The integrin specificity of human recombinant osteopontin.

The ability of full-length human recombinant osteopontin (OPN) to support the adhesion of various alphav integrin-expressing cell lines was determined in order to characterize its integrin selectivity. The identity of this protein was assessed by cDNA sequence and mass spectroscopic analysis, and confirmed as full-length OPN. Neither the human embryonic kidney 293 cell line, which expresses the alphavbeta1 integrin, nor the human colonic adenocarcinoma HT-29 cell line, which expresses the alphavbeta5 integrin, were able to adhere to OPN; both of these cell lines are deficient in the beta3 subunit. In contrast, an alphavbeta3 integrin-expressing cell line, SK-MEL-24, was able to adhere to OPN in an arginine-glycine-aspartic acid dependent manner. In addition, this OPN-mediated cellular adhesion was completely blocked with an anti-alphavbeta3 integrin antibody (LM609), confirming that only the alphavbeta3 integrin mediated this cellular adhesion. These data demonstrate that, at least among the alphav integrins, only the alphavbeta3 is able to support cellular adhesion to osteopontin. This finding may have implications for the design of therapeutics targeting OPN-integrin interactions.

Influence of antiresorptive agent OST-766 on signal transduction pathways involved in parathyroid hormone action.

The effects of OST-766, an inhibitor of vacuolar H+-ATPase activity, on adenylyl cyclase and phospholipase C activity were explored in the osteoblast cell line ROS 17/2.8. In fresh homogenates of ROS 17/2.8 cells, OST-766 inhibited adenylyl cyclase activity (ACA) in response to guanine nucleotide and forskolin but had no effect on basal ACA. OST-766 enhanced the basal generation of IP2, but not that formed in response to Ca2+ or guanine nucleotides. In marked contrast, incubation of intact ROS 17/2.8 cells with OST-766 for at least 48 hours resulted in an increase in basal ACA as well as in response to PTH, guanine nucleotides and forskolin. Under similar conditions, the compound also increased IP1, IP2 and IP3 generation in response to guanine nucleotides and Ca2+. Levels of the guanine nucleotide binding proteins Gs and Gi were also increased in OST-766-treated cells. The results suggest that the actions of this H+-ATPase inhibitor include effects on osteoblasts through PTH-sensitive signal transduction pathways.

Hypotensive response to atrial natriuretic factor in conscious renal hypertensive beagles.

The hemodynamic responses to i.v. infusion of 0.3 and 0.6 microgram/kg per min of human atrial natriuretic factor (hANF [102-126]) in intact, conscious, one-kidney, perinephritic, hypertensive beagles were examined and compared with the responses in ganglionic-blocked dogs. Blood pressure and heart rate were not affected but plasma ANF-like immunoreactivity was increased by as much as 627%. After hexamethonium (20 mg/kg, i.v.) blockade, a dose-dependent hypotensive response of up to 29 mmHg with no change in heart rate was observed. It is concluded that the compensatory mechanisms of the neurally mediated baroreflex system masked the depressor actions of hANF.

LHRH and analogs: contraceptive and therapeutic considerations.

This paper reviews the developments of the LHRH analogs (agonists and antagonists) in terms of their conceptive, contraceptive and therapeutic utility. It draws upon the mass of data derived from pre-clinical and clinical studies, in both sexes, reinforcing, in particular, the application of the natural LHRH in treating a spectrum of infertile conditions. This profertility approach is contrasted by utilization of the paradoxical antireproductive effect of the agonists as contraceptives, and as a novel therapy for precocious puberty, endometriosis and hormone-dependent tumors, such as prostatic and mammary carcinoma. The LHRH antagonists, by virtue of their singular anti-LHRH properties, are presently being evaluated as antifertility agents, and for the above steroid-dependent pathologies.

Cyclic response of hypophysectomized rats to ovulation induced by LHRH agonist: mediation by prostaglandins.

Further confirmation that the LHRH/LHRH agonist-induced ovulation in the hypophysectomized (hypx) rat is due to a direct ovarian effect and not mediated by LH release from residual pituitary tissue or other CNS sites is provided by the persistence of this effect despite concomitant median eminence lesion or passive immunization to LH. Adrenalectomy did not affect the ovulatory activity of the LHRH agonist, D-Trp6-N alpha MeLeu7-DesGly10-Pro9-NHEt-LHRH (Wy-40,972), in the hypx rat. Prior administration of a potent LHRH antagonist blocked ovulation induced in hypx proestrous rats by Wy-40,972 but not by LH-S19. Ovulation can be induced by Wy-40,972 one day earlier (e.g. metestrus) in the intact rat than it can in the hypx rat. Results in the hypx metestrous rat indicate that the ovulatory responsiveness of the intact rat at this stage of the cycle may occur by complementary action of Wy-40,972-stimulated endogenous LH release and a direct ovarian effect of the agonist. Prostaglandins (PG) are involved in the ovulatory mechanism of Wy-40,972 in the hypx proestrous rat as evidenced by the dose-dependent inhibition of this effect by PG synthetase inhibitors, indomethacin and Fentiazac. Moreover, there were significant increases in ovarian concentrations of PGF2 alpha and PGE2-PGE1 in response to Wy-40,972 that could be prevented by indomethacin. However, exogenous administration of either of these PG's was not effective in inducing ovulation in the hypx rat.

