL) in the sensitivity of the various smooth muscles to different agonists (Horrobin et al., 1973; Muriuki et al., 1974; Lipton et al., 1978). Lower concentrations of PRL produced potentiation of pressor responses to norepinephrine and angiotensin, while depression was observed with higher concentrations when these were applied to the superior mesenteric vascular bed (Mtabaji et al., 1976; Manku et al., 1979) and other vascular smooth muscles (Manku et al., 1973; Horrobin et al., 1974a). Similar biphasic effects of PRL on the contractile responses of cardiac muscle preparations (Nas-

Cholinergic receptor

sat et al., 1974; Horrobin et al., ]974b) have also been shown. Enhanced gastric emptying and increased gut motility in lactating mice were reported by Adams et al. (1976). Mainoya (1976) showed that, in rodents, the length of the gut and its absorptive capacity for various ions, amino acids and sugar were substantially increased during lactation. These reports favoured the suggestion that higher level of PRL may possibly have a direct effect on gastrointestinal function and motility. Since there is no literature available on the in vitro effect of PRL on the responses of intestinal smooth muscle, experiments were designed to investigate the probable role of PRL on gastrointestinal function using guinea pig isolated ileum as a model.

* To whom correspondence should be addressed. 0 014-2999/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press

12 2. Materials and methods

2.1. Tissue The ileum was dissected out from freshly killed male guinea pigs (200-300 g). Segments of mid-ileum (3-4 cm) were mounted in an organ bath of 30 ml capacity under a resting tension of 0.5 g in normal Ringer solution (mM: NaC1 154.0; KC1 5.4; CaC12 2.4; NaHCO3 6.0; dextrose 11.0) which was continuously aerated and maintained at 37°C. Isotonic contractions were recorded on a kymograph drum after an initial equilibration for a period of 30 min.

2.2. Drugs and hormone Acetylcholine chloride (ACh, E. Merck), histamine acid phosphate (E. Merck), serotonin creatinine sulphate (5HT, E. Merck), prostaglandins El, F2~ (PGE1, PGF2a, Upjohn Co.), nicotine (BDH), neostigmine methyl sulphate {National Pharmaceuticals), atropine sulphate (E. Merck), 1-acetyl-2-(8chloro-10,11,dihydrodibenz(b,f) (1,4)oxazepine-10 carbonyl) hydrazine (SC-19220, G.D. Searle & Co.), morphine sulphate (Govt. Opium& Alkaloid Works, Ghazipur, India) and chymotrypsin (Sigma} were used in this study. Ovine prolactin was obtained from Ferring AB, Sweden and from the National Pituitary Agency, NIH, NIAMDD, U.S.A. The prostaglandins and SC-19220 were dissolved in ethanol (0.01%) and further dilutions were made with 0.02% sodium carbonate solution. All other drugs and PRL were dissolved in Ringer solution. The concentrations of various drugs and hormone used in the present study are expressed in terms of salt per ml of the bathing medium.

N.P. PILLAI ET AL. 5HT and PRL, and the EDs0 values were calculated graphically. Single dose-responses were obtained with PRL, ACh, PGEI, PGF2~ alone as well as in the presence of either SC19220, a prostaglandin antagonist (10 pg) (Bennet and Posner, 1971) or atropine (5 ng) maintained in the bathing medium after initial exposure for a period of 15 min. Each experiment was replicated in at least six animals. A contact time of 30 sec was allowed for each agonist with an interval of 5 min between successive responses. PRL and ACh responses were assessed after exposing the tissues to neostigmine (10 pg). To elucidate the involvement of ACh release in PRLinduced contractions, nicotine which acts through ACh release (Day and Vane, 1963) was included in the study. Responses to PRL and nicotine were assessed before and in the presence of morphine (1.5 ~g; 15 min) which inhibits ACh release (Paton, 1957). In addition, pA2 values were determined for atropine against PRL as well as ACh following the method described by Schild (1947}. The contractile effect of two PRL preparations (NIH and Ferring) were compared by 2 + 2 assay using a cross-over design. PRL solutions were either denaturated by heating up to 60°C for 2 h or exposed to proteolytic digestion with chymotrypsin (25 pg) in 0.1 ml of sodium phosphate buffer (0.1M, pH 8.0) for 3 h at room temperature. Contractile responses were again tested after the above procedures. The effect of PRL on the nicotinic receptor was studied on a frog rectus muscle preparation incubated in Frog Ringer solution (mM: NaC1 110.0; KC1 1.86; CaC12 0.81; NaH2PO4 0.06; NaHCO3 2.4 and dextrose 11.0) with or without neostigmine. All the experimental data were analyzed statistically using Student's t-test.

