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The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs

D Wu, I J Clarke, and C Chen

Abstract

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P<0·001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP–protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP–PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP–PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP–PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Journal of Endocrinology (1997) 154, 219–230

DOI:
https://doi.org/10.1677/joe.0.1540219
Volume 154: Issue 2,
Pages:
219–230 (12 total)
Restricted access

Signal transduction systems employed by synthetic GH-releasing peptides in somatotrophs

C Chen, D Wu, and I J Clarke

Introduction

Around the time when the endogenous hypothalamic releasing factor for growth hormone (GH) was isolated, a new range of synthetic peptides were also shown to be specific releasors of GH. These peptides were originally developed in the late 1970s by Bowers et al. (1980) who synthesized the enkephalin analogue Tyr-d-Trp-Gly-Phe-Met-NH2 and found that it specifically stimulated GH secretion. Although this pentapeptide was a weak GH secretagogue it was the first non-GH-releasing hormone (GHRH) found to act on the anterior pituitary to specifically release GH and was used as a model to design more potent forms of GH-releasing peptides (GHRPs) (Momany et al. 1981). The most potent of the first generation analogues was GHRP-6 (Bowers et al. 1984, Momany et al. 1984), a hexapeptide which stimulates GH release in a variety of animal species and in man (Bowers et al. 1984, 1991, Wu et al. 1994a). Since then, a range

DOI:
https://doi.org/10.1677/joe.0.1480381
Volume 148: Issue 3,
Pages:
381–386 (6 total)
Free access

Characterization of the concentration gradient of prostaglandin H synthase 2 mRNA throughout the pregnant baboon uterus

WX Wu, GC Smith, J Rose, and PW Nathanielsz

The present study was designed to determine the effect of the spatial gradient from the cervix to the uterine fundus on the control of local prostaglandin H synthase (PGHS) 2 mRNA expression. We performed total cesarean hysterectomies during the last trimester in 12 pregnant baboons, 7 not in labor and 5 in labor, and examined PGHS2 mRNA expression throughout the uterus. PGHS2 mRNA abundance was quantified by in situ hybridization and northern blot analysis in the uterine fundus, lower uterine segment and the different segments of the cervix. Quantitative northern blot and in situ analysis demonstrated a gradient of PGHS2 mRNA expression, with the highest levels at the level of the lower portion of the cervix and decreased expression through the mid- and upper portion of the cervix and lower uterine segment; the lowest levels of expression were seen in the uterine fundus. Moreover, cellular localization of PGHS2 mRNA and protein demonstrated high levels of expression in the cervical glandular epithelial cells with only occasional staining of smooth muscle cells in pregnant baboons. Decreased PGHS2 mRNA concentration gradient from the cervical external os to the fundus suggests that prostaglandin (PG) production in the uterus and cervix strongly depends on anatomical relations. This increased local PG production activity may be critical to pregnancy-associated lower uterine segment elongation, cervical softening and effacement in primate labor. These data provide a compelling biological basis for the use of PGHS2 inhibitors in the prophylaxis of preterm birth and cervical incompetence.

DOI:
https://doi.org/10.1677/joe.0.1820241
Volume 182: Issue 2,
Pages:
241–248 (8 total)
Restricted access

The Effect of GH-Releasing Peptide-2 (GHRP-2 or KP 102) on GH Secretion from Primary Cultured Ovine Pituitary Cells Can be Abolished by a Specific GH-Releasing Factor (GRF) Receptor Antagonist

Danxing Wu, Chen Chen, Kazuo Katoh, Jin Zhang, and Iain J. Clarke

Abstract

A newly synthesised GH-releasing peptide, KP 102 (also named GHRP-2), was studied in an in vitro perifusion system of primary cultured ovine anterior pituitary cells. Application of KP 102 to the perifusion medium caused a dose-dependent increase in GH secretion. Dose-response relationships indicated that KP 102 had similar potency to GRF and was 10-fold more potent than earlier generations of GH-releasing peptide (GHRP-6 and GHRP-1) tested in same system. The response to a second application of KP 102 given within 1 h of initial application was significantly lower than the response to the first application. When KP 102 (or GRF) was applied first and then GRF (or KP 102) given 1 h later, the second response was not attenuated. When GRF and KP 102 were coadministered, an additive effect on release of GH was obtained. The effect of maximal dose of KP 102 (100nM) on GH release was totally abolished by [Ac-Tyr1, d-Arg2] GRF 1-29 (1μM) which is believed to be a specific antagonist for the GRF receptor. Blockade of Ca2+ channels by Cd2+ (2mM) diminished the basal GH secretion and abolished the increase in GH release in response to KP 102 (100nM). These data suggest that the action of KP 102 is blocked by a GRF receptor antagonist and therefore acts through a different receptor to that employed by earlier generations of GH-releasing peptides. GH release in response to KP 102 involves an increase in Ca2+ influx and there is no cross-desensitization between KP 102 and GRF responses.

