The extracellular Ca++ -independent component of luteinzing hormone (LH) secretion is manifest under the steroidal conditions known to induce gonadotrophine-releasing hormone (GnRH) self-potentiation

Quartered pituitaries obtained from intact males or gonadectomized males and females (72h) ± estradiol (24h) and/or testosterone (72h) implants were perifused at 0.25 ml/minute with Ca++ -free medium at 37°C, and sequential effluent fractions collected every 10 minutes, in an attempt to determine if the steroidal conditions known to induce or prevent GnRH self-potentiation would activate or inhibit the extracellular Ca++ -independent component of luteinizing hormone (LH) secretion. Continuous infusions (4h) of 1nmol GnRH or 1µmol of phorbol 12-myristate 13-acetate (PMA) did not stimulate LH secretion from the pituitaries of castrated males, intact males, estradiol-treated intact males or ovariectomized females. In contrast, estradiol induced delayed (20-30 minutes), protein synthesis-dependent components of LH secretion in response to both GnRH and PMA from pituitaries of gonadectomized males and females. Implantation of testosterone capsules immediately following gonadectomy resulted in an inhibition of the estradiol- induced GnRH- and PMA-stimulated responses from pituitaries of castrated or ovariectomized animals. These results suggest that estradiol can induce extracellular Ca++ -independent components of LH secretion from pituitaries of gonadectomized animals; responses which depend on de novo protein synthesis and which could involve protein kinase C. Additionally, the effects of estradiol are prevented by testosterone, indicating that this component of LH secretion is only apparent under the steroidal conditions known to facilitate GnRH self-potentiation.

The extracellular Ca++ -independent component of luteinzing hormone (LH) secretion is manifest under the steroidal conditions known to induce gonadotrophine-releasing hormone (GnRH) self-potentiation

Quartered pituitaries obtained from intact males or gonadectomized males and females (72h) ± estradiol (24h) and/or testosterone (72h) implants were perifused at 0.25 ml/minute with Ca++ -free medium at 37°C, and sequential effluent fractions collected every 10 minutes, in an attempt to determine if the steroidal conditions known to induce or prevent GnRH self-potentiation would activate or inhibit the extracellular Ca++ -independent component of luteinizing hormone (LH) secretion. Continuous infusions (4h) of 1nmol GnRH or 1µmol of phorbol 12-myristate 13-acetate (PMA) did not stimulate LH secretion from the pituitaries of castrated males, intact males, estradiol-treated intact males or ovariectomized females. In contrast, estradiol induced delayed (20-30 minutes), protein synthesis-dependent components of LH secretion in response to both GnRH and PMA from pituitaries of gonadectomized males and females. Implantation of testosterone capsules immediately following gonadectomy resulted in an inhibition of the estradiol- induced GnRH- and PMA-stimulated responses from pituitaries of castrated or ovariectomized animals. These results suggest that estradiol can induce extracellular Ca++ -independent components of LH secretion from pituitaries of gonadectomized animals; responses which depend on de novo protein synthesis and which could involve protein kinase C. Additionally, the effects of estradiol are prevented by testosterone, indicating that this component of LH secretion is only apparent under the steroidal conditions known to facilitate GnRH self-potentiation.

Adenosine 3',5'-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone.

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 mumol/l cycloheximide. Forskolin (1 mumol/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.

Calcium mobilization is a prerequisite for the expression of phorbol ester-stimulated luteinizing hormone secretion from pituitaries of male and acutely ovariectomized rats.

Ionomycin, which mobilizes Ca2+, and phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), were used to compare the effects/interactions of Ca2+ and PKC on luteinizing hormone (LH) secretion from pituitaries of intact male and acutely ovariectomized (72 h) rats. Quartered pituitaries from donor animals were perifused at 0.25 ml/min and sequential effluent fractions were collected every 10 min. Continuous administration (4 h) of 1 nmol of gonadotropin-releasing hormone (GnRH) resulted in an increase in LH secretion. Cycloheximide (5 mumol) dissociated the GnRH-stimulated LH responses into protein synthesis-independent and -dependent components. While ionomycin (10 mumol) stimulated LH secretion from pituitaries of both sexes by protein synthesis-independent mechanisms, PMA (1 mumol) and the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (PDD), were ineffective secretagogues. However, PMA (but not PDD) interacted synergistically with ionomycin and GnRH to augment LH secretion by protein synthesis-dependent mechanisms. These results suggest a similarity in the effects/interactions of Ca2+ and PKC in gonadotropes from male and ovariectomized rats. If the effects of PMA can be attributed to PKC activation, then it also appears that Ca2+ mobilization is necessary for the manifestation of PKC as a mediator of LH secretion from these gonadotropes. While PKC activity can be divorced from the protein synthesis-independent component of LH release (this component appears to be mediated by Ca2+ mobilization), the enzyme might be involved in amplifying the response to Ca2+ mobilization through synergistic protein synthesis-dependent mechanisms.

