Myometrial Membranes Research Articles

Reconstitution of the myometrial oxytocin receptor into proteoliposomes

The requirements for regaining high-affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse-phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio-beads SM-2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high-affinity binding of [3H]oxytocin and 125I-oxytocin antagonist to reconstituted proteoliposomes. Binding of [3H]oxytocin but not of the antagonist was dependent on the presence of Mn2+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68-80 kDa.

Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium.

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectin-affinity chromatography. Human myometrial membrane preparations contained variable amount (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for 125I-CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd = 1.2 nM). The proteins in the myometrial membrane preparation were cross-linked to 125I-[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-kDa and 105-120-kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic-peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps-solubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66-70 kDa to 33 kDa.

GRP-preferring bombesin receptors increase generation of inositol phosphates and tension in rat myometrium.

In the estrogen-treated rat myometrium, bombesin (Bn) and related agonists triggered contraction and the increased generation of inositol phosphates. The relative order of potencies was identical for both responses: Bn = gastrin releasing peptide (GRP) = litorin = neuromedin C >> neuromedin B. Two specific GRP-preferring receptor antagonists, namely [D-Phe6]Bn-(6-13) methyl ester and [Leu14,psi 13-14]Bn were inhibitory for both Bn-mediated tension and generation of inositol phosphates. [125I-Tyr4]Bn bound to myometrial membranes with high affinity (Kd = 104 pM) to a single class of sites in a saturable and reversible manner. The relative potencies for inhibiting binding were GRP = litorin = [Tyr4]Bn (Ki = 0.4 to 0.6 nM) >> neuromedin B (Ki = 10.3 nM). The high affinity displayed by [D-Phe6]Bn-(6-13) methyl ester (Ki = 2.8 nM) and [Leu14,psi 13-14]Bn (Ki = 35 nM) for competing for [Tyr4]Bn binding supported the involvement of a GRP-preferring Bn receptor. Guanine nucleotides decreased the binding of [125I-Tyr4]Bn and accelerated the rate of ligand dissociation, reflecting the coupling of receptors to guanine nucleotide regulatory proteins (G proteins). The results demonstrate that rat myometrium expresses functional GRP-preferring Bn receptors whose activation stimulates the phospholipase C pathway, pertussis toxin-insensitive event that contributes to Bn-mediated uterine contractions.

Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1‐receptor subtype

1. The pharmacological profile of valsartan, (S)-N-valeryl-N-([2'-(1H-tetrazol-5-yl)biphenyl-4-yl]-methyl)-vali ne, a potent, highly selective, and orally active antagonist at the angiotensin II (AII) AT1-receptor, was studied in vitro and in vivo. 2. Valsartan competed with [125I]-AII at its specific binding sites in rat aortic smooth muscle cell membranes (AT1-receptor subtype) with a Ki of 2.38 nM, but was about 30,000 times less active in human myometrial membranes (AT2-receptor subtype). 3. In rabbit aortic rings incubated for 5 min with valsartan, at concentrations of 2, 20 and 200 nM, the concentration-response curve of AII was displaced to the right and the maximum response was reduced by 33%, 36% and 40%, respectively. Prolongation of the incubation time with valsartan to 1 h or 3 h, further reduced the maximum response by 48% or 59% (after 20 nM) and by 59% or 60% (after 200 nM) respectively. After 3 h incubation an apparent pKb value of 9.26 was calculated. Contractions induced by noradrenaline, 5-hydroxytryptamine, or potassium chloride were not affected by valsartan. No agonistic effects were observed in the rabbit aorta at concentrations of valsartan up to 2 microM. 4. In bovine adrenal glomerulosa, valsartan inhibited AII-stimulated aldosterone release without affecting the maximum response (pA2 8.4). 5. In the pithed rat, oral administration of valsartan (10 mg kg-1) shifted the AII-induced pressor response curves to the right, without affecting responses induced by the electrical stimulation of the sympathetic outflow or by noradrenaline. Animals treated with valsartan 24 h before pithing also showed significant inhibition of the response to AII. 6. In conscious, two-kidney, one-clip renal hypertensive rats (2K1C), valsartan decreased blood pressure in a dose-dependent manner after single i.v. or oral administration. The respective ED30 values were 0.06 mg kg-1 (i.v.) and 1.4 mg kg-1 (p.o.). The antihypertensive effect lasted for at least 24 h after either route of administration. After repeated oral administration for 4 days (3 and 10 mg kg-1 daily), in 2K1C renal hypertensive rats, systolic blood pressure was consistently decreased, but heart rate was not significantly affected. 7. In conscious, normotensive, sodium-depleted marmosets, valsartan decreased mean arterial pressure, measured by telemetry, after oral doses of 1-30 mg kg-1. The hypotensive effect persisted up to 12 h after 3 and 10 mg kg-1 and up to 24 h after 30 mg kg-1. 8. In sodium-depleted marmosets, the hypotensive effect of valsartan lasted longer than that of losartan(DuP 753). In renal hypertensive rats, both agents had a similar duration (24 h), but a different onset of action (valsartan at 1 h, losartan between 2 h and 24 h).9. These results demonstrate that valsartan is a potent, specific, highly selective antagonist of AII at theAT1-receptor subtype and does not possess agonistic activity. Furthermore, it is an efficacious, orally active, blood pressure-lowering agent in conscious renal hypertensive rats and in conscious normotensive,sodium-depleted primates.

