Search

In a previous study, we have demonstrated that infusion of neuropeptide Y (NPY) into the stalk-median eminence (S-ME) of gonadectomized rhesus monkeys stimulated LHRH in a dose-dependent manner. This finding led us to address the following questions: 1) What are the characteristics of NPY release in vivo? 2) How does NPY release relate to LHRH release? 3) Is endogenous NPY essential to pulsatile LHRH release? To answer these questions, three experiments using push-pull perfusion were performed in adult gonadectomized rhesus monkeys. Perfusate samples from the S-ME were collected at 10-min intervals for 6 to 12-h periods, and the concentrations of LHRH and NPY in perfusates were determined by RIA. In Exp I, the release pattern of NPY and LHRH in the S-ME was independently determined in a group of 11 conscious monkeys: NPY release in the S-ME was pulsatile with an interpulse interval of 44.9 +/- 3.3 min (n = 11). This interpulse interval was similar to that seen for LHRH release (43.8 +/- 1.1 min, n = 7). Exp II was designed to determine whether NPY pulses and LHRH pulses occur synchronously and to examine whether NPY release in the S-ME is correlated with circulating LH pulses. NPY and LHRH concentrations in aliquots of the same perfusate sample from the S-ME and circulating LH levels were concurrently measured in 8 monkeys sedated with Saffan. It was found that NPY pulses were temporally correlated (P less than 0.001) with LHRH pulses, which were also temporally correlated (P less than 0.001) with LH pulses. Moreover, NPY pulses were correlated (P less than 0.05) with LH pulses. NPY peaks preceded LHRH peaks by 4.5 +/- 0.6 min, LHRH peaks preceded LH peaks by 5.5 +/- 0.6 min, and NPY peaks preceded LH peaks by 9.7 +/- 0.8 min. In Exp III, the role of endogenous NPY in LHRH release was evaluated by infusing a specific antiserum to NPY into the S-ME during push-pull perfusion in 8 conscious monkeys. Infusion of a specific antiserum to NPY into the S-ME at 1:100 and 1:1000 dilutions suppressed pulsatile LHRH release significantly (P less than 0.05). Infusion of nonimmune serum as a control was without effect. These results are summarized as follows: 1) NPY release in the S-ME is pulsatile, 2) NPY pulses occur synchronously with LHRH and LH pulses, and 3) immunoneutralization of endogenous NPY in the S-ME suppresses pulsatile LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

