Calmodulin and protein kinase C activation duplicates the biphasic secretion of luteinizing hormone

doi:10.1016/0303-7207(89)90042-7

Molecular and Cellular Endocrinology

Volume 66, Issue 1, September 1989, Pages 1-8

https://doi.org/10.1016/0303-7207(89)90042-7 Get rights and content

Abstract

lonomycin, which activates the Ca²⁺-calmodulin system, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to investigate potential roles of these systems as mediators of the biphasic secretion of luteinizing hormone. Quartered pituitaries from diestrous II female rats were perifused at 37° C, and sequential effluent fractions collected every 10 min. Gonadotropin-releasing hormone administration resulted in a biphasic response: an initial, protein synthesis-independent secretion, followed 60 min later by a secondary, augmented, protein synthesis-dependent component, lonomycin-stimulated gonadotropin secretion was immediate and partially independent of protein synthesis, whereas the PMA-induced secretion was delayed (approximately 70 min), and was completely dependent on protein synthesis. Simultaneous infusions of ionomycin and PMA resulted in an initial, protein synthesis-independent response followed by the secondary, augmented, protein synthesis-dependent component, which exhibited synergistic interactions between calmodulin and PKC. These results suggest that calmodulin mediates the initial, protein synthesis-independent secretion, PKC mediates part of the secondary, augmented response, while calmodulin and PKC synergize to mediate the remaining component of the secondary response.

