Rat ovarian granulosa cell as a site of endothelin reception and action: attenuation of gonadotropin-stimulated steroidogenesis via perturbation of the A-kinase signaling pathway.

Endothelins (ETs) are a family of vasoactive peptides that may be involved in granulosa cell (GC) luteinization or follicular maturation. However, the precise role of ET in ovarian physiology remains unknown. We have investigated whether the rat GC is a site of ET reception and have characterized the antigonadotropic effect of ET in cultured GC from immature rats. Two major ET binding species (52 and 30 kDa) were observed after cross-linking of GC membranes with radiolabeled ET-1, although the smaller protein may represent a degradative product. Unlabeled ET-1, ET-2, or ET-3 were equipotent in displacing radiolabeled ET-1 from these putative ET receptors, with EC50s of 0.3-0.7 x 10(-9) M. Similarly, ET-1, ET-2, and ET-3 were equipotent (EC50s of about 10(-10) to 10(-9) M) in inhibiting the FSH-supported accumulation of progesterone. ET-1 (10(-7) M) also inhibited (> 90%) FSH-supported estrogen accumulation. Maximum progesterone inhibition (> 90%) by ET-1 (10(-7) M) was achieved throughout the range of FSH does and cell densities tested and by 48 h or 72 h of culture. ET-1 was not cytotoxic in the dose range tested. Forskolin-stimulated progesterone accumulation was similarly inhibited by ET-1, suggesting that ET-1 inhibits cAMP-mediated (e.g., FSH or forskolin-stimulated) progesterone accumulation. ET-1 inhibited (74%) the FSH-stimulated accumulation of cAMP, suggesting that it acts at sites related to cAMP generation or degradation. In addition, ET-1 inhibited 8-bromo-cAMP-generated progesterone accumulation (60%), suggesting that it also acts at sites distal to cAMP generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin-1 as a luteinization inhibitor: inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation.

Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.

PDGF induces tyrosine phosphorylation in osteoblast-like cells: relevance to mitogenesis.

We have studied the association between protein tyrosine phosphorylation and the mitogenic effect induced by platelet-derived growth factor (PDGF) in the osteoblast-like cell line MC3T3-E1. PDGF caused a dose-dependent increase in [3H]thymidine incorporation in MC3T3-E1 cells, reaching a plateau at 10 ng/ml. Vanadate, a potent phosphatase inhibitor, induced a twofold increase in thymidine incorporation. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation. Genistein, a tyrosine kinase inhibitor, inhibited in a dose-related manner (2-20 microM) the mitogenic effect induced by either PDGF or the combination of vanadate and PDGF. These observations suggest that tyrosine kinases are involved in mediating the mitogenic effect of PDGF in these cells. PDGF treatment of MC3T3-E1 cells and subsequent immunoblotting with antiphosphotyrosine antibodies resulted in a marked phosphorylation of the PDGF receptor. Vanadate had a lesser effect on PDGF receptor phosphorylation, but given together with PDGF it induced a significant increase in the intensity of receptor phosphorylation. Preincubation with genistein abrogated these effects. Taken together, these findings indicate a direct correlation between thymidine incorporation and tyrosine phosphorylation in MC3T3-E1 cells and suggest that tyrosine phosphorylation plays a role in PDGF-induced mitogenic activity in osteoblast-like cells.

Preparation and application of radioiodinated sulfhydryl reagents for the covalent labeling of SH-proteins present in minute quantities.