Modulation of human myometrial PGE 2 receptor by GTP characterization of receptor subtype

We studied PGE 2 specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE 2 ≥ PGE 1 ⪢ PGF 2α > Iloprost ≥ Carbacyclin ⪢ ZK 110841 (PGD 2 analogue). These relative affinities indicated that the receptor was of the EP type. In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE 2 binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10 −4 sec −1 (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10 −4 sec −1 half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10 −9 M to 4.96 ± 1.25.10 −9M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10 −4M. GppNHp and GTPγS were effective at 1.10 −5M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE 2 binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla. Competition studies with PGE 2 analogues (sulprostone, 17-phenyl-ω-trinor PGE 2, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP 3 subtype.

Oxytocin receptors and prostaglandin release in rabbit amnion.

Besides stimulating uterine myometrial and mammary myoepithelial cell contraction, oxytocin (OT) causes the release of prostaglandins (PGs) from uterine endometrium/decidua and amnion cells. Lacking information about OT receptors eliciting PG release, we don't know how they are related to OT receptors involved in smooth muscle contraction. The amnion offers great potential for characterizing OT receptors associated with PG release, as the amount of iodinated OT antagonist ([125I]OTA) bound to rabbit amnion membranes during labor is among the greatest of any tissue yet studied, reaching about 10 pmol/mg membrane protein. The relative affinities of several OT analogues for binding sites on amnion membranes are the same as those on decidual membranes. There are differences in the ligand profile between amnion and myometrium, but they could be due to the additional presence of vasopressin receptors on myometrial membranes. An increase in the sensitivity of PGE2 release from amnion cells in culture to OT and analogues accompanies the rise in OT receptor concentration at the end of gestation. Increases in [125I]OTA binding in vivo can be mimicked with cultured amnion cells by addition of agents that elevate intracellular cAMP levels. Based on the time course and inhibition of the increase with cycloheximide, cAMP might induce OT receptor gene expression. The increase also is reflected by a marked elevation in the covalent labeling of a 50-kDa electrophoretic band with a photoactivated derivative of [125I]OTA. Because of the homogeneity of cell types in the amnion, the ease of culturing amnion cells, and the high concentration of OT receptors that can be induced, this tissue should be very useful in characterizing OT receptors associated with PG synthesis.

Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus.

