Simone Harbon

Carbachol and oxytocin stimulate the generation of inositol phosphates in the guinea pig myometrium

In the guinea pig myometrium prelabelled with myo‐[2‐3H]inositol, carbachol and oxytocin enhanced a concentration‐dependent and rapid release of IP3 which preceded that of IP2 and IP1. The specific receptor‐mediated phospholipase C activation degrading PIP2 to IP3 did not require the presence of extracellular Ca2+. The ionophore A23187 as well as K+ depolarization failed to increase inositol phosphate accumulation. It is proposed that IP3 could have a role in the contraction of uterine smooth muscle elicited by the activation of muscarinic as well as of oxytocin receptors.

Cyclic adenosine 3′, 5′ monophosphate levels in rat myometrium under the influence of epinephrine, prostaglandins and oxytocin. Correlations with uterus motility

Summary L-epinephrine and the prostaglandins of the E series (PGE 1 and PGE 2 ) greatly stimulate the activity of adenylcyclase in rat myometrium. It is demonstrated that the same adenylcyclase, but different hormone receptors are involved in these stimulations. Oxytocin and prostaglandin F 2 α (PGF 2 α ) have no effect nor do they inhibit the stimulation induced by the former agonists. The latter agents strongly contract the rat uterus, an action which is antagonized by epinephrine and N 6 -2′-O-dibutyryl cyclic AMP, but not by PGE 1 nor PGE 2 . It is concluded that the intracellular level of cyclic AMP is not the sole parameter regulating uterine motility.

Characterization of G proteins in rat myometrium A differential modulation of Gi2α and Gi3α during gestation

Myometrial membranes, obtained from estrogen‐dominated (day 0) fat uteri, were immunoblotted with antiserum (SGI), which recognizes the α subunits of both Gi1 and Gi2, with antiserum (I.E2) specific for Gi2α, and with 13B antiserum specific (Gi3α. The data revealed the absence of detactable levels of Gi1α and the simultaneous presence of Gi2α and Gi3α as Gi subunits in rat myometrium. The expression of Gi proteins during gestation (days 0, 12, 21) was studied with the above antibodies. No qualitative change in the nature of Giα species was observed during gestations Gi1α remained undetectable. Gi2α and Gi3α were both present on days 12 and 21. Of significance was the increase (100%) in the amount of Gi2α at midgestation (day 12) compared to days 0 and 21. A different pattern was observed with Gi3α, which decreased with advancing gestation (day 0> 12> 21). Immunodetection of β subunits of G proteins indicated the presence of a 35/36 kDa doublet on days 0, 12 and 21, with an increase at midgestation. The simultaneous increase in Gi2α and β subunits may provide an explanation for the previously demonstrated alteration in adenylate cyclase stimulability detected at midgestation.

Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes

Immunoblotting of rat myometrial membranes with an antiserum (SG1) which recognizes the α‐subunits of both Gi1 and Gi2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for Gi2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OC1, IM1, ON1) and polyclonal (RV3) antisera, all of which recognize rat brain Go, produced a more complex pattern. Antisera OC1 and ON1 recognized a single polypeptide which essentially comigrated with rat brain Go. In contrast, antisera RV3 and IM1 did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G‐protein, related immunologically to Go, in rat myometrial membranes.

Myosin light chain phosphorylation in intact rat uterine smooth muscle. Role of calcium and cyclic AMP

SummaryMyometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two-dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium (0.8mm) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a β-adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproterenol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 μm) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation-dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium.