In this study we have searched for sulfhydryl reagents which can be radiolabeled and detect minute quantities of SH-proteins. Iodoacetamidotyramine reacts with sulfhydryls at a low rate, having a pseudo-first order rate constant, kappa obs = 3 +/- 0.2 M-1 s-1, at neutral pH. In contrast, N-ethylmaleimide-containing reagents, such as tyrosine-MIB and tyramine-MIB were three orders of magnitude more reactive in alkylating sulfhydryls. Pseudo-first order rate constants, kappa obs, were in the range of 5200-5700 M-1 s-1. Therefore, a simple and convenient procedure was designed for the synthesis and the radioactive labeling of tyramine-MIB. Simplification was attained by virtue of the specific-'affinity' adsorption of [125I]tyramine-MIB (and not the other intermediates) to small Sephadex G-10 column and its elution with ethanol. [125I]Tyramine-MIB was stable for weeks in dried form and for hours in acidic to neutral aqueous solutions. The reagent, when radiolabeled to high specific activity (0.5 Ci/mumol), detected sulfhydryl proteins at concentrations as low as 1-10 pM. The applicability of the reagent in studying biological systems was demonstrated by adding it to intact adipocytes and the consequent labeling of a single protein with an apparent Mr = 32,000, which is most likely an externally oriented surface plasma membrane SH-protein. [125I]Tyramine-MIB reactivity and sensitivity exceeds that of protein-tyrosyl radioiodination by the chloramine-T procedure and is expected to assist in studying minute quantities of SH-proteins.

Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells.

The mitogenic activity of endothelin (ET) was studied in osteoblast-like cells, MC3T3-E1. [3H] Thymidine incorporation induced by ET was markedly lower than that of platelet-derived growth factor (PDGF). ET synergistically stimulated [3H] thymidine incorporation induced by PDGF with an apparent ED50 value of 2.5 nM. Treatment of MC3T3-E1 cells with ET and subsequent immunoblotting of the cell extracts with antiphosphotyrosine antibodies followed by labeling with [125I] protein A resulted in the identification of several phosphotyrosine-containing proteins. The intensity of these labeled phosphoproteins significantly increased when the cells were treated with a combination of ET and PDGF. Genistein, an inhibitor of tyrosine kinases, blocked [3H] thymidine incorporation as well as protein tyrosine phosphorylation stimulated by either ET, PDGF or the combination of ET and PDGF. These findings suggest that tyrosine phosphorylation could play a role in the comitogenic activity of ET in osteoblast-like cells.

Biotinylated endothelin as a probe for the endothelin receptor.

Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using 125I-labeled ET-1 revealed IC50 values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.

Direct evidence for multiple endothelin receptors.

Competition binding experiments and peptide mapping techniques were employed in order to directly address the possible existence of endothelin (ET) receptor subtypes in the atria. Competition binding assays for 125I-labeled ET-1 or 125I-labeled ET-3 to bovine atrial membrane preparations suggest the existence of two ET receptor subtypes, one of which binds ET-1 and ET-3 with a similar affinity while the other shows preference for ET-3. However, cross-linking experiments of both peptides to this tissue resulted in the identification of a single 50-kDa protein. To identify directly the existence of multiple ET receptors, peptide mapping of cross-linked 125I-labeled ET-1 or 125I-labeled ET-3 receptors was conducted. Different peptide maps were obtained only under conditions that preferentially label one receptor subtype. These results indicate, for the first time, the existence of two ET receptor subtypes in the atria which differ from each other in both their binding characteristics and primary structure.

Neuroendocrine peptides in milk.

Milk, which is a mammal-specific biologic fluid, contains several neuroendocrine peptides at concentrations higher than those found in plasma. These neuroendocrine peptides can be synthesized or processed in the mammary gland or excreted into milk through various pathways. In addition, certain milk proteins, notably casein, can be enzymatically processed to release "exorphins," peptides with opioid activities. In suckling mammals, hormones and neuropeptides are absorbed through the gastrointestinal tract and appear intact in the plasma. This absorption is age dependent and could have physiologic significance in neonatal development.

Identification of a single binding protein for endothelin-1 and endothelin-3 in bovine cerebellum membranes.