1. We have investigated the role of changes of potassium efflux in the inhibition of uterine force produced by cyanide. K+ efflux (86Rb) was measured from pregnant and non-pregnant rat myometrial strips during metabolic inhibition with cyanide and following manoeuvres to displace intracellular pH (pHi). 2. Cyanide greatly reduced or abolished spontaneous contractions. If the membrane was depolarized directly at this stage (by elevating external K+) then contraction redeveloped. This suggests that the initial depression of force is due to a failure of membrane excitation. 3. Cyanide reversibly increased 86Rb efflux (30-35%) in both pregnant and nonpregnant uteri and contraction was reduced. The increase in 86Rb efflux with cyanide was not secondary to changes of membrane potential as it also occurred in both high-K+ and Ca(2+)-free solutions. 4. Glibenclamide (20 microM), an antagonist of K+ATP channels, reduced the cyanide-evoked increase of 86Rb efflux by about 50%. The glibenclamide-insensitive component of efflux persisted in a Ca(2+)-free solution. Despite its action on 86Rb efflux, glibenclamide did not restore contraction. 5. Intracellular pH falls during metabolic inhibition. We therefore investigated whether reducing pHi (in the absence of cyanide) had an effect on 86Rb efflux. Application of the weak acid butyrate (60 mM, at constant external pH, 7.4) had no significant effect on 86Rb efflux. Thus it is unlikely that the acidification in hypoxia contributes to the increased K+ efflux. 6. Intracellular alkalinization produced by the weak base trimethylamine (60 mM) increased the frequency of uterine contraction and the 86Rb efflux. However, there was no effect on the 86Rb efflux in a Ca(2+)-free solution. The increased efflux is therefore presumably a consequence of the increased frequency. 7. It is concluded that metabolic inhibition produced by cyanide, produces an increase in K+ efflux from the myometrium. Part of this efflux is glibenclamide sensitive. This increased K+ efflux will lead to hyperpolarization of the myometrial membrane and thus decrease excitation. Thus reduced surface membrane excitability will contribute to the fall of force in hypoxia; specifically it may cause the initial loss of spontaneous contractions in the uterus.

Endothelin‐1 receptors in the human myometrium: evidence for different binding properties in post‐menopausal as compared to premenopausal and pregnant women

We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n = 12), myomatous premenopausal women (n = 14) and pregnant women (n = 14), using 125I-labelled endothelin-1. The mean (+/- SD) maximal receptor density (Bmax) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 +/- 196, 1116 +/- 201 and 490 +/- 145 pmol/g protein, respectively). Receptor affinity (Kd) did not differ significantly between these groups. Among the pregnant women, mean Bmax and Kd values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.

The identification of a human myometrial corticotropin-releasing hormone receptor that increases in affinity during pregnancy.

The concentration of immunoreactive, placentally derived CRH is increased in the peripheral circulation during the third trimester of human pregnancy. However, the function of this placental CRH is entirely unknown. A number of observations have led us to believe that CRH might influence myometrial contractility and, hence, parturition via specific receptor mechanisms. 1) In idiopathic preterm labor, plasma immunoreactive-CRH concentrations are significantly elevated compared to control values. 2) CRH and oxytocin exhibit a marked synergistic effect on myometrial contractility which is prostaglandin dependent and can be inhibited by the CRH receptor antagonist [alpha-helical CRF-(9-41)]. In view of this, we searched for specific CRH-binding sites in myometrial tissue obtained at biopsy from pregnant (cesarian section) and nonpregnant (hysterectomy) patients. To test for the presence of CRH receptors, we prepared myometrial membranes and performed binding studies using [125I]tyr-o-CRH as a ligand. The binding was found to be pH, time, temperature, and divalent cation concentration dependent and was fully reversible on addition of 1 microM unlabeled ovine CRH. In both tissues, there was a single, specific, homogenous, high affinity population of CRH receptors. Scatchard analysis of the specific binding sites revealed dissociation constants of 250-300 and 30-60 pM for the nonpregnant and pregnant myometrium, respectively. This compares with dissociation constants of 130 pM (rat anterior pituitary receptor) and 100 pM (human CRH-binding protein). This would mean that in the nonpregnant state, the equilibrium for binding is in favor of the binding protein, but during the later stages of pregnancy, the change in affinity of the receptor alters the binding in favor of the myometrial receptor.

Properties of large-conductance K+ channels in human myometrium during pregnancy and labour.

