CALCIUM REQUIREMENT IMPORTANCE OF

PROSTAGLANDINS

OF UTERINE CONTRACTION INDUCED BY PGEl: INTRACEJ,IULAR CALCIUM STORES.

Villar, A.: D'Ocon, M,P. and Anselmi, E.

Departamento de Farmacognosia y Farmacodinamia Facultad de Farmacia. Avda. Blasca Ibdiiez, 13 46010 Valencia. SPAIN.

ABSTRACT

high The contraction of the rat uterus in response to PGEl in

K+ medium and in Ca-free solution which contained EDTA has been investigated in order to examine whether excitation-contrac- tion coupling involves the release of Ca from an intracellular store.

In uterus maximally contracted by K+, cumulative concentra- tions of PGEl (1.25 - 20 ng/ml) caused maintained concentration-de- pendent contraction. PGEl induced sustained contraction of rat uter- us in Ca-free medium after incubation with 3mM EDTA for 50 min. In these conditions the involvement of extracellular Ca is highly un- likely. The PGEl-induced contraction could be repeated without ex- posure to external Ca ions and with only sligth reduction in magni- tude. The PGE concentrations required to elicit uterine contraction in Ca-free so ution were about 1000 times higher than the effective 1 doses in KCl-depolarized uterus.

In conclusion, the present investigation shows that Ca influx is not essential for PGE1-induced contraction of rat uterus, although extracellular Ca enhances it presumably by increasing the free Ca levels in the cytosol.

INTRODUCTION

It is widely believed that prostaglandins E produce a con- traction of smooth muscle by increasing free-cytoplasmic Ca concen- tration. This change may be due to a mobilization of the Ca ions via three different mechanisms: (a) increased Ca inward current through voltage-dependent channels (1,2); (b) Ca influx through receptor-operated channels (2) and/or (c) release of intracellular Ca (3,4,5).

In the present study we have attempted to separate these pro- cesses by investigating the uterine response to PGEl with and with- out extracellular Ca.

METHODS

Preparation of uterine horns: female Wistar rats weighing 150

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to 200g were given estradiol benzoate (5mg/kg); 24h later then were killed by a blow on the head and exsanguinated. One uterine horn was removed and mounted in a 30ml organ bath chamber filled with Jalon or Locke Ringer's solution bubbled with a mixture of 95% O2 and 5% CO2 at 31°C.

1.

2.

3.

ET&mental procedure

PGE res 4.

cumulative doses: the uterine horn was suspended under a lng load of lg in Jalon Ringer solution and equilibrated for

20min. Concentration-response curves were made by addition of cumulative doses of PGEl (0.25 - 4 ug/ml) (6).

Depolarized uterus: the uterus first was immersed in Jalon Ringer solution and equilibrated for 20min with a resting load of lg. KC1 was then added to give a final concentration of 56mM. This caused a rapid contraction followed by a slight relaxation and then a prolonged contraction plateau. Cumulative doses of PGEl were then administered.

Ca-free solution (7): the uterine horn was equilibrated for lh in Locke Ringer solution with a resting load of 0,5g, Then the solu- tion was replaced by Ca-free solution with 3mM EDTA and incubation was continued for 50min to deplete the tissue Ca. Subsequently, the solution was replaced by Ca-free solution containing 1mM EDTA and after 20 to 30 min cumulative doses of PGEl were added.

Isometric responses were measured using a recorder (680M HP) with an amplifier (8805C HP) and a force displacement transducer (Gould Statam UC2).

Solutions

:‘t Jalon Ringer solution (mM): NaCl (154,); KC1 (5.63); CaC12 (0.648); NaHC03 (5.95); glucose (2.77).

St Locke Ringer solution (mM): NaCl (154,); KC1 (5.63); CaC12 (2.16); MgC12 (2.10); NaHC03 (5.95); glucose (5,551. Ca-free solution had the same composition except for the omission of CaC12 and the addition of EDTA 3mM or 1mM.

Drugs used: PGEl was purchased from Sigma Chemical Company.

RESULTS

1. Contractile responses to PGEl cwnulative doses in Jalon Ringer solution

Contractile responses were obtained when cumulative doses of PGEl were added but increase in concentration was not accompanied by a proportional increase in uterine contraction in any of 12 experi- ments (Fig. 1).

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J 1.5g

I

h Zmin t tttt

0.25 0.512 4

ughnt PFE, [bath concMo111

Figure 1

Cumulative dose-response curve to PGEl in Jalon Ringer solution (a representative experiment).

2. ContractiZe effect of PGEl en KCZ-depotariaed rat uterue

The KCl-depolarized rat uterus is highly sensitive to PGEI. The threshold contractile response was obtained with PGEl 1.25 ng/ml The EC value for PGEl was 3.8 i: 0.5 ng/ml with cumulative addition (mean sOs.e., n=6) (Fig. 2).

1.5g

Concentration-response curve to PGEl in KCl-depolarized rat uterus, Maximal contraction induced by KC1 was 2.50 i 0.19 g (mean f s.e,, n=6).