A variant form of dipeptidyl peptidase II (DPP II), initially reported under the name of "DPP V" (Eisenhauer, D. A., and McDonald, J. K. (1982) Fed. Proc. 41, 507), was detected in aqueous extracts of porcine ovaries on the basis of a markedly enhanced action on prolyl bonds. This porcine form of DPP II, which was most sensitively assayed on Phe-Pro-2-naphthylamide (Phe-Pro-NNap), was purified 1400-fold to a specific activity of 28 mumol/min/mg of protein (pH 6.0, 37 degrees C) from an aqueous extract of hog ovaries taken during pregnancy, when ovarian levels of DPP II are some 3- to 8-fold higher. Purification involved ammonium sulfate fractionation, molecular exclusion chromatography, chromatofocusing, affinity chromatography on concanavalin A-Sepharose 4B, and high performance ion exchange chromatography. The purified enzyme, which was apparently electrophoretically homogeneous at pH 3, 7, and 8.8 (pI = 5.0), was shown to be an Mr = 110,000 glycoprotein containing about 2% carbohydrate (primarily mannose) and less than 1% sialic acid, and to consist of two noncovalently linked Mr = 54,000 subunits. A serine catalytic mechanism was supported by inhibitor studies and by common mobilities seen during electrophoresis for (histochemically detected) Phe-Pro-arylamidase activity and the [14C]diisopropyl fluorophosphate-labeled enzyme. In the standard fluorometric assay at 37 degrees C, Phe-Pro-NNap (0.2 mM) was hydrolyzed optimally at pH 6.0 (Km = 45 microM; kcat = 54 s-1). In comparison to the rate seen on Lys-Ala-NNap, the usual DI P II assay substrate, rates seen on Phe-Pro-, Lys-Pro-, and Arg-Pro-NNap were about 8-, 4-, and 2-fold higher, respectively. No action occurred on N-blocked derivatives or on Pro-NNap. Action on oligopeptides appeared to be limited to tripeptides, in particular those containing proline or alanine in the P1 position, i.e. Phe-Pro-Ala (100%), Lys-Ala-Ala (35%), Ala-Ala-Ala (33%), and Gly-Pro-Ala (26%). Only a trace of activity was seen on Phe-Pro-Ala-Ala, and none on Z-Phe-Pro-Ala or Ala-Ala-Ala-OMe. Evidence for the lysosomal localization of DPP II included sedimentability and latency and its distribution, coincident with acid phosphatase, in two distinct isopycnic regions following equilibrium density centrifugation. Lysosomal heterogeneity was suggested by this dual isopycnic banding.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcitonin gene-related peptide (CGRP) has been found in nerves that innervate the rat ovary. In this study we used indirect immunohistochemical methods to investigate the normal distribution of CGRP-immunoreactive fibers in the prepubertal rat ovary and to determine the route by which these fibers reach the gland. Additional experiments were performed to examine the possible colocalization of CGRP with immunoreactivities of substance P-, neuropeptide Y-, and tyrosine hydroxylase. Potential effects of CGRP on estradiol and progesterone secretion were explored with cultured granulosa cells and short-term incubation of whole ovaries in vitro. In ovaries with intact nerves, CGRP-labeled fibers were observed in dense plexuses surrounding the ovarian vasculature. Additional fibers were found occasionally in the interstitial tissue and in the vicinity of ovarian follicles. Surgical transection of the plexus nerve completely eliminated CGRP immunoreactivity. Section of the superior ovarian nerve or the abdominal vagal trunks had no discernible effect on CGRP labeling. Additional studies revealed coexistence of CGRP and substance P in several axons. Neither tyrosine hydroxylase nor neuropeptide Y-immunoreactivity was co-localized in CGRP-containing fibers. CGRP (10(-10) - 10(-6) M) had no effect on the basal or follicle-stimulating hormone-stimulated release of estradiol or progesterone from ovaries of pregnant mare's serum gonadotropin-treated rats or from cultured granulosa cells from hypophysectomized, diethylstilbestrol-treated rats. We conclude that CGRP-containing nerves enter the ovary via the plexus nerve and are probably involved in the regulation of vasomotor function.

NPY exhibits a broad distribution throughout the body. NPY has been localized in neurons that synthesize norepinephrine or epinephrine and also in many cell bodies which are not catecholaminergic. Coexistence of NPY and several other peptides has also been observed. Accordingly, NPY displays a wide variety of functional activities depending on its location and coexistence with other substances, especially catecholamines. NPY exerts direct effects at several targets and also modulates the cellular response to catecholamines and to peptides such as LHRH. It is reasonable to expect that NPY will modulate the pre- and postjunctional effects of catecholamines at many of their targets in view of the distribution of NPY in central catecholaminergic neurons and throughout the preaortic and sympathetic chain ganglia. Virtually nothing is known at the time of this writing about NPY receptors and postreceptor transduction mechanisms at different sites of NPY activity. Equally mysterious are the pre- and postjunctional receptor-coupled transduction mechanisms which are involved in the modulation of catecholaminergic or other peptidergic effects by NPY. The distribution of NPY and its involvement in cardiovascular, GI, endocrine, and neuroendocrine systems suggest that NPY may be an extremely important regulator of a spectrum of physiological functions.

Relaxin, a peptide hormone responsible for the widening of the birth canal in mammals, has been purified from the ovaries of pregnant hogs. The amino acid sequences of its constituent A and B chains were determined, and the positions of the disulfide cross-links were established. Relaxin was shown to be identical to insulin with respect to its disulfide bond distribution, but significant homology was lacking in other positions. These findings suggest that relaxin and insulin were derived from a common ancestral gene. Since the intrauterine mode of propagation is synonymous with the development of mammals, the genetic distance between insulin and relaxin should therefore permit an estimate of the earliest possible time of commitment of one evolutionary branch to the development of mammals. This event was estimated to have occurred about 5 X 10(8) years ago.