Competition binding experiments of [125I]endothelin-3 ([125I]ET-3) to bovine cerebellum membrane preparations in the presence of either ET-3 or ET-1 have indicated the presence of a single class of high affinity binding sites for these two peptides in the brain. Cross-linking of [125I]ET-3 to cerebellum membrane preparations with dissuccinimidyl suberate (DSS) resulted in the labeling of two bands with apparent mol wt of 52 and 30 kDa. Under these conditions the labeling intensities of these two bands were similar. However, addition of 5 mM EDTA to the protease inhibitor mixture during membrane preparations as well as the binding and cross-linking reaction increased the labeling of the 52-kDa protein while reducing the labeling of the 30-kDa protein. Peptide map comparisons of the 52- and 30-kDa protein bands using Staphylococcus aureus V8 protease and papain revealed that the 30-kDa band is a proteolytic degradation product of the 52-kDa protein. These results suggest that the 52-kDa protein represents the specific binding protein of ET-3, and thus, the apparent mol wt of the ET receptor is 50 kDa, subtracting the mol wt of the iodinated ET. Since cross-linking of [125I]ET-1 to cerebellum membrane preparations revealed the same two bands of 52 and 30 kDa, peptide mapping of the 52-kDa proteins, cross-linked with either [125I]ET-1 or ET-3, was conducted. Under these experimental conditions, identical peptide fragments were generated by both Staphylococcus aureus V8 protease and papain. These results suggest that ET-1 and ET-3 bind to a common brain binding protein with an apparent mol wt of 50 kDa.

Photoaffinity labeling of pituitary gonadotropin-releasing hormone receptors in goldfish (Carassius auratus).

Receptors for GnRH were labeled by use of an iodinated (125I) photoreactive GnRH derivative [D-Lys6-azidobenzoyl]-GnRH. This derivative was found to bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. The binding affinity of [D-Lys6-azidobenzoyl]-GnRH was found to be greater than that of D-Lys6-GnRH, but lower than a superactive fish GnRH agonist [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH (sGnRH-A). Analysis of the photoaffinity-labeled goldfish pituitary GnRH receptors by SDS-PAGE and autoradiography indicated the presence of three labeled proteins displaceable by unlabeled sGnRH-A. The first and the most prominently labeled band was a 71,000-Mr protein, the second a 51,000-Mr protein, and the third a minor band of 130,000 Mr. Displacement characteristics of the 71,000- and 130,000-Mr bands were consistent with those of the low-affinity binding sites; displacement of the iodinated ligand from these proteins was achieved only in the presence of 10(-6) M sGnRH-A. The 51,000-Mr band had characteristics similar to those of the high-affinity site; displacement of the labeled ligand was achieved in the presence of 10(-9) M sGnRH-A. These findings provide for the first time some biochemical characterizations of pituitary GnRH receptors in a nonmammalian vertebrate.

Purification of gonadotropin releasing hormone receptors using the avidin-biotin technique.

The avidin-biotin technique has been applied to the purification of gonadotropin releasing hormone (GnRH) receptors from other solubilized membrane proteins. The following steps were involved in this approach: (a) solubilization of rat pituitary GnRH receptor with the zwitterionic detergent CHAPS, 3-[(3-cholamidopropyl)-di-methylammonio]-1-propane sulfonate, (b) equilibration of the solubilized GnRH receptor with [biotinyl-D-Lys6]GnRH immobilized on avidin-agarose; and (c) elution of the receptors with high salt and GnRH analogues. Following two cycles of affinity chromatography the GnRH receptor was purified to homogeneity. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract and the calculated purification was approximately 10,000 to 15,000 fold. The development of a two step affinity chromatography for the purification of GnRH receptors can be used for detailed studies on the structure and function of the receptor. These studies will advance our understanding of the molecular basis of GnRH action.

Bovine cerebellum endothelin receptor: solubilization and identification.