Estradiol modulation of PMA--and ionomycin-stimulated LH secretion from pituitaries of castrated rats.

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 microM), and was necessary for the manifestation of PMA-stimulated (1 microM) LH secretion. Cycloheximide (5 microM) 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.

Forskolin but not cholera toxin bypasses flufenamate-induced inhibition of cAMP production in anterior pituitaries.

The pharmacological activators of adenylyl cyclase (cholera toxin and forskolin) were utilized in the present study to determine whether they could bypass the inhibitory effects of flufenamate on cAMP production in rat hemipituitaries. During 2 hr incubations, 10 microM flufenamate inhibited gonadotropin-releasing hormone (GnRH)-stimulated (25 nM) cAMP production. Flufenamate did not affect GnRH-receptor interactions as evidenced by its inability to significantly affect either the binding affinity or the binding capacity for GnRH. Additionally, flufenamate inhibited the cholera toxin-stimulated cAMP production, but was ineffective against forskolin-induced activation of adenylyl cyclase. These results indicate that forskolin can be used to restore cAMP production in the presence of flufenamate. Since GnRH and cholera toxin stimulate cAMP production via the GnRH receptor and the Gs protein respectively, and forskolin exerts its stimulatory effects via the catalytic component, the data are consistent with the possibility that flufenamate exerts its inhibitory effect at the level of the Gs protein.

The use of flufenamate and forskolin to evaluate the role of cAMP in gonadotropin-releasing hormone-stimulated luteinizing hormone secretion from pituitaries of ovariectomized rats.

Flufenamate which inhibited gonadotropin-releasing hormone (GnRH)-stimulated cAMP production in pituitaries from ovariectomized (72 hr) rats, was used to determine whether ovariectomy induces a change in the role of cAMP as a mediator of luteinizing hormone (LH) secretion. Additionally, the study evaluated the practicability of utilizing forskolin to restore intracellular cAMP concentrations in the presence of flufenamate. Infusions of flufenamate to perifused pituitary tissue blocks did not affect the protein synthesis-independent component of GnRH-stimulated LH secretion, but completely inhibited the protein synthesis-dependent component. Dibutyryl cAMP (dbcAMP) and forskolin potentiated the GnRH-stimulated responses, and restored the LH secretion inhibited by flufenamate, even though these agents were ineffective secretagogues when administered singly. The LH responses affected by forskolin were dependent on protein synthesis, while dbcAMP affected both the protein synthesis-dependent and -independent components of GnRH-stimulated LH secretion. Since the effects of dbcAMP on the protein synthesis-independent component of LH secretion might be due to interactions with GnRH receptors, the results suggest that forskolin might be a better choice for restoring intracellular cAMP levels in the presence of flufenamate when assessing the role of cAMP in gonadotropes. The study also indicates that ovariectomy does not result in a change in the role of cAMP, which appears to be a pivotal, but indirect mediator of the protein synthesis-dependent component of GnRH-stimulated LH secretion.

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

Ionomycin, 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 degrees 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. Ionomycin-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.

Dependency of phorbol ester-induced gonadotropin secretion on estradiol.

Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) was used to investigate the estradiol dependency of PKC-stimulated luteinizing hormone (LH) secretion from perifused anterior pituitaries. Infusions of PMA stimulated LH secretion from diestrous II, ovariectomized + estradiol-treated, and orchidectomized + estradiol-treated quartered pituitaries, by protein synthesis-dependent mechanisms. In contrast, pituitaries from intact, orchidectomized males, or ovariectomized females were unresponsive to PMA. Interestingly, dispersed male pituitary cells differed from male pituitary tissue blocks, in that the dispersed cells responded to PMA with increased LH secretion. These results indicate that PKC's ability to directly stimulate LH secretion is dependent on de novo protein synthesis and estradiol. Moreover, the effects of estradiol on PKC-stimulated secretion form at least one basis for the estradiol-induced increased responsiveness of gonadotrophs to GnRH. Additionally, it appears that dispersed pituitary cells may not respond to activators of PKC in a physiological manner.

The phorbol ester-induced extracellular Ca2+-independent release of LH is dependent on estradiol and de novo protein synthesis.