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Adenosine 3′,5′-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone
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An in vitro perifusion system was used to ascertain the role of cAMP in the genesis of the self-priming effect of gonadotrophin-releasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 μmol/l cycloheximide. Forskolin (1 μmol/l) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial 10-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskolin + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in cAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on cAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that cAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon.
Effects of administration of oxytocin in association with gonadotropin-releasing hormone on luteinizing hormone levels in rats in vivo
1995, Peptides
Gonadotropin-releasing hormone (GnRH) and oxytocin both stimulate the secretion of luteinizing hormone (LH), although with different characteristics. Therefore, interaction between oxytocin and GnRH in the control of LH may be postulated. We developed models for investigating whether oxytocin can modulate GnRH action on LH in vivo. Pentobarbitone is known to pharmacologically isolate the pituitary from hypothalamic GnRH. We found that after pentobarbitone anesthesia of female rats at proestrus, oxytocin caused a synergistically enhanced LH response to administered GnRH (p < 0.04). In a second series of experiments, female proestrous rats were anesthetized with althesin. This anesthetic allows transport of endogenous GnRH from the hypothalamus to the pituitary. In control animals, which received no exogenous hormone, there was a surge in the mean LH concentration on the evening of proestrus, indicating the presence of endogenous GnRH activity. Thus, the novel model enables detection of interactions of administered hormones with endogenous GnRH. Administration of GnRH plus oxytocin in the afternoon of proestrus caused a reduction (p < 0.01) in the mean level of LH observed in the evening. The reduction was larger than if GnRH alone was administered. Following althesin anesthesia, rats sometimes had low LH levels on the afternoon of proestrus. There was a statistically significant difference between the number of rats that received oxytocin plus GnRH and had low LH levels and the number with low LH levels in the control group (p < 0.02). Neither of the hormones administered alone had a significant effect. Thus, it appears that oxytocin accentuated the effect of GnRH in reducing LH concentrations in althesin-anesthetized rats. If althesin anesthesia was embloyed one estrous cycle (4 days) before hormone administration, there was no difference in the evening LH surge observed in control and experimental groups. However, oxytocin synergistically enhanced the acute LH response to exogenous GnRH (p < 0.03). Our results demonstrated interaction of oxytocin with GnRH in vivo, revealing potential for complex modulation by oxytocin of GnRH control of LH.
The involvement of dihydropyridine-sensitive calcium channels in phorbol ester-induced luteinizing hormone and growth hormone release
1993, Molecular and Cellular Endocrinology
We examined the role of voltage-activated, L-type, Ca²⁺ channels in phorbol ester-induced luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue. The L-type Ca²⁺ channel inhibitor, nimodipine (NMD), inhibited phorbol 12,13-dibutyrate (PDBu)-induced GH release but had no significant effect on LH release. The L-type Ca²⁺ channel activator BAY K 8644 had no effect on PDBu-induced GH release but potentiated PDBu-induced LH release. In contrast, 60 mM K⁺-induced LH and GH release were inhibited by NMD, whereas BAY K 8644 had no effect. When PDBu and either K⁺ or BAY K 8644 were used together, they acted synergistically to evoke levels of LH release greater than addition of release caused by each secretagogue alone. However, the release of GH was additive with PDBu and either K⁺, BAY K 8644.
The protein kinase C (PKC) inhibitor staurosporine inhibited both PDBu-induced LH release and GH release. A structurally different PKC inhibitor, H7, significantly inhibited PDBu-induced LH release but had no effect on PDBu-induced GH release. Both staurosporine and H7 inhibited LH release induced by PDBu and BAY K 8644 together. In contrast, although staurosporine inhibited GH release induced by PDBu and BAY K 8644, H7 significantly potentiated this response. A difference in the action of these two inhibitors was also apparent on K⁺-induced hormone release where staurosporine partially blocked K⁺-induced LH and GH release but H7 had no effect on the release of either hormone.
Data obtained in ⁴⁵Ca²⁺ influx experiments further suggested that a staurosporine-sensitive, but H7-resistant, PKC-like kinase may tonically maintain L-channels in a voltage-sensitive state, as down-regulation of PKC in dispersed anterior pituitary cells by long term PDBu treatment caused a significant reduction in K⁺-induced ⁴⁵Ca²⁺ influx. We conclude that phorbol ester-induced GH release, but not LH release, is a result of L-type Ca²⁺ channel activation which may occur by means of alterations in the channel itself to increase its responsiveness to a given depolarisation.
Estradiol modulation of PMA - and ionomycin-stimulated LH secretion from pituitaries of castrated rats
1993, Cell Calcium
Quartered pituitaries from castrated (72 h) ± estradiol (E₂)-treated (24 h) rats were used in a perifusion system to investigate estradiol modulation of ionomycin and ionomycin + PMA stimulated LH secretion. Estradiol enhanced the LH responses to GnRH (1 nM) and ionomycin (10 μM), and was necessary for the manifestation of PMA-stimulated (1 μM) LH secretion. Cycloheximide (5 μM) inhibited the E₂-enhanced responses to GnRH, ionomycin and PMA. The protein synthesis inhibitor also partially suppressed the GnRH response from pituitaries of castrates, but was totally ineffective against the ionomycin-induced LH secretion. Protein synthesis-dependent, synergistic interactions between PMA and ionomycin were evident from pituitaries of castrates (even though PMA alone was an ineffective secretagogue). Synergistic interactions were not apparent from pituitaries of castrated + E₂-treated rats. These results indicate that: (i) estradiol enhances the responsiveness of male gonadotropes to ionomycin and PMA by protein synthesis-dependent mechanisms which appear to mask their synergistic interactions; and (ii) increases in cytoplasmic Ca²⁺ might be a prerequisite for an expression of the involvement of PKC as a mediator of LH secretion in the absence of high concentrations of estradiol.
The priming effect of luteinizing hormone-releasing hormone (LHRH) but not LHRH-induced gonadotropin release, can be prevented by certain protein kinase C inhibitors
1992, Molecular and Cellular Endocrinology
The priming effect of LHRH in vitro (which results in increased responsiveness of gonadotropes to both LHRH receptor-mediated and receptor-independent stimuli) is brought about by an unknown mechanism. The present results indicate that induction of the LHRH priming effect is inhibited in a concentration-dependent manner by the protein kinase C (PKC) inhibitors Staurosporine, K252a, H7 and by the novel highly-selective PKC inhibitor, Ro 31-8220. In contrast, a range of other compounds that are relatively selective inhibitors of other kinases such as tyrosine kinases and Ca²⁺/calmodulin-dependent kinases were unable to prevent priming. The PKC inhibitors prevented priming without affecting initial LHRH-induced gonadotropin secretion. Thus, the priming-elicited increment in secretion was selectively removed, restoring hormone release to the level measured during an initial response to LHRH. Similar results were obtained on different days of the estrous cycle where the magnitude of the priming effect varies. Experiments on the time course of PKC inhibitor action revealed that the critical period was in the induction of the priming effect, not its expression. The PKC inhibitors had neither acute nor delayed effects on gonadotropin secretion induced by ionomycin. Staurosporine, K252a and Ro 31-8220 inhibited LHRH priming with identical potencies to their inhibition of phorbol ester-induced gonadotropin secretion. The reduced potency of H7 seen on LHRH priming compared to phorbol ester-induced gonadotropin release parallels results seen with this inhibitor on phorbol ester-induced secretion of growth hormone (Johnson and Mitchell (1989) Biochem. Soc. Trans. 17, 751–752) and on the pharmacological characteristics of PKCs partially purified from anterior pituitary tissue. In all aspects of this study, effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion appeared to be entirely similar.
The Secretory Granule and the Mechanism of Stimulus-Secretion Coupling
1992, Current Topics in Cellular Regulation
Secretion is a fundamental cellular process. For a granule-bearing secretory cell, the stimulation of its specific surface receptors will trigger a chain of events culminating in the exteriorization of the secretory granule and its contents. This process, also known as exocytosis, can assume a regulatory and/or a communicative role depending on the mediators involved. As the initiation of a secretory process is also linked to the synthesis of a wide range of primary and secondary messenger molecules, the understanding of this process is paramount to the understanding of the mechanism of cellular regulation. This chapter describes the mechanism of stimulus–secretion coupling. The examination of the literature in this field reveals a concentration of research efforts aimed at understanding the mechanism of signal transduction. For a secretory cell, such effort may lead to the understanding of how a signal resulting from the stimulation of cell surface receptors can be transmitted across the plasma membrane and traverse the cytosol to cause the activation and the exteriorization of a secretory granule and its contents. The literature on the mechanism of signal transduction has implicated many phospholipid-related events and a plethora of phospholipid-derived metabolites in the mechanism of transmembrane signaling.

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^☆: This work was supported by grants from the Alberta Heritage Foundation for Medical Research and the Canadian Medical Research Council, MA-9600. A preliminary report was presented at the Annual Meeting of the Society for the Study of Reproduction, Seattle, WA U.S.A., August 1988.

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Research PaperCalmodulin and protein kinase C activation duplicates the biphasic secretion of luteinizing hormone☆

Abstract

Mol. Cell. Endocrinol.

Mol. Cell. Endocrinol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Methods Enzymol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Endocrinology

Nature

Endocrinology

Endocrinology

Am. J. Physiol.

FEBS Lett

Research Paper
Calmodulin and protein kinase C activation duplicates the biphasic secretion of luteinizing hormone☆