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The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.

The contractile effect of okadaic acid (OA) and its derivatives was investigated in the rat uterus. OA (20 microM) induced a transient contraction which, after plateauing, slowly decreased. The structurally related compound okadanol (20 microM) failed to induce any significant contraction. Conversely, the synthetic compound methyl okadaate (20 microM) and the naturally occurring ester 7'-hydroxy-4'-methyl-2'-methylen-hept-4'(E)-enyl okadaate (20 microM) were as active as the free acid. The OA-induced contraction was unaffected in the presence of neomycin (5 mM), mepacrine (30 microM), 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (10 microM), calphostin C (3 microM) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (30 microM). The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (100 microM) did not modify the amplitude of the OA-induced contraction but significantly increased the rate of tension decay. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (1 mM) significantly reduced the peak amplitude of the contraction. Staurosporine (0.03-0.1 microM) did not modify the contractile component of the OA-induced response but inhibited the subsequent decrease in tension. In freshly dispersed myometral cells loaded with the fluorescent Ca++ indicator indo 1, OA did not produce any significant increase in [Ca++]i. OA (5- to 90-min contact) also failed to modify the intracellular levels of arachidonic acid, compared with basal values. These data suggest that in the rat uterus 1) the contractile effect of OA (20 microM) is specifically mediated by inhibition of protein phosphatases type 1 and/or 2A and is related to a direct interaction with the contractile machinery; 2) the decreasing phase of the OA-induced mechanical response could be mediated by a staurosporine-sensitive protein kinase different from protein kinase C.

The effects of ouabain and K(+)-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K+ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20-30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K(+)-free solution induced either rhythmic waves, which ceased after 8-10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 +/- 10 mg after 180 min exposure). Nifedipine (0.3 microM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K(+)-free solution. Ouabain (0.1-1 mM) or K(+)-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca(2+)-free, 3 mM EGTA-containing solution. Cromakalim (10 nM-0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca2+ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 microM), ouabain (0.3 mM), K(+)-free-solution and cromakalim (10 microM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K(+)-free solution and cromakalim (10 microM) decreased the Ca(2+)-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca(2+)-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca(2+)-free solution containing 60 mM K+. In conclusion, these results suggest that the response induced by ouabain or K(+)-free solution in estrogen-primed rat myometrium involves Ca2+ influx through potential-operated calcium channels but not Ca2+ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K(+)-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K+, due to the inhibition of the electrogenic Na+ pump.

The effects of the aqueous extract of leaves of Bridelia atroviridis (Bridelia), a small African tree, on the mechanical activity of rat uterus were studied. The aqueous extract of leaves of B atroviridis administered in a concentration-dependent manner (5 x 10(-6)-1.2 x 10(-3) g/ml) induced contractions that were antagonized by various calcium entry blockers (nifedipine, diltiazem, manganese chloride). In absence of external calcium ions, repeated applications of a supramaximal concentration of Bridelia (1.2 x 10(-3) g/ml) evoked sustained and repeated contractions the amplitude of which was congruent to 20% of those obtained in the physiological external calcium concentration. Bridelia-induced contractions in calcium-free medium were inhibited by isoprenaline (8 x 10(-7) M), caffeine (15 x 10(-3) M) and trifluoperazine (10(-5) M). Contractile responses induced by Bridelia in both calcium-containing and calcium-free media were antagonized by prior incubation of uterus with phorbol 12, 13-dibutyrate (6 x 10(-7) M), cholera toxin (6 x 10(-8) M) or pertussis toxin (5 x 10(-7) g/ml). These results show that Bridelia has a potent uterotonic action in the rat. The cellular basis of this action appears to be complex, and involves various mechanisms including calcium mobilization from both intra and extracellular compartments and activation of phospholipase C through a G-protein.

The effects of phorbol 12, 13-dibutyrate (PDB) on mechanical activity in the pregnant rat uterus were investigated in isolated strips. In Ca(++)-containing solution, PDB (2.5 x 10(-8) to 10(-6) M) increased in a concentration-dependent manner the amplitude of the electrically induced contraction, but had no effect on the resting tension. PDB (10(-7) M) had a dual action, stimulatory then inhibitory, on contractions evoked by K(+)-rich (40 mM K+) solution or oxytocin. The inhibitory effect appeared more rapidly and the percentage of inhibition was increased for 10(-6) M PDB, which in addition abolished completely oxytocin-induced contraction after 20 min of application. PDB also reduced the amplitude of transient contraction evoked by oxytocin in Ca(++)-free solution. In saponin-skinned strips, 10(-7) M PDB increased the contraction induced by pCa ranging from 7 to 6, whereas 10(-6) M PDB reduced all Ca(++)-activated contractions from pCa = 7 to pCa = 5. PDB had no significant effect on the Ca(++)-uptake and the Ca(++)-release mechanisms of the intracellular Ca(++)-store. All of the effects of PDB were antagonized by the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (2 x 10(-5) M). In addition, the inactive phorbol 13,20-diacetate (10(-8) to 10(-6) M) had no effect on the mechanical activity in uterus. These results suggest the existence of different sites of action of PDB in rat uterus, via the activation of protein kinase C: 1) contractile machinery; 2) potential-dependent Ca channels; and 3) phospholipase C.

