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The reported effects of corticotrophin-releasing hormone (CRH) on human myometrium support the existence of specific receptors for the hormone in this tissue. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) technique to study the expression of mRNA coding for the CRH R1 and R2 receptors. RT-PCR of total RNA from both nonpregnant and pregnant myometrium using specific primers resulted in amplification products of the expected sizes for the R1 alpha and R2 alpha CRH receptors. The identity of these amplification products was confirmed by specific restriction digests and sequencing. Immunohistochemistry using a rabbit antibody raised against a specific domain of the CRH R1 receptor demonstrated that the R1 mRNA is translated into protein and confirmed that it is the uterine smooth muscle cells from both nonpregnant and pregnant women that bear this receptor. Our results suggest that CRH may play a role in human pregnancy at the myometrium.
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The precise factors involved in the transition of the relaxed pregnant uterus to the contractile state at the onset of parturition remain unclear, but it is accepted that cAMP-generating pathways contribute to uterine relaxation. We have previously reported an increased expression of the adenylyl cyclase (AC)-stimulating protein Galphas in human myometrium during gestation, with a corresponding increase in GTP-stimulated AC activity. However, little is known about the predominating AC isoforms expressed during pregnancy. This information is important, because although all AC isoforms are stimulated by Galphas, their regulation by other signalling molecules is very different. In the present study we have identified the isoforms of AC expressed in both pregnant and non-pregnant myometrium by mRNA analysis and immunoblotting. mRNA encoding for AC I, II, III, VIII and IX was present in non-pregnant and pregnant myometrium, and in cultured myometrial cells. Differing levels of AC protein could be detected in myometrial plasma membranes, with decreased levels of Group 1 (isoforms I, III and VIII) and Group 4 (IX) ACs allied with increased levels of Group 2 (II, IV and VII) and 3 (V and VI) ACs during pregnancy. These findings imply a role for Group 2-activating pathways, e.g. G-protein betagamma-subunits and protein kinase C, in the maintenance of uterine quiescence, whilst suggesting a lesser involvement of calcium-calmodulin complex, an activator of Group 1 AC isoforms, in uterine relaxation during gestation. These data may provide an alternative pharmacological approach for the attenuation of preterm labour.
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There is evidence for hormonal receptor desensitisation in human myometrium, but little is known about the mechanisms involved in the loss of myometrial response to agonists such as beta(2)-adrenergic agonists, prostaglandin gamma and oxytocin. It is well known that the receptors for these hormones are coupled to G-proteins. The first step of receptor desensitisation is the phosphorylation of activated receptors by a G-protein-coupled receptor kinase (GRK). GRKs are members of a multigene family and the various subtypes differ in their localisation, regulation and mode of action. We have used Western blotting and reverse transcription PCR to identify the GRKs present in human myometrium from pregnant and non-pregnant women as well as in cultured human myometrial cells. We have found that human myometrium expresses the GRK subtypes 2, 4gamma, 5 and 6. On the other hand, GRK3 and the isoforms GRK4alpha, beta and delta were not found in myometrial tissue. Our data indicate that GRK2 is only expressed in pregnant term myometrium and is not found in non-pregnant tissue. Moreover, GRK6 appears to be expressed at a much higher level in pregnant term tissue than in non-pregnant myometrium. Our observations suggest that GRK2 and GRK6 may contribute to the regulation of uterine contractility at term. Further work is necessary to determine whether GRKs and receptor desensitisation play a role in disorders of uterine contractility.
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ABSTRACT
Phosphoinositide hydrolysis is important in mediating the actions of oxytocin and prostaglandin (PG) F2α on uterine contractions during labour. We have measured the effect of oxytocin, PGF2α and other agents on the formation of inositol phosphates (IPs) in cultured human myometrial cells labelled with [3H]inositol and on changes in intracellular free Ca2+ concentration ([Ca2 + ]i) in cells loaded with Fura-2.
Oxytocin induced the formation of [3H]IPs in a concentration-dependent manner with an EC50 (concentration of agonist producing 50% of the maximal response) of 1·4 ±0·5 nmol/l (mean ± s.e.m.). The maximal response was obtained with 1 μmol oxytocin/l and represented a stimulation of 670% over basal. PGF2α also stimulated the formation of [3H]IPs and the response at 1 μmol/l was a 204% stimulation over basal. The effects of PGF2α were independent of extracellular Ca2 + but the effect of oxytocin was reduced with low extracellular Ca2 +. Cyclic AMP formation, induced by forskolin or PGE2, had no effect on the stimulated levels of [3H]IPs. Pertussis toxin (PT) reduced the oxytocin-stimulated formation of [3H]IPs in a concentration-dependent manner. The maximal effect of PT resulted in an 80% reduction in the formation of [3H]IPs. However, PGF2α stimulation was not affected by PT treatment.
To analyse the action of PT further, we studied its effect on oxytocin-induced changes in [Ca2 + ]i. The basal [Ca2 +]i was 112 ±4 nmol/l (n=225 cells) and was not affected by PT treatment (109 ± 3 nmol/l; n= 200 cells). In the absence of PT, 1 μmol oxytocin/l increased [Ca2 + ]i to a peak of 522 ±26 nmol/l, and in PT-treated cells, the [Ca2 + ]i peak was reduced to 348 ± 16 nmol/l. Similar inhibitory effects of PT were obtained at oxytocin concentrations ranging from 1 to 100 nmol/l.
Our data suggest that in human myometrial cells, the oxytocin-induced production of [3H]IPs and increase in [Ca2 + ]i are mediated by a PT-sensitive G-protein. However, a significant fraction of the oxytocin response appears to be mediated by a PT-insensitive G-protein, possibly a member of the Gq family.
Journal of Endocrinology (1993) 136, 497–509
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Abstract
We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 × 103 binding sites/cell, but this was time-dependently reduced to 27 × 103 sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.
Journal of Endocrinology (1997) 154, 7–18