DOI:
https://doi.org/10.1677/joe.0.140R009
Volume 140: Issue 2,
Pages:
R9–R13
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EFFECTS OF OESTROGEN AND PROGESTERONE ON THE DEVELOPMENT, OVIDUCTAL TRANSPORT AND UTERINE RETENTION OF EGGS IN HYPOPHYSECTOMIZED PREGNANT RATS

J. T. WU, Z. DICKMANN, and D. C. JOHNSON

SUMMARY

Hypophysectomy of rats on day 1 of pregnancy resulted in expulsion of most eggs from the uterus between days 5 and 6, delayed entry of eggs into the uterus, and retarded development of eggs.

Expulsion of eggs was prevented with daily injections of 2 mg progesterone. Other regimes of progesterone administration were also beneficial, but not as effective as daily injections.

Daily injections of 2 or 4 mg progesterone, or of progesterone plus oestrone, restored egg transport to normal, whereas a single injection of progesterone did not. Daily injections of small amounts of oestrone caused total disappearance of eggs from the reproductive tract.

The development of eggs was significantly improved with daily injections of progesterone plus oestrone, but not with progesterone alone.

Combined treatment with progesterone and oestrone caused premature shedding of the zona pellucida in mature blastocysts, young blastocysts, and morulae.

DOI:
https://doi.org/10.1677/joe.0.0510569
Volume 51: Issue 3,
Pages:
569–574 (6 total)
Restricted access

EFFECTS OF OVARIECTOMY OR HYPOPHYSECTOMY ON DAY ONE OF PREGNANCY ON DEVELOPMENT AND TRANSPORT OF FERTILIZED RAT EGGS

J. T. WU, Z. DICKMANN, and D. C. JOHNSON

SUMMARY

The development of fertilized eggs and their transport through the oviduct were studied in rats ovariectomized or hypophysectomized on day 1 of pregnancy. When killed on day 5 of pregnancy, 100%, 66% and 27% of the eggs were in the uterus of intact controls, ovariectomized and hypophysectomized rats, respectively. Thus both ovariectomy and hypophysectomy retarded transport of a proportion of the eggs. Ninety-nine per cent, 72% and 78% of the eggs were in the blastocyst stage in intact controls, ovariectomized and hypophysectomized rats. The remainder of the eggs were either morulae or degenerated.

Blastocysts and morulae recovered from the hypophysectomized rats were transferred to the uteri of pseudopregnant recipients: 52–57% developed into normal term foetuses compared to 57% for intact controls.

In hypophysectomized rats killed in the morning of day 6, the number of eggs recovered was greatly reduced as compared with rats killed on day 5. This loss of eggs was prevented by placing a ligature at the cervical end of the uterus in the morning of day 5. It was therefore concluded that the eggs had been expelled from the uterus into the vagina between days 5 and 6.

DOI:
https://doi.org/10.1677/joe.0.0490507
Volume 49: Issue 3,
Pages:
507–513 (7 total)
Free access

Expression of a novel factor, short-type PB-cadherin, in Sertoli cells and spermatogenic stem cells of the neonatal rat testis