The conversion of the electrically silent pregnant uterus to highly excitable at term represents a dramatic physiological event which is poorly understood. Here we provide the first description, from single-channel recordings, of a large conductance (212 pS) calcium-activated potassium channel (BKCa) in human pregnant myometrium which, in labour tissue, is either absent or has been considerably altered in its physiological and pharmacological properties. In the latter, the K+ channels have an identical conductance (221 pS) and K+ selectivity to BKCa channels but exhibit no Ca2+ or voltage sensitivity. We have termed these BK channels. Furthermore, the activity of the BKCa channel from pregnant tissue is inhibited by internal application of Ba2+ but not tetraethylammonium (TEA), whereas the activity of the BK channel is sensitive to internal TEA but not Ba2+. The role of the BKCa channel may be to suppress myometrial activity during gestation whereas BK channel activity may be important in providing a Ca(2+)-independent K+ conductance which would allow cytoplasmic Ca2+ levels to rise without activating a counteracting Ca(2+)-dependent outward current, normally provided by the BKCa channels which, by its very nature, would tend to oppose depolarization. The findings suggest that K+ channels may have an important role in determining the functional activity of the myometrium.

Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes

Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes

Endothelin in rabbit uterus during pregnancy.

Specific immunostaining for endothelin-1 (ET-1) was observed in the uterine mucosa of pregnant and nonpregnant rabbits. During gestation, giant cells immunopositive for ET-1 were identified in either endometrium or myometrium. In the latter, they were located in close proximity to the muscular fibers. High-affinity (dissociation constant = 0.25 nM) high-capacity [maximal binding (Bmax) = 7 pmol/mg protein] receptors for ET-1 were present in myometrial membranes of estrous rabbits. During pregnancy the concentration of ET-1 receptors progressively decreased (Bmax on day 29 = 4 pmol/mg protein), rising again at the time of spontaneous delivery. Conversely, ET-1 receptors in the aorta did not change during pregnancy and parturition. The presence of giant cells immunopositive for ET-1 in close proximity to the myometrial cells, together with the evidence of an increase in ET-1 receptors in myometrium of parturient rabbits, suggests a paracrine role for endometrial ET-1 during delivery. Because we previously demonstrated that oxytocin releases ET-1 from endometrial cells in primary culture and that endometrial and myometrial oxytocin receptors abruptly rise at the time of parturition, we propose that ET-1 might participate in the complex cell-to-cell interactions that occur during labor.

Characterization of oxytocin receptors in rabbit amnion involved in the production of prostaglandin E2.

We characterized oxytocin (OT) receptors in purified plasma membranes from amnion, decidua, and myometrium of late pregnant rabbits using an iodinated OT antagonist (OTA). Saturation studies showed similar Kd values for specific binding sites in the 100- to 250-pM range in all three tissues. OT receptor concentrations in decidua and myometrium did not change until the day of labor (day 31), when they rose about 2.5- and 18-fold, respectively. Increases in amnion receptors were first apparent on day 28 and continued to maximal levels on day 31. There was an increase of about 230-fold from day 26 to labor, reaching 9.5 pmol/mg protein. Competition studies using analogs showed that ligand specificities of amnion and decidual membranes were indistinguishable. Those of myometrial membranes were somewhat different, possibly owing to the presence of both AVP receptors and OT receptors in the myometrium. Binding of OTA corresponded to the OT-induced release of prostaglandin E2 (PGE2) by amnion cells in culture. The effects of OT were dose dependent, agonist specific, and selectively inhibited by OTA. Amnion cells from days 22 and 28 did not respond significantly to either OT or phorbol 12-myristate 13-acetate (PMA), but cells from day 30 pregnant rabbits responded strongly to both. In contrast, calcium ionophore stimulated comparable amounts of PGE2 release from cells cultured on day 22, 28, or 30. These studies show that specific, high affinity OT receptors are associated with the release of PGE2 from rabbit amnion cells. Increases in amnion OT receptor and protein kinase-C activity precede by several days the increases in receptor concentrations in decidua and myometrium, suggesting important roles for the amnion and OT in the initiation of labor in rabbits.

Down-regulation of myometrial atrial natriuretic factor receptors by progesterone and pregnancy and up-regulation by oestrogen in rats.