Endothelin receptors were solubilized from bovine cerebellum membrane preparations in an active form by using the zwitterionic detergent CHAPS, [3-(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid. The solubilized receptors displayed high affinity, saturability, and specificity. The dissociation constant (Kd) for endothelin was 7 +/- 2 nM, and the number of binding sites was 600 +/- 200 fmol/mg protein. These results are similar to those obtained for the membrane-bound receptor and suggest that during solubilization the binding characteristics of the receptor are preserved. Attempts to purify the solubilized receptors in an active form using affinity chromatography techniques, i.e. Affi-gel 15 column coupled to endothelin, were not successful. Nevertheless, identification of the receptors was achieved by affinity chromatography of the solubilized proteins and subsequent iodination. Autoradiographic analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major protein with an apparent mol wt of 50 kD. Taken together with our previous findings, this result suggests that the 50-kD band represents the endothelin receptor.

Identification of endothelin receptors by chemical cross-linking.

Specific binding sites for human/porcine endothelin have been found in rat brain membrane preparations using an [125I]endothelin. The apparent Kd value was 3 +/- 2 nM with a Bmax value of 2,200 +/- 200 fmol/mg protein. Chemical cross-linking of [125I]endothelin to rat brain membranes by using the cross-linking reagent disuccinimidyl suberate (DSS) revealed two specific proteins of Mr = 52,000 and Mr = 30,000. The same results were obtained by photoaffinity labeling of [125I]azidoendothelin to rat brain membranes. The labeling of the two proteins was inhibited in a dose-dependent manner by unlabeled endothelin but not by unrelated peptides. Peptide map comparisons of the Mr = 52,000 and Mr = 30,000 protein bands using Staphylococcus aureus V8 protease and papain revealed that the Mr = 30,000 band is a proteolytic degradation product of the Mr = 52,000. The apparent mol wt of the endothelin receptor is, therefore, 50,000, subtracting the mol wt of the iodinated endothelin.

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein.

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.

Competitive interaction between endothelin and sarafotoxin: Binding and phosphoinositides hydrolysis in rat atria and brain.

Binding studies with the structurally similar vasoconstrictor peptides 125I-endothelin and 125I-sarafotoxin b, the former of mammalian origin and the latter derived from snake venom, reveal their mutually exclusive binding to rat atrium and various regions of the rat brain. In these tissues endothelin, like sarafotoxin, induces phosphoinositide hydrolysis which is in part Ca2+-independent. It is suggested that endothelins and sarafotoxins share common binding sites and mechanisms of action.

Isolation and identification of neuroendocrine peptides from milk.

Hormone, hormone-like substances, and neuroendocrine peptides are natural constituents of milk, and they may have an important physiological and pharmacological role in neonate development. The concentration of these peptides in milk greatly exceeds those in serum and implies an active concentration mechanism in the mammary gland. The large quantities in which milk can be supplied and the development of highly efficient procedures for the purification of peptides suggest that milk may prove to be an excellent source for identifying as yet "unknown" hormones and neuroendocrine peptides.

Identification and characterization of melanotropin binding proteins from M2R melanoma cells by covalent photoaffinity labeling.

In this study, two melanotropin binding proteins from M2R melanoma cells have been identified based on the photochemical cross-linking of [125I]iodinated porcine beta-MSH ([ 125I]iodo-beta-MSH) to melanoma cell membranes, using N-hydroxysuccinimidyl-azidobenzoate. Autoradiography of photoaffinity-labeled M2R membrane protein, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the specific labeling of two separate bands with an apparent molecular mass of 43 and 46 kilodaltons, respectively. Photoaffinity labeling of both bands was of near-equal intensity and could be inhibited, in a dose-dependent manner, by the addition of unlabeled beta-MSH before photolysis. In addition, agents known to inhibit the binding of beta-MSH to its cellular receptor, such as EGTA, GTP, guanosine 5'-O-(3-thio)triphosphate, and a synthetic analog of the calmodulin-binding domain of myosin light chain kinase-M5, were all found to specifically inhibit the labeling of these two protein bands by the azido derivative of [125I]iodo-beta-MSH. In contrast, addition of a nonrelated peptide, vasoactive intestinal peptide, had no effect upon the labeling of these melanotropin-binding proteins. On the basis of these results we suggest that the two proteins may function as the binding domain(s) of the cellular receptor for melanotropins, or may represent entire receptor moieties themselves.