1. The effect of caffeine on mechanical activity was studied in pregnant rat myometrium. 2. In muscle cells with intact plasmalemmae, caffeine (0.1-50 mM) produced no contraction whatever the experimental conditions. 3. Caffeine (0.1-10 mM) inhibited, in a concentration-dependent manner, contractions induced by electrical stimulation, potassium-rich (60 mM K+) solution, sodium-free solution or oxytocin (22.5 nM). 4. In Ca2(+)-free solution, various substances (oxytocin, sodium orthovanadate and prostaglandin E2) evoked sustained contractions that were suppressed by caffeine (5-10 mM). When caffeine (greater than 5 mM) was applied during Ca2(+)-loading of the tissue (2.1 mM Ca2+, 5 min) in the presence of a K(+)-rich solution, the subsequent transient contraction induced by a short application (10s) of oxytocin (22.5 nM) in Ca-free solution was reduced (63 +/- 3.5% reduction for 20 mM caffeine, n = 4). 5. In saponin-skinned strips, application of caffeine (5-10 mM) during loading of the Ca2(+)-store increased the subsequent contraction induced by myo-inositol 1,4,5 trisphosphate (IP3, 10 microM). Caffeine (10-30 mM) decreased calcium-activated contractions in skinned fibres lacking a functional internal Ca-store. This effect was reduced by the cyclic AMP-dependent protein kinase inhibitor Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp (8 microM). 6. In conclusion, it is suggested that the inability of caffeine to cause spasm of rat myometrium is due to the absence of a caffeine-sensitive calcium-release channel in the sarcoplasmic reticulum. Relaxant effects of caffeine can be explained by mechanisms leading to a decrease in both the cytoplasmic free Ca2+ concentration and the Ca2 +-sensitivity of the contractile machinery.

Chemically skinned fibers were prepared from pregnant rat myometrium. In the presence of a low EGTA concentration (0.1 mM), caffeine (0.1-50 mM) produced no contraction whereas both inositol-1,4,5,-triphosphate and the ionophore A23187 evoked contractile responses insensitive to cyanide and azide. Furthermore, caffeine antagonized the maximal contractions induced by pCa = 5 (in the presence of 4 mM EGTA). It is concluded that caffeine inhibits some mechanism of the contraction in rat myometrium and thus that caffeine is not a good tool for the study of intracellular Ca stores in this smooth muscle.

The effects of varying the sodium gradient-either by lowering [Na+]o or by increasing [Na+]i on the electromechanical properties of pregnant rat uterine smooth muscle were studied. In normal tissues, complete removal of external sodium ions (choline, Tris or sucrose as substitutes) induced a strong and maintained contraction which was dependent on the presence of extracellular calcium ions, and was sensitive to Ca2+-antagonist drugs (Nifedipine; D 600, Mn2+). Electrical recordings showed that the membrane was transiently hyperpolarized (-10 +/- 2.4 mV, n = 20); after 1 minute depolarization accompanied by a spontaneous spike discharge occurred. Partial withdrawal of external sodium ions resulted in following changes in twitch contractions evoked by electrical stimulation: a linear relationship was found between the time constant of twitch relaxation and the external Na-concentration. In Na-rich tissues, where the Na/K pump was blocked, or in the presence of monensin, Na-free solutions (whatever the substitute, even K+ ions) again triggered strong contractions entirely dependent on external calcium but rather insensitive to Ca-antagonists. The Na-free (K+) induced contraction was larger than the Na-free (choline or Tris)-induced contraction. It was concluded that the sodium gradient was an important factor for the regulation of contractile activity of uterine smooth muscle. Na-Ca exchange appeared to mediate twitch relaxation in normal tissues and was responsible for Ca-influx in Na-rich tissues.

The effects of angiotensin II (A II) were studied on membrane potentials, ionic currents and isometric contractions in uterine smooth muscle strips from pregnant rats. In reference solution, A II in a low concentration (5 x 10(-9) M) exerted no effect on the resting potential or the action potential amplitude but significantly increased the rate of repetitive firing. However, in tetraethylammonium chloride (TEA)-containing solution, an increase in the action potential amplitude was always observed. The inward and outward currents were increased simultaneously, so that it was necessary to block the outward current with TEA ions in order to measure the enhancement of the inward current. The reduction of the deactivation time constants of the outward current as well as the increase in the inward current intensity could account for the acceleration of rhythmic activity in the presence of the peptide. The mechanical response to A II consisted of early tetanus which was followed by increased twitch contractions. The calcium contractures were also increased in depolarized strips. The tetanus could be dependent on the increase in and prolongation of repetitive firing while the enhancement of twitch contractions and calcium contractures could be explained by the increase in inward current intensity.