We previously reported that pregnancy and progesterone treatment abolish and ovariectomy reduces the tocolytic activity of atrial natriuretic factor (ANF) on rat uterus. The present study was carried out to determine whether the hormonal state of the animal influences myometrial ANF receptors. A single high-affinity binding site for 125I-labelled ANF(1-28) was detected in myometrial membrane preparations partially purified by discontinuous sucrose gradient centrifugation. The specifically bound ANF could be displaced completely by unlabelled ANF(1-28) (biologically active circulating form of ANF), partially (approximately 50%) by the clearance receptor (ANF-R2) ligand, ANF(4-23), and insignificantly by angiotensin II, vasopressin, carbachol and progesterone. Maximum binding capacities (fmol/mg protein +/- S.E.M.) of myometrial membrane preparations for ANF were as follows: vehicle-treated cyclic rats, 30.2 +/- 5.9 (n = 6); oestrogen-treated virgin rats, 55.8 +/- 7.1 (n = 8); progesterone-treated virgin rats, 21.9 +/- 2.4 (n = 6); 20-day pregnant rats, 18.9 +/- 4.3 (n = 4); ovariectomized rats, undetectable (n = 3); ovariectomized rats treated with oestrogen, 50.2 +/- 4.1 (n = 3); virgin rats treated with progesterone antagonist RU 486, 64.5 +/- 5.2 (n = 3). Relative to cyclic rats, oestrogen and RU 486 caused a significant (P less than 0.05) increase and progesterone and pregnancy caused a significant (P less than 0.05) decrease in ANF myometrial receptors. The hormonal state did not change dissociation constants for ANF binding to myometrial preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and enzymatic deglycosylation of the myometrial oxytocin receptor using a radioiodinated photoreactive antagonist.

To identify and characterize oxytocin receptors, a 125I-labeled photoreactive oxytocin antagonist was synthesized. The specific oxytocin antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-methyltyrosine,4-threonine,8- ornithine,9-tyrosylamide]oxytocin ([Mca,Tyr(O-Me)2,Thr4,Orn8,Tyr9-NH2]oxytocin) described by Elands et al. (Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski, K., Manning, M., and Sawyer, W. H. (1987) Eur. J. Pharmacol. 147, 192-207) bound to the guinea pig uterine oxytocin receptor with high affinity (apparent Kd = 0.74 nM). The introduction of a 4-azidophenylamidino group at Orn8 resulted in the photoreactive ligand [Mca1,Tyr(O-Me)2,Thr4,Orn(4-azidophenylamidino)8,Tyr9- NH2]oxytocin, which retained the high binding affinity (Kd = 0.69 nM) of the parent compound. The photoreactive antagonist monoiodinated at Tyr9 had approximately double (Kd = 0.39 nM) the affinity of the photoreactive antagonist and several times that of oxytocin (Kd = 2.6 nM) for the guinea pig uterine oxytocin receptor. In photo-affinity labeling experiments using myometrial membranes obtained from guinea pigs during late pregnancy, the 125I-labeled photoreactive antagonist specifically labeled a protein with an apparent molecular mass of between 68 and 80 kDa: the labeling of this protein was completely suppressed by a 100-fold molar excess of oxytocin and oxytocin receptor-specific agonists, but not by vasopressin analogues specific for V1 or V2 receptors or by other peptide hormones. The ability of oxytocin to suppress labeling was decreased in the presence of guanosine 5'-O-(thiotriphosphate) or in the absence of Mn2+. Digestion of the photolabeled oxytocin receptor with endoglycosidase F gave rise to a protein with an apparent molecular mass of 38 +/- 2 kDa. The endoglycosidase F effect and the lack of endoglycosidase H action show that the myometrial oxytocin receptor is highly glycosylated with asparagine-linked complex oligosaccharide chains. Our results suggest that the radioiodinated photoreactive oxytocin antagonist could be a helpful tool in the isolation and further characterization of the oxytocin receptor.