2.3. Experimental procedure

3. Results

Cumulative dose-response curves were obtained (Van Rossum and Van Den Brink, 1963) with histamine, ACh, PGE1, PGF2a,

PRL produced contractions of the ileum immediately on its addition to the bath. PRL from two different sources (NIH& Ferring)

13

PROLACTIN-INDUCED I L E A L CONTRACTION

produced almost equal heights of contraction when tested in the same tissues in both cumulative and single doses. Further, when a 2 + 2 assay was used, there was no significant difference in p o t e n c y for this effect b e t w e e n P R L preparations. However, there was a wide animal to animal variation in this contractile p h e n o m e n o n as shown b y the EDs0 values (mean + S.E.M.) for P R L (table 1). This effect of P R L was compared with the contractions produced b y histamine, ACh, 5HT, PGE1, PGF2a. Their EDs0 values and the maximal responses obtained are presented in table 1. The maximal contractile effect for P R L was lower than that of other agonists and was 44% of that of the histamineinduced responses. Single dose~responses were recorded in several tissues. Fig. 1 depicts the effect observed in one of the sensitive tissues. SC19220 p r o d u c e d reversible inhibition of responses to b o t h PGs w i t h o u t affecting those to P R L and ACh. Atropine markedly inhibited the responses to b o t h P R L and ACh with a lesser reduction in the responses to PGs. This ~ inhibition was reversible as the responses could be restored to their original heights on washing. These experiments were repeated in 6 tissues and the analysis of these single doseresponse data is shown in table 2. TABLE 1 Comparison of PRL with various agonists on guinea pig isolated ileum. The values are mean +- S.E.M. of the number of experiments given in parentheses, Agonist

EDs0 (ng/ml)

Maximal contraction

~30S •

•

•

o

o

•

•

00$¢I

i

OWl

D al 0

?

ATO

D m 0 WO(2 t O

Fig. 1. Effect of SC-19220 and atropine on ACh-, PG- and PRL-evoked responses in guinea pig isolated ileum. • ACh 2 ng; [] PGEI 10 ng; • PGF2~ 10 ng; o PRL 100 ng; SC SC-19220 10 p g ; W wash; AT atropine 5 ng.

TABLE 2 Effect of atropine and SC-19 220 on the contractile effect induced by PRL, ACh and PGs in guinea pig isolated ileum. Each value shown below represents the average of at least six experiments. Height of responses in mm (mean + S.E.M.)

Agonists

ACh 2 ng PGEI 10 ng PGF2a 10 ng PRL 1 pg

Control

SC-19 220 (10 pg)

Atropine (5 ng)

46.7 24.2 21.3 34.9

44.3 4.8 7.4 32.8

10.8 14.8 13.2 12.0

_+2.4 + 4.1 -+ 3.2 -+ 6.6

+ 2.8 + 2.0 b -+ 3.5 b -+ 5.7

_+1.7 + 2.6 -+ 2.5 -+ 2.0

b a a b

a p < 0.05 and b p < 0.01 compared to respective control value.

Efficacy -a

(ram) Histamine (9) ACh (10) 5 HT (6) PGEI (5) PGF2~ (5) PRL (9)

3.9 5.4 203.3 98.5 121.6 214.2

-+ 0.6 -+ 0.3 -+ 26.0 + 12.5 -+ 15.6 + 79.4

118.6 102.0 99.8 99.1 94.7 52.4

-+ 5.0 + 3.2 + 4.1 + 4.7 + 3.8 -+ 10.4

a For each agonist, efficacy was calculated as: maximal contraction of agonist x 100. maximal contraction of histamine

100 86 84 84 80 44

e •

4A

O

No~$

5'

Fig. 2. Effect o f neostigmine and morphine on ACh-, nicotine- and PRL-evoked responses in guinea pig isolated ileum. • ACh 2 ng; • nicotine 100 ng; o PRL 1 #g; N neostigmine (10 pg) and M morphine (1.5 #g); W wash.

14

N.P. P I L L A I E T AL.

TABLE 3 Effect of incubation with neostigmine and morphine o n t h e r e s p o n s e s t o ACh, n i c o t i n e a n d P R L in guinea pig isolated ileum. Each value r e p r e s e n t s t h e average o f a t least six e x p e r i m e n t s . Agonists

ACh 2 ng N i c o t i n e 100 ng P R L 1 ~g

H e i g h t o f c o n t r a c t i o n s in m m ( m e a n + S.E.M.) Control

Neostigmine ( 1 0 pg)

Morphine (1.5/Jg)

39.6 + 1.8 22.2 -+ 2.2 35.8 + 5.9

73.5 -+ 4.6 b 42.6 -+ 2.5 b 75.6 + 6.3 b

37.9 -+ 2.2 7.3 -4"1.5 b 24.8 -+ 3.1 a

a p < 0.05 a n d b p < 0.01 c o n t r o l value.

compared

t o respective

Fig. 2 represents the effect of neostigmine and morphine on PRL-evoked responses, while table 3 depicts the average results of similar experiments in several tissues. Contractile • responses elicited by PRL were significantly potentiated in the presence of neostigmine, as were the ACh- and nicotine-induced responses (P < 0.01). Prior incubation with morphine in the bathing fluid reduced (P <: 0.05) the responses to PRL, while the nicotine-induced responses were markedly inhibited (P < 0.01). Morphine pretreatment, on the other hand, failed to affect the responses to exogenous ACh as shown in the same figure. As the responses to PRL were potentiated by neostigmine and inhibited by atropine, pA2 values were determined in at least six different tissues obtained from different animals. The pA2 values (mean +-S.E.M.) for atropine against PRL and for atropine against ACh responses were 8.79-+ 0.15 and 8.98-+ 0.30 respectively. The difference between these two values was not statistically significant. Prior heating or proteolytic digestion of PRL with chymotrypsin abolished its contractile effect on ileum. Even in a higher concentration (1.0 mg), PRL did not elicit any contractile effect

either in normal or neostigmine-treated frog rectus abdominis muscle preparations.