Resistance of the mouse to the antifertility effects of LHRH agonists.

In the mouse, the LH-releasing activity of the LHRH agonist, D-Trp6-N alpha-MeLeu7-DesGly10-Pro9-NHEt-LHRH (Wy-40,972), was established by its ability both to induce ovulation when administered at 1600 hours on the the second day of diestrus and to elevate serum LH in adult males. While Wy-40,972 was only slightly less active in terms of these and points than it was in the rat, the predictive and possibly causal association between LH-releasing and antifertility activity established for this LHRH analog in the rat could not be clearly identified in the mouse. A total daily dose of 1000 microgram Wy-40,972/mouse was required to completely inhibit pregnancy during days 1-7 of pregnancy and produced only partial inhibition during days 7-12. This dose represents, on a body weigh basis, 8250 times the 100 percent effective pregnancy-terminating dose for the rat during equivalent intervals. The resistance of the mouse to the antifertility activity of Wy-40,972 was found not to be restricted to this particular LHRH analog or to the reproductive state. Administration of another potent LHRH analog, D-Ala6-DesGly10-Pro9-NHEt-LHRH (Wy-18,481), to adult male mice at a dose of 100 microgram/mouse/day for up to 14 days had no inhibitory effect on the weights of the testes or sex accesory organs. This dose of Wy-18,481 is 7500 times that necessary for significant reduction of these reproductive organ weights in rats within 7 days of treatment. Investigation as to the nature of the mouse's apparently divergent response to the LHRH agonists may further elucidate the antifertility mechanism of such compounds in susceptible species.

Reproductive pharmacology of LHRH and agonists in females and males.

PIP: This report reviews research supporting the anti-reproductive pharmacologic characteristics of LHRH (luteinizing hormone releasing hormone) and its agonist analogues, and their relevance to fertility regulation in the clinic. Approximately 1000 derivatives of LHRH have been synthesized since 1971. LHRH and agonistic derivatives have the ability to induce the release of pituitary LH and FSH (follicle stimulating hormone), and ovulation in a variety of animal models. These agents have been shown to produce predictable postcoital contraceptive effects, such activity and potency having been related to its basic agonist properties. This class of peptides also have the ability to 1) retard puberty; 2) disrupt the estrous cycle (delay onset of estrus and mating); 3) induce premature ovulation; 4) induce luteolysis; 5) cause ovarian and uterine regression; 6) reduce fecundity in inseminated animals; and 7) inhibit ovarian/uterine stimulation which occurs with human chorionic gonadotropin. These effects are reversible because once treatment is withdrawn, normal breeding processes resume quickly. Several LHRH agonists are also being tapped for use as a potential luteal phase-inhibiting/menses-inducing approach to contraception. In the male, however, the agonists cannot function as contraceptives due to the inappropriate dissociation between testosterone production and spermatogenesis. The antireproductive mechanisms of LHRH agonists can be traced to the 1) hypersecretion of LH; 2) dysphasic FSH and distorted prolactin secretion; 3) decrease in gonadal LH, FSH and prolactin receptors; and 4) inhibition of steroidogenesis and eventual disruption of the reproductive continuum. They may also be useful as anti-tumor agents in steroid-dependent mammary gland and prostatic neoplasms. Toxicologic, pathologic and ancillary pharmacologic studies involving varied dosing regimens show encouraging potential of selected agonists as contraceptive agents with no related side effects.^ieng

Luteinising hormone-releasing and anti-fertility properties of a glucagon-selective somatostatin analogue.

The hypothalamic tetradecapeptide, somatostatin (SRIF), inhibits the secretion of growth hormone (GH) and numerous other hormones, including insulin and glucagon. Attempts to use SRIF as an adjunct in the treatment of diabetes mellitus met with limited success due to its short biological half-life and the undesirable diabetogenic activity of its insulin-lowering properties. Efforts at synthesis have yielded SRIF derivatives with prolonged GH-lowering activity which did not suppress glucagon or had equivalent insulin-inhibiting activity as well as several short-acting compounds with the appropriate glucagon specificity. A dodecapeptide analogue [des-Ala, Gly] His-D-Trp-SRIF (Wy-41, 747) has been identified that combines selective inhibition of GH and glucagon release with prolonged activity. However, in routine pharmacological tests chronic treatment of mature rats with Wy-41, 747 produced anti-reproductive effects resembling those described for luteinising hormone (LH)-releasing hormone (RH) and its agonists. We report here that Wy-41, 747, unlike SRIF and other of its analogues tested, releases LH, induces ovulation and inhibits pregnancy when administered before or after implantation; these properties are traditionally associated with the separate LH-releasing class of peptides.