Smooth muscle fibers isolated from pregnant rat myometrium were skinned by saponin treatment. Properties of the contractile system and the involvement of an intracellular source of activator calcium were studied. (1) In the presence of 4 mM total EGTA, skinned fibers contracted in a concentration-dependent manner to micromolar applications of calcium ions. Rat myometrium exhibited a high calcium sensitivity (80% of the maximum contraction was achieved in the presence of 10(-6) M Ca2+). (2) Several divalent cations induced concentration-dependent contractile responses in skinned uterine muscle. The rank order of potency was: Ca2+ greater than Mn2+ greater than Sr2+ greater than Ba2+. All these divalent cation-activated contractions were antagonized by trifluoperazine in a concentration-dependent manner. (3) Pretreatment of skinned fibers with cAMP (5 X 10(-5)-5 X 10(-4) M) depressed cation-activated contractions. This effect was dependent on the free cation concentration. (4) In the presence of a low EGTA concentration (0.1 mM) the pCa-tension curve was shifted to the right with a 2.5-fold increase in the Ca-concentration required to induce half-maximum contraction. (5) After Ca-loading (10(-6) M Ca2+ for 3 min in low EGTA-containing solution), total replacement of K+ ions by choline induced a small and tonic contraction. In these conditions, the ionophore A23287 (5 X 10(-8) M-5 X 10(-5) M) and inositol 1,4,5-triphosphate (InsP3; 2 X 10(-6) M-2 X 10(-5) M) also produced contractions of skinned uterine fibers even in the presence of NaN3 (5 mM) and of NaCN (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of acetylcholine (10(-4) M), prostaglandin E2 (10(-6) M), vanadate (5 X 10(-4) M) and fluoride (10(-2) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. All four substances produced maintained contractions which could be initiated repeatedly after exposure to Ca2+-free solution for more than 1 h, without a significant decrease. The largest contractions were obtained with vanadate and the smallest ones with acetylcholine. The tension was usually 7-30% of the control contraction triggered by an action potential in Ca2+ containing solution. Maintained contractions induced by fluoride were unaffected by isoprenaline while those induced by acetylcholine, prostaglandin E2 and vanadate were completely relaxed. Prostaglandin E2- and vanadate-induced contractions were slightly reduced by Na+ removal or by adding Ca2+ antagonists. In contrast, contractions induced by acetylcholine were suppressed in Na+-free solution and largely inhibited in the presence of Ca2+ antagonists. The depolarization induced by acetylcholine in Ca2+-free solution was strongly dependent on the external Na+ concentration. The relationship between the size of the acetylcholine-induced depolarization and the membrane potential (shifted by constant currents) was linear, giving an apparent reversal potential for acetylcholine close to zero potential. In Ca-free solutions and in the presence of atropine, Na+ action potentials of long duration can be evoked which produced contractions of the same order of magnitude as those initiated by acetylcholine-induced depolarizations. 7 These results are consistent with the hypothesis that the maintained contractions in Ca2+-free solutions induced by several stimulants could be related to Ca2+-independent mechanisms (fluoride) or Ca2+ release from an intracellular store. This latter mechanism would include both pharmacomechanical (prostaglandin E2, vanadate) and electromechanical (acetylcholine) coupling.

The effects of acetylcholine (ACh, 10(-4)M) and angiotensin II (Ang II, 10(-6) M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+-free EGTA-containing solutions. Both ACh and Ang II produced transient contractions, the amplitude of which can be taken as a measurement of the amount of Ca2+ present in a drug-sensitive Ca2+ store. The degree of filling of this store depended on the external Ca2+ concentration, and on the presence of contractile responses during the Ca2+ loading period. The existence of two pathways (either direct or transcytoplasmic) is suggested for Ca2+ uptake into the internal Ca2+ store. The rate of filling of the Ca2+ store in 2.1 mM-Ca2+-containing solution was faster (time to half-maximal response, t 1/2 = 29 +/- 2.2 s, n = 4) than the rate of depletion in Ca2+-free solution (t 1/2 = 3 +/- 0.3 min, n = 3). The gradual depletion of this store was much slower at 18 degrees C than at 35 degrees C, and in the presence of vanadate which is known to inhibit Ca2+-ATPases. Methoxyverapamil (D600, 10(-6)-10(-5) M) had no appreciable effect on the direct Ca2+ uptake or on the release of Ca2+ from the store by ACh and Ang II. Mn2+ (10(-3) M) completely inhibited the direct pathway to the internal Ca2+ store and also reduced the release of Ca2+. ACh and Ang II induced repetitive depolarizations close to zero potential which did not parallel the transient contractions as a function of the time of perfusion in Ca2+-free solution. Applications of 2 mM EGTA, 135 mM K+ or Ca2+ antagonists which suppressed or reduced the drug-induced depolarizations did not affect appreciably the drug-induced contractions. These results suggest that myometrial cells have an intracellular Ca2+ store sensitive to different stimulus substances. This store is not affected by depolarization of the plasma membrane and is certainly different from that described in voltage-clamp experiments.