4. Discussion

Several peptides such as bradykinin, substance P, vasopressin, angiotensin, neurotensin (NT), thyrotrophin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) are known to exert contractile activity on isolated ileal smooth muscles (Kitabgi and Freychet, 1978; Cohen and Landry, 1980; Furukawa et al., 1980). These studies have also shown that VIP, TRH and NT exert their contractile effects through a cholinergic mechanism. The present study revealed a similar contractile effect of PRL on the guinea pig ilum. The NIH and the Ferring PRL showed no significant difference in the contractile pattern elicited or in potency. PRL from Ferring is known to contain few contaminants (Karmali and Horrobin, 1976), while earlier batches of the NIH preparation contained small amounts of both oxytocin and vasopressin i.e. 0.5-1.5 #g/mg of PRL (Vorherr et al., 1978). Both oxytocin (Syntocinon, Sandoz) and lysine vasopressin (Sigma) in the concentration ranges likely to be present in the PRL preparations failed to produce any effect on the ileum. Since denaturation and proteolytic digestion resulted in the loss of contractile activity it can be suggested that this effect was due to an inherent property of the PRL molecule itself. The contractions produced by PRL were effectively inhibited by atropine and potentiated by neostigmine. This suggests the involvement of muscarinic receptors in the PRL-evoked contractile phenomenon. Moreover, pA2 values for atropine against both PRL and ACh were similar confirming the above suggestion. This finding is in agreement with the report of Adams et al. (1976) who observed that atropine antagonised the increased gut motility in lactating mice where a higher level of PRL has been recorded. We have also observed that PRL itself, in higher

PROLACTIN-INDUCED ILEAL CONTRACTION

than physiological plasma concentrations, enhanced intestinal transit in mice (Unpublished observation). However, earlier studies have shown that the effect of PRL on rat superior mesenteric vascular bed was accompanied by an increase in the output of PG-like substances (Horrobin et al., 1974a; Mtabaji et al., 1976). This effect may have been due to a PRL-enhanced synthesis and release of PGE1 (Manku et al., 1979). Rillema (1975) has also observed that mammary RNA synthesis stimulated by PRL could be blocked by agents which inhibit PG synthesis and that the inhibition could be reversed on addition of PGF2~ to the medium. PGs of both E & F series are known to be present in the ileal smooth muscle and to contract the ileum as does ACh (Kadlec et al., 1974; Bennett et al., 1975; Stockley, 1979). In the present study, however SC-19220 a specific blocker of PGs failed to affect the contractile responses to PRL, suggesting that PGs may not be involved in this action of PRL. The slight reduction in PG responses produced by atropine may have been due to the fact that PGs partly exert their contractile effect through a cholinergic mechanism (Bennett et al., 1975). The lack of any contractile effect of PRL in frog rectus abdominis muscle preparations indicates that PRL has no effect on skeletal muscle nicotinic receptors. Preincubation of the ileal tissues with morphine markedly antagonised nicotinic responses in the ileum by inhibiting the release of ACh. The slight reduction in PRL responses in the presence of morphine suggests that PRL-induced contractions may be partly mediated through ACh release. To summa_rise, PRL produces contractions of guinea pig isolated ileum partly by direct muscarinic receptor stimulation and partly through ACh release as did NT, TRH and VIP. This suggests that there may be a common process for these peptides to elicit muscarinic receptor stimulation in the gastrointestinal tract. Normal physiological plasma concentrations

15

of PRL remain in the range of 5-28 ng/ml (Friesen and Hwang, 1973; Frantz, 1978). Higher levels are encountered in lactation, stress, surgery, pituitary adenoma, galactorrhea amenorrhea of varied causes, and also on prolonged treatment with various drugs such as phenothiazines, s-methyl DOPA, reserpine and combined contraceptive pills. Friesen and Hwang (1973) have detected as much as 10 #g PRL in the plasma of patients with pituitary or hypothalamic tumors. As PRL-induced contractile responses were elicited by higher concentrations of PRL, the increased gut motility and enhanced gastric emptying seen during lactation and other hyperprolactinemic states (Adams et al., 1976) may be attributed to this cholinomimetic activity of PRL on intestinal tract.

Acknowledgements The generous supply of ovine prolactin (batch P-S-13) by NIH, NIAMDD, Bethesda, Maryland, U.S.A., M/s. Ferring AB, Malmo, Sweden and Dr. David F. Horrobin, Institute for Innovative Medicine, Montreal, Canada is gratefully acknowledged. Our thanks are also due to Dr. John E. Pike of Upjohn Co., U.S.A. for the gift of prostaglandins and Dr. Tony B. Martinez of G.D. Searle & Co., U.S.A. for SC-19220 used in the present study.

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