Phorbol 12-myristate 13-acetate (PMA) was used to determine whether the PMA-induced extracellular Ca2+-independent release of LH was dependent on sex, estradiol and de novo protein synthesis. Infusions of gonadotropin-releasing hormone (GnRH) or PMA in a perifusion system stimulated a partial secretion of LH from diestrous II and ovariectomized + estradiol-treated female pituitaries (responses inhibited by cycloheximide). In contrast, PMA was ineffective in stimulating PRL secretion from these pituitaries, as well as LH secretion from male or ovariectomized female pituitaries. These results indicate that the PMA-stimulated extracellular Ca2+-independent secretion of LH is a specific process which is dependent on sex, estradiol and de novo protein synthesis, and mimics the characteristics of the GnRH-stimulated responses.

Sex differences in the extracellular Ca2+-independent release of LH and FSH.

The present study was undertaken to investigate the effects of sex and estrous cycle on the manifestation of the extracellular Ca2+-independent component of gonadotropin secretion. Quartered pituitaries from male, ovariectomized (OVX) females +/- estradiol (E2) implants, and mature females at each stage of the estrous cycle were perifused with Ca2+-free medium. Gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from male and OVX pituitaries was inhibited in Ca2+-free medium. In contrast, only a partial inhibition was obtained from OVX + E2 or regularly cycling female pituitaries. This extracellular Ca2+-independent component of gonadotropin secretion was lowest at estrus and increased progressively during the estrous cycle. Estradiol replacement in OVX animals resulted in a response similar to that obtained on proestrus. These results indicate that the extracellular Ca2+-independent component of LH and FSH release is only manifest from intact female and not male pituitaries, and is dependent on estradiol.

Cyclic AMP indirectly mediates the extracellular Ca2+-independent release of LH.

This study was undertaken to determine whether cyclic AMP mediates the extracellular Ca2+-independent component of luteinizing hormone (LH) release. Gonadotropin releasing hormone (GnRH) increased cAMP production in female pituitaries incubated in Ca2+-free medium containing 3-isobutyl-1-methylxanthine (IBMX), but was ineffective in male pituitaries. The increases in female pituitaries were inhibited by flufenamate (flu). GnRH-stimulated LH secretion from male pituitaries was completely inhibited in Ca2+-free medium, whereas only a partial inhibition was obtained from female pituitaries, a response prevented by cycloheximide. Infusions of flu completely inhibited the extracellular Ca2+-independent release of LH, while dibutyryl cAMP (dbcAMP) partially restored this component of LH secretion. The dbcAMP-restored response was dependent upon protein synthesis. These results suggest that (i) the extracellular Ca2+-independent component of LH release is indirectly mediated by cAMP through the stimulation of de novo protein synthesis, and (ii) extracellular Ca2+ is required for the activation of adenylate cyclase in male but not in female gonadotrophs.

Evidence for cAMP as a mediator of gonadotropin secretion from female pituitaries.

Sodium flufenamate, which inhibited gonadotropin-releasing hormone (GnRH)-stimulated increases in adenosine 3',5'-cyclic monophosphate (cAMP), was used to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion. Quartered pituitaries from diestrous II female rats were perifused at 37 degrees C, and sequential effluent fractions were collected every 10 min. Administration of GnRH resulted in a characteristic biphasic response for both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas 5 microM cycloheximide inhibited the secondary augmented responses (phase II) of both hormones. Infusions of 0.1 mM flufenamate inhibited GnRH-stimulated gonadotropin secretion in a manner similar to that of cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate resulted in the restoration of LH and FSH secretion. The dibutyryl cAMP-restored response appeared to be protein synthesis dependent and specific for cAMP. These results suggest that although the cyclic nucleotide is not involved in the acute release of LH and FSH, it does appear to play a pivotal but indirect role in phase II release of the hormones, by effects involving the stimulation of de novo protein synthesis.

Evidence for cAMP as a mediator of gonadotropin secretion from male pituitaries.

The purpose of this study was to use sodium flufenamate, a compound that inhibits gonadotropin-releasing hormone (GnRH)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in the pituitary, to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion from male pituitaries. Quartered male pituitaries were perifused at 37 degrees C and sequential effluent fractions collected every 10 min. Infusions of GnRH resulted in a twofold increase in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Cycloheximide, 5 microM, completely inhibited the GnRH-stimulated LH and FSH secretion. Infusions of 0.1 mM flufenamate had similar effects on gonadotropin secretion as cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate restored the secretory responses of both hormones. The flufenamate-inhibited GnRH stimulated LH and FSH release, which was restored by DBcAMP and appeared to be protein synthesis dependent and specific for cAMP. These results suggest an indirect role for cAMP as a mediator of gonadotropin secretion from male pituitaries. However, in contrast to female pituitaries, the secretion of these hormones from male pituitaries is completely dependent on cAMP and de novo protein synthesis.