Modulation of cyclic AMP content of the rat myometrium: Desensitization to isoproterenol, PGE2 and prostacyclin

Exposure of the oestrogen-dominated rat myometrium to either isoproterenol or PGE2 resulted in a rapid but transient accumulation of cyclic AMP, with a progressive loss of responsiveness to the corresponding agonist. Induction of refractoriness was a time- and dose-related phenomenon. In the earliest time, desensitization was agonist-specific but was followed, with continued exposure, by a cross desensitization between isoproterenol and PGE2 and vice versa. Differential time courses for development and reversal of specific and heterologous refractoriness indicate at least 2 different processes for the 2 phenomena, the non-specific type being possibly mediated by cyclic AMP. Exposure to isoproterenol or PGE2 also caused an attenuated cyclic AMP response to prostacyclin (PGI2). Kinetics for PGE2-induced desensitization to PGI2 were comparable to that of an agonist-specific refractoriness, indicating that PGE2 and PGI2 may share common receptor sites. PGF2 alpha, PGD2 and 6-keto PGF1 alpha, which contract the myometrium but are ineffective on adenylate-cyclase activity, did not promote cyclic AMP refractoriness to PGE2, PGI2 or isoproterenol. Isoproterenol also caused refractoriness to its own relaxing activity, whereas PGE2 did not affect isoproterenol-induced relaxation despite a marked attenuation of the beta-adrenergic response to cyclic AMP. These results provide further evidence for the non-exclusive role of cyclic AMP in mediating uterine relaxation.

Contribution of lipoxygenase and cyclooxygenase metabolites in the modulation of cyclic nucleotides in the guinea pig mymotrium. Differential effects of carbachol and ionophore A23187

Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A23187, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachidonic acid, A23187 and PGI2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2-3 fold) in the generation of PGI2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI2 was involved. Omission of Ca2+ or addition of mepacrine or p-bromophenacylbromide abolished the stimulatory effects of A23187 and carbachol on cyclic GMP as well as the A23187-induced elevations in both PGI2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.

Differential Expression of Inositol 1,4,5-Trisphosphate Receptor Types 1, 2, and 3 in Rat Myometrium and Endometrium During Gestation1

Abstract The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP3Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF4− was enhanced with advancing gestation. 3[H]IP3 binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP3R as well as their corresponding proteins, IP3R-1, IP3R-2, and IP3R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP3Rs increased with advancing gestation, except for IP3R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP3Rs. The difference noted between the two uterine tissues suggests that IP3Rs may have cell-specific functions.

Modulation of rat myometrial guanylate cyclase activities by sodium nitroprusside and unsaturated fatty acids

Abstract Guanylate cyclase activities are present in both soluble and particulate fractions of rat myometrial extract. Triton, slightly stimulated the soluble (50%) while markedly increasing (1000%) the particulate activity. Both fractions appear to be regulated independently. Predominantly, the soluble form was activated by sodium nitroprusside, involving interactions with SH-groups. On the other hand, the particulate form was stimulated by a series of unsaturated fatty acids and their hydroperoxides. The latter activation appears to result from direct hydrophobic effects rather than peroxide or free radical generation.

Receptors and Signal Transduction in the Myometrium

In the myometrium, as in many other smooth muscle preparations, Ca2+ and cAMP, the two major intracellular second messengers, exert opposite effects at the level of contractility. The necessity of calcium for uterine contraction has long been recognized, the role of Ca2+ being obligatory, whether the stimulus is hormonal or voltage-induced. On the other hand, cAMP has been shown to contribute to uterine relaxion (Hardman 1981, Do Khac et al. 1986b, Diamond 1990). The increase in intracellular Ca2+ evoked by stimulatory agonists is considered to originate at least in part from intracellular stores (Van Breemen and Saida 1989; Somlyo and Himpens 1989; Mironneau et al. 1984). In this regard, the phosphoinositide-phospholipase C transducing mechanism that is consistently associated with Ca2+-mobilizing receptors (Berridge and Irvine 1984; Berridge 1987) has been demonstrated to be activated by contracting agonists in different myometrial preparations (Marc et al. 1986, 1988; Anwer et al. 1989; Goureau et al. 1990). Additionally, a number of recently reported findings provide satisfactory correlations between the increased generation of inositol phosphates, the ability of inositol 1,4,5-trisphosphate, InsP(1,4,5,)P3, to release Ca2+ from intracellular stores, and the accompanying Ca2+-induced uterine contractions (Carsten and Miller 1985; Marc et al. 1988;Kanmuraetal. 1988).