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Antagonist binding reveals two heterogenous B 2 bradykinin receptors in rat myometrial membranes

In rat myometrial membranes, two bradykinin binding sites with K I values of 18 pM and 5.6 nM were identified. Three potent bradykinin antagonists were tested for their ability to compete for [ 3H]bradykinin binding. Two of them, D-Arg[Hyp 3, Thi 5,8,D-Phe 7]bradykinin and [Hyp 3,Thi 5,8,D-Phe 7]bradykinin, also bound to both the high- (K H) and the low-affinity (K L) site whereas [Thi 5,8,D-Phe 7]bradykinin identified only the low-affinity bradykinin receptor. There is a close correlation between the antagonistic potencies and the K L site affinities.

Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides.

We have previously reported that [3H]bradykinin [( 3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (Bmax) of 1119 +/- 160 fmol/mg of protein, with an equilibrium dissociation constant (KD) of 314 +/- 70 pM and with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[beta gamma-imido]triphosphate (Gpp[NH]p, [3H]BK bound to the soluble receptors with a KD of 929 +/- 129 pM and a Bmax. of 706 +/- 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and Bmax., which was half-maximal at 0.5 microM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G-)protein.

High-affinity bradykinin receptor-catalyzed G protein activation in rat myometrium

Binding of the labelled GTP analog, guanosine-5'-O-(3-thiotriphosphate ([35S]GTP[S] to G proteins was studied in rat myometrial membranes in the presence of GDP (1 microM). Binding was stimulated by bradykinin at subnanomolar concentrations. while oxotremorine increased binding of [35S]GTP[S] to myometrial membranes at micromolar concentrations. The bradykinin-induced stimulation was antagonized by the receptor antagonist, [D-Arg-(Hyp3, Thi5,8, D-Phe7)]bradykinin. Addition of NaCl (150 nM) decreased control binding and abolished the stimulatory effect of bradykinin. On the other hand, addition of CaCl2 (5 mM) had no effect on control binding but also prevented the bradykinin-induced increased in [35S]GTP[S] binding. Saturation experiments revealed that activation of the bradykinin receptor leads to about a three-fold increase in the apparent GTP[S] binding affinity of about 30% of the total GTP[S] binding sites measured in these membranes. The results provide evidence for a high-affinity bradykinin receptor in rat myometrial membranes which interacts with and activates G proteins. This receptor action, which appears to be under the control of both sodium and calcium ions, is catalytic and leads to a large signal amplification, in that one agonist-liganded bradykinin receptor can apparently activate up to 100 G proteins.

Regulation by progesterone of the high-affinity state of myometrial beta-adrenergic receptor and of adenylate cyclase activity in the pregnant rat.

The effects of pregnancy or progesterone dominance on the beta-adrenergic responsiveness of the uterus were studied in myometrial membranes from mid- and late-pregnant rats (day 15 and on the 16th h of day 22 of pregnancy respectively) or 24 h after administration of progesterone. Levels of the high (RH)- and low (RL)-affinity states of the beta-adrenergic receptor were determined by competition experiments between 125I-labelled cyanopindolol binding and the selective beta-agonist isoproterenol. The ratio KL/KH (respective dissociation constants) was determined since it also reflects the degree of formation of the high-affinity state of the beta-adrenergic receptor. From day 15 to the 10th h of day 22 of pregnancy, two distinct affinity states were apparent: 80-55% RH (KH = 0.31-0.21 microM) and 45-20% RL (KL = 14-5 microM) with a ratio of KL/KH of 55-34. In the last 6 h before birth, beta-adrenergic receptors underwent uncoupling which was paralleled by decreased responsiveness of myometrial adenylate cyclase to isoproterenol (maximum velocity (Vmax) = 17 +/- 3 vs 44 +/- 3 fmol cyclic AMP/10 min per mg protein on day 15). At this stage of pregnancy, previous exposure to progesterone resulted in a 1.8-fold increase in 125I-labelled cyanopindolol-binding sites (Bmax) and the reappearance of the high-affinity state (67% RH, KH = 0.19 +/- 0.04 (S.E.M.) microM, ratio KL/KH = 81.1 +/- 16.9).(ABSTRACT TRUNCATED AT 250 WORDS)