J Wu, Jester WF Jr, AL Laslett, A Meinhardt, and JM Orth

In the rodent testis, contact-mediated interactions between gonocytes, or neonatal stem cells, and Sertoli cells are critical for development. Previously, we showed that the neural cell adhesion molecule (NCAM) serves as a Sertoli cell-gonocyte attachment factor in neonates. Its expression decreases dramatically by 1 week of age and eventually disappears in vivo, and appears to be down-regulated by thyroid hormone (tri-iodothyronine (T(3))). In this study, we used a cDNA microarray to screen for additional adhesion factors which might be important in testes of developing rats and detected expression of a novel factor, short-type PB-cadherin (STPB-C). Next, RT-PCR was used to generate cDNA for STPB-C from total RNA isolated from co-cultures, cDNA was cloned into pPCR-Script Amp SK(+) cloning vector, and plasmid DNA was isolated and sequenced to confirm the fidelity of the STPB-C cDNA portion of the plasmid. In situ hybridization analyses of testicular sections indicated that STPB-C expression in neonates is localized in the cytoplasm of many, but not all, gonocytes and in the cytoplasm of most of the surrounding Sertoli cells. Parallel hybridizations carried out on co-cultures also demonstrated a strong cytoplasmic signal in some gonocytes and in the great majority of the Sertoli cells of the underlying monolayer. With Northern analyses we found that STPB-C is expressed in vivo at high levels between days 1 and 5, with a subsequent large drop by day 10 and thereafter, suggesting that its expression may be associated with Sertoli or germ cell differentiation. Subsequent analyses of co-cultures exposed under a variety of conditions to T(3) suggest that, unlike NCAM, STPB-C is not regulated by this hormone. Next, we studied production of STPB-C protein by using an antiserum recognizing a peptide sequence unique to this factor in Western blotting and in immunolocalization. Signal was detected both intracellularly and at cell surfaces in most Sertoli cells and many gonocytes, although many of the latter cell type were also found to be negative for the protein, suggesting a potential role for STPB-C in survival and further development of some of these germ cells from which all subsequent spermatogenic cells originate.

DOI:
https://doi.org/10.1677/joe.0.1760381
Volume 176: Issue 3,
Pages:
381–391 (11 total)
Restricted access

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3′,5′-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs

D Wu, C Chen, J Zhang, C Y Bowers, and I J Clarke

Abstract

The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,d-Arg2]-GRF 1–29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep.

Journal of Endocrinology (1996) 148, 197–205

DOI:
https://doi.org/10.1677/joe.0.1480197
Volume 148: Issue 2,
Pages:
197–205 (9 total)
Free access

Intrauterine growth restriction alters term fetal baboon hypothalamic appetitive peptide balance

Cun Li, Thomas J McDonald, Guoyao Wu, Mark J Nijland, and Peter W Nathanielsz

Neurons controlling appetite are located in the hypothalamic arcuate nuclei (ARH). Offspring appetite regulation has been shown to be modified by dysregulation of ARH nuclear development. Most ARH developmental studies have been in altricial rodents whose hypothalamic development is predominantly postnatal. In primates including humans, much development of hypothalamic appetite regulatory centers occurs before birth. We hypothesized that i) appetitive peptides are abundantly expressed by 90 percent gestation (0.9G), ready for postnatal function; ii) by 0.9G, intrauterine growth restriction (IUGR) increases the orexigenic:anorexigenic peptide ratio; iii) IUGR increases fetal glucocorticoid receptor (GR) expression; and iv) IUGR decreases STAT3, which signals inhibition of appetite. We developed a fetal baboon IUGR model resulting from reduced maternal nutrition. Pregnant baboons were fed ad libitum, controls (CTR; n=24), or 70% CTR diet to produce IUGR (n=14). C-section was performed at 0.9G. In CTR (n=7) and IUGR (n=6) fetal brains, ARH appetite regulatory peptides (neuropeptide Y (NPY) and proopiomelanocortin (POMC)) were quantified immunohistochemically. Fetal plasma cortisol was raised in IUGR fetuses. We observed that NPY and POMC were well expressed by 0.9G. IUGR increased NPY, GR, and active phosphorylated GR and decreased POMC and phosphorylated form of STAT3. We conclude that IUGR dysregulates ARH development in ways that will reset the appetitive neuropeptide balance in favor of increased appetite drive in postnatal life. We postulate that changes in peptide abundance are in part due to increased fetal cortisol and ARH GR. These changes may contribute to predisposition to obesity in IUGR offspring.

DOI:
https://doi.org/10.1530/JOE-13-0012
Volume 217: Issue 3,
Pages:
275–282 (8 total)
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Isolation of FSH from bovine pituitary glands

J.-B. Wu, P. G. Stanton, D. M. Robertson, and M. T. W. Hearn

ABSTRACT

An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5·7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay.

SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19·5 kDa and 15·8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with α- and β-subunits, and a third minor (< 20%) sequence consistent with the α-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators.

Journal of Endocrinology (1993) 137, 59–68

DOI:
https://doi.org/10.1677/joe.0.1370059
Volume 137: Issue 1,
Pages:
59–68 (10 total)