Effects of prolactin on testicular regression and recrudescence in the golden hamster.

Transfer of adult male hamsters from a long to a short photoperiod causes testicular atrophy, which is accompanied by decreases in testicular LH receptors and in plasma PRL and testosterone levels. A decrease in plasma gonadotropin levels is also frequently observed. When the decline in peripheral PRL concentration is prevented by transplantation of homologous pituitary(ies) under the kidney capsule, testicular atrophy is delayed and incomplete. This appears to be due to the maintenance of testicular LH receptors by PRL secreted from the grafts and probably also to the stimulation of GSH release from the in situ pituitary. In hamsters maintained in a long photoperiod, ectopic pituitary homografts can increase testicular LH receptor levels, concentrations of testosterone and FSH in the plasma, and the weights of the testes and the seminal vesicles. In contrast to the findings obtained in rats and mice, chronic hyperprolactinemia in the male hamster does not inhibit gonadotropin release or interfere with copulatory behavior. These findings are consistent with our earlier suggestion that PRL plays an important part in the regulation of gonadal function in the male hamster but indicate that testicular atrophy in a short photoperiod cannot be explained solely by a reduction in PRL release.

Agonist (ovulation induction) and post-coital contraceptive properties of [D-Ala6] and [D-Trp6]-LHRH series.

Seven derivatives of LH-RH, representing the [D-Ala6] or [D-Trp6] series, with or without a Fujino modification, were evaluated for ovulation-inducing (agonist and post-coital contraceptive activity in rats. Six of these analogues had a high degree of agonist and pregnancy-terminating potency. In general, several modifications can result in a particular series of composite molecules that possess a biologic potency greater than each of its predecessors; this correlation of structure with activity was more consistent in the [D-Ala6]-series than in the [D-Trp6]-series. The relationship between structural modifications, resistance to enzyme degradation (based on literature reports) and increased biologic potency is discussed.

Testicular LH binding in the hamster: modification by photoperiod and prolactin.

Suppression of testicular weight and activity induced in the hamster by light deprivation can be partially reversed by treatment with prolactin (PRL). The present study investigates the possibility that the stimulatory effect of PRL in this preparation may be mediated through increased LH binding. Hamsters exposed to 5 h light per day for two months to induce gonadal atrophy were injected daily for 2 1/2 weeks with saline, 250 mug PRLP, 20 MUG LH+150 mug FSH, or PRL+LH+FSH. Short light control animals exhibited significantly less LH binding than controls on 14 h of light per day. Treatment with LH+FSH had no effect on LH binding while PRL alone or in combination with LH+FSH increased binding to levels greater than the long light controls. Peripheral testosterone concentrations reflected the level of LH binding. Scatchard analysis indicates that the decreased binding in the short-day animals is due to reduced LH receptor numbers and that PRL treatment elevates receptor levels thereby increasing LH binding. These results suggest that the mechanism by which PRL stimulates testicular function in hamsters with regressed gonads is through increased binding of endogenously produced LH.

Serum gonadotropins and follicular development in the Syrian hamster.

Hamster serum gonadotropins were measured by RIA at 4 h intervals during the estrous cycle. On the afternoon of proestrus both LH and FSH exhibited a surge, but unlike the situation in the rat and mouse FSH returned to "baseline" with LH by early evening of proestrus. Shortly following this return FSH concentrations increased and reached a second peak by noon on estrus which was equal in magnitude and longer in duration than that occurring on proestrus. FSH fell to its lowest levels on diestrus 2 (D2) and early proestrus. Serum gonadotropins were measured by RIA 6 h following unilateral ovariectomy on D2. A slight elevation of LH resulted while FSH increased to a level equal in magnitude to that found during the proestrous surge. In intact females administration of a total of 45 mug FSH in 5 injections on D2 resulted in ovulation of twice the normal number of eggs. The t1/2 of this rat FSH in the male hamster was found to be 122 minutes. The low levels of FSH during the cycle between D2 and proestrus, the large increase in serum FSH following unilateral ovariectomy, and the "doubled" ovulation in intact hamsters following the administration of FSH on D2, suggest that the serum FSH concentration on D2 is critical in determining the number of follicles which will be available for the subsequent ovulation.