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3, Maintained contractile responses of rat uterus induced by PGEl in Ca-free solution

In Ca-free EDTA-containing solution, contractions were ob- tained when cumulative doses of PGEl were added (0.05; 0.1; 0.2; 0.4 0.8) ug/ml (8 experiments) but they were not obtained with low con- centrations of PGE (1.25; 2.5; 5; 10; 20) ng/ml (9 experiments). The amplitude of t & ese contractions was similar for all concentra- tions of PGE as long as t h

and when the full response was reached, it was sustained e preparation was exposed to the agonist (Fig. 3).

The tissue relaxed to its former length after washing out PGEl and a maintained contraction could be repeated by adding now PGE in this case, O.O5ug/ml produced a contraction that was 70-90% o *

but the

maximum obtained with cumulative addition up to 0.8ug/ml PGEl.

Figure 3 Maintained contraction induced by PGEl in Ca-free EDTA-containing solution.

DISCUSSION

The present results show that PGEl can contract rat uterus in Jalon Ringer solution. The observation that PGEl also contracts KCl-depolarized uterus suggests an increase in the cytoplasmic Ca concentration independent of change in membrane potential.

It is generally accepted that this contractile response is triggered by an increase in the free-cytoplasmic concentration of calcium ions (1,2,8). The present results demonstrate sustained PGEl-induced contraction of rat uterus in Ca-free medium, even in the presence of EDTA. In these conditions extracellular Ca is highly unlikely to contribute in PGE1-induced contraction.

PGEl may therefore release Ca ions from the intracellular stores which are proposed sites for excitatory drugs in the uterus

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(7,B) and other smooth muscles (2,5). This suggestion is based on previous reports that PGs inhibit ATP-dependent Ca binding in mi-

crosomal fractions (3,4,9) with increase in intracellular free Ca- levels.

The maintained contraction induced by PGEl could be repeated without exposure to external Ca ions and with a slight reduction in magnitude. Perhaps Ca ions are mobilized from an intracellular store able to resequester the same Ca with very little loss from the cell, or perhaps the intracellular store is large enough even with losses to produce maintained contractions for prolonged periods of time.

The existence of sustained PGEl-induced contraction of uterus in Ca-free medium suggests that external Ca does not play an essen- tial role in the genesis of contractions. Effective concentrations

of PGEl (0.25 - - 4 ug/ml) in presence of external Ca were the same as effective PGE. concentrations in Ca-free solution although the low magnitude of iontractions in this case indicates that extracellular Ca enhances the contraction by increasing the free Ca levels in the cytosol. The hypothesis proposed by Droogmans and Casteels (10) to explain noradrenaline-induced contractions may also be useful in explaining our results. These authors suggested that receptor-opera- ted channels and agonist-sensitive Ca stores can be integrated into a single mechanism; receptor activation releases calcium from its storage sites and the depletion of this Ca compartment causes an in- crease in the Ca-permeability. The concomitant influx of Ca through that pathway increases the contractile response in Ca-containing me- dium.

This suggestion can also explain the ability of low concentra- tions of PGEl to elicit contractSons in uteri maximally contracted by KU, by opening receptor-operated Ca-channels and thus permitting additional influx of Ca.

REFERENCES

1.

2.

3.

4.

Adelstein, R.S. and Hathaway, D.R. Role of calcium and cyclic adenosine 3'-5' monophosphate in regulating smooth muscle con- traction. Am. J. Cardiology 44,: 703, 1979. -

Bolton, T.B. Mechanisms of action of transmitters and other subs- tances on smooth muscle. Physiol. Rev. 59: 606, 1979. -

Carsten, M.E. Sarcoplasmic reticulum from pregnant bovine uterus: Prostaglandins and calcium. Gynec. Invest. 4: 95, 1973. -

Carsten, M.E. and Miller, M.D. Effects of prostaglandins and oxy- tocin on calcium release from a uterine microsomal fraction. J. Biol. Chem. 252: 1576, 1977.

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5.

6.

7.

8.

9.

Mironneau, C,; Mironneau, M. and Savineau, J.P. Maintaiqgd con- tractions of rat uterine smooth muscle incubated in a Ca -free solution, Br. S. Pharmac, 82: 735, 1984.

Van Rossum, J.H. Cumulative dose-response curves, II Technique for the making of dose-response curves in isolated organs and the evaluation of drug parameters. Arch, Int. Pharmacodyn. 143: 299 1963.

Sakai, K.; Yamaguchi, T. and Uchida, M, Oxytocin-induced Ca-free contraction of rat uterine smooth muscle, Effects of divalent ca- tions and drugs. Arch. Int. Pharmacodyn. 250: 40, 1981.

Daniel, E.E. and Janis, R.A. Calcium regulation in the uterus. Pharmac. Therap. 1: 695, 1975. -

Carsten, M.E. Prostaglandins and oxytocin: Their effects on uter- ine smooth muscle. Prostaglandins 5: 33, 1974,

fO_..Droogmans, G. and Casteels, R. "Electromechanical and Pharmaco- mechanical coupling in smooth muscle:' In: Pharmacology of the Vascular Wall (J. Esplugues, ed.) University Press, Valencia, Spain, 1984, p.65.

Editor: A. Bennett Received: 2-12-84 Accepted: 6-25-85

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