Pituitary LH responsiveness to GnRH in vitro as related to GnRH receptor number.

The objective of this in vitro study was to determine whether the increase in the augmented phase of the biphasic luteinizing hormone (LH) response to gonadotrophin-releasing hormone (GnRH) and its enhancement by estradiol (E2) were associated with GnRH-stimulated increases in pituitary GnRH receptor concentration. Pituitary glands from 72 h ovariectomized (OVX), OVX + E2, or proestrous rats were perifused continuously with GnRH (12 ng/h). LH release was measured at 10-min intervals, and pituitary GnRH-binding capacity (GnRH-BC) was assessed at 0, 40, 80, 120, and 240 min after addition of GnRH. All treatment groups exhibited a biphasic pattern of LH release; initial (20-70 min) and augmented (120-240 min) mean rates of LH secretion (micrograms/h) were 1.78 and 3.92 (OVX), 6.40 and 16.67 (OVX + E2), and 2.79 and 18.64 (proestrus), respectively. Total LH release was significantly greater in the OVX + E2 and proestrous groups (44.0 and 45.8 micrograms) vs. the OVX group (12.4 micrograms). Throughout the GnRH infusion period, GnRH-BC did not change significantly in any of the treatment groups with the exception of the OVX group in which there was a transient small decrease at 80 min post-GnRH infusion. There were no significant differences between treatment groups in GnRH-BC at any time after infusion of GnRH. These results demonstrate that the acute and augmented phases of GnRH-stimulated LH release and the enhancement of this biphasic response by E2 occurs independent of any increase in GnRH-BC and suggest that these events are mediated by postreceptor mechanisms.

Anterior pituitary hormonal regulation of testicular gonadotropin-releasing hormone receptors.

Testicular GnRH receptors are increased 2-fold 1 day after hypophysectomy and remain elevated for up to 6 days. Consequently, the present study was undertaken to determine which pituitary hormone(s) regulated testicular GnRH receptors. Adult male rats were hypophysectomized and injected sc every 8 h for 2 days with LH, FSH, GH, PRL, estradiol, testosterone, 5 alpha-dihydrotestosterone, and vehicle (controls), beginning within 5 h of surgery. The animals were decapitated on the third day after hypophysectomy, the testes were excised, and interstitial tissue was teased from seminiferous tubules before storage at -70 C until assayed. The analog D-Ala6-des-Gly10-GnRH ethylamide was used to assess GnRH receptors on 10,800 X g-membrane fractions of interstitial tissue. The administration of GH, PRL, and FSH at concentrations which maintained LH receptors in adult or immature hypophysectomized rats did not prevent the increase in GnRH receptors, whereas LH replacement prevented the rise in GnRH analog binding in a dose related manner. LH also reduced preexisting posthypophysectomy increases in GnRH receptor concentrations. Injections of estradiol (5 micrograms/day) partially inhibited the posthypophysectomy increase in GnRH receptors, whereas the androgens 5 alpha-dihydrotestosterone and testosterone were ineffective. These results indicate that LH can regulate testicular GnRH receptors. Since GnRH directly inhibits testosterone secretion, inhibition of testicular GnRH receptors by LH may be one of the mechanisms by which LH replacement enhances testosterone production after hypophysectomy.

Testicular GnRH receptors--characterization and localization on interstitial tissue.

Testicular GnRH membrane receptors were demonstrated using the non-degradable GnRH analog D-Ala6des-Gly10 GnRH ethylamide (D-Ala6) as ligand. Displaceable 125I D-Ala6 binding was present on crude membranes prepared from whole testes and interstitial tissue but not on the fractions from seminiferous tubules. 125I D-Ala6 binding to interstitial tissue was specific as only unlabeled D-Ala6 analog and synthetic GnRH inhibited binding of D-Ala6 tracer. Scatchard analysis of the analog data revealed a single high affinity binding site (Ka = 7 x 10(9) M-1) with a binding capacity of 200 +/- 10 (SE) fmol/mg membrane protein. In vivo treatment of both intact and hypophysectomized adult male rats with synthetic GnRH (6.6 microgram every 8 hr for 3 days) resulted in a 2-fold increase in GnRH binding capacity without change in receptor affinity. These results indicate that specific high affinity GnRH receptors are present only on interstitial tissue membrane fractions and receptor numbers are increased by a direct action of GnRH on the testis.