A study on the prolactin releasing mechanism using an in vitro perfusion system with a cell column of cultured rat anterior pituitary cells

It is well-known that the hypothalamus predominantly exerts an inhibitory control on prolactin secretion and that dopamine (DA) is the main prolactin inhibiting factor (PIF). In addition, the hypothalamus contains prolactin-releasing factors (PRF). Thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) and peptide-histidine-isoleucine (PHI) are the components of PRF. However, the detailed mechanism by which the peptides release prolactin (PRL) at the pituitary level is still unknown. Therefore, in this paper, an in vitro perifusion system using the cell column of cultured rat pituitary cells attached on Cytodex beads was employed to investigate the mechanism of PRL release. The rat anterior pituitary cells were isolated using collagenase, and the dispersed pituitary cells were cultured with swollen Cytodex beads in Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum at 37 degrees C in 5% CO2 and 95% air for 2--3 days. The cultured anterior pituitary cells attached on Cytodex beads were packed in a column and perifused with DMEM at a constant flow rate of 0.4 ml/min using a peristaltic pump. The following results were obtained. A five minute perifusion with 100 pg/ml to 100 ng/ml TRH caused a significant increase of PRL in a dose-related manner. A continuous perifusion with 2 ng/ml or 10 ng/ml DA inhibited PRL release in a dose-related manner. When TRH at a dose of 1 ng/ml, 10 ng/ml or 100 ng/ml was perifused for 120 min at a rate of 0.4 ml/min, a large amount of PRL was released during the early period of the TRH infusion, and then the PRL release gradually decreased to the basal levels in spite of the continuous TRH infusion. An additional TRH, of which the concentration was ten-fold higher than the TRH level in the continuous infusion, when added at the end of the continuous TRH infusion, had no effect on PRL release. On the other hand, a 5 minute TRH infusion given at 30 min after the end of the continuous TRH infusion caused a significant increase in PRL release. A continuous perifusion with 1 mM 8-bromo-cyclic AMP caused a small but continuous PRL release. An additional continuous 8-bromo-cyclic AMP infusion during the late period of a continuous TRH infusion caused a continuous PRL release similar to that induced by the continuous infusion of cyclic AMP only. A short period perifusion with 1 X 10(-9)M to 1 X 10(-7)M of vasoactive intestinal polypeptide (VIP) enhanced a significant increase of PRL release in a dose-related manner, but the amounts of PRL release induced by VIP were smaller than those induced by TRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro studies on basal and stimulated prolactin release by rat anterior pituitary: a possible role for calmodulin

The mechanism by which PRL is released from mammotrophs is a calcium-dependent process. Although calcium seems to function as a second messenger, its regulatory mechanism in PRL release has not been clarified. The binding of calcium to calmodulin and the activation of calmodulin-dependent enzymes have been suggested to be important steps during stimulus-secretion coupling in various cells. In the present work we investigated the in vitro effect of penfluridol, a potent neuroleptic that also possesses the ability to inhibit calmodulin's biological activity, on basal and stimulated PRL release. The effect of pimozide and haloperidol on basal PRL release was also investigated. Penfluridol, pimozide, and haloperidol inhibited basal PRL secretion in a dose-related manner, with the EC50 ranging from 0.5-1 microM for penfluridol to 1-2 microM for pimozide and more than 3 microM for haloperidol. These concentrations are similar to those necessary for the inactivation of calmodulin-dependent enzymes in vitro. Ionophore A-23187, a compound whose ability to mobilize extracellular calcium is not affected by neuroleptics, stimulated PRL secretion in vitro. This effect, however, was blocked by penfluridol pretreatment. The site of action of penfluridol may occur after calcium mobilization, with calmodulin a possible target for penfluridol's inhibitory action on PRL secretion. TRH, K+, (Bu)2cAMP, and theophylline, compounds that affect calcium mobilization, also significantly stimulated PRL release. The coincubation of varying concentrations of penfluridol with 70 nM TRH, 50 mM K+, 3 mM (Bu)2cAMP, or 5 mM theophylline resulted in a dose-related inhibition of secretagogue-stimulated PRL secretion. Perifusion of dispersed anterior pituitary cells with 1 microM penfluridol reduced the ability of 70 nM TRH to stimulate PRL release by approximately 50%, whereas removal of the penfluridol perifusion allowed the cells to again be fully responsive to TRH. These results are consistent with the hypothesis that calmodulin is involved in the stimulus-secretion coupling of PRL.     

Effect of serotonin on basal and TRH-induced release of prolactin from rat pituitary glands in vitro

The effect of serotonin on the release of prolactin (PRL) was studied in vitro. Anterior hemipituitary glands from ovariectomized rats were incubated for 1 h in the presence of different doses of serotonin. Serotonin added into the culture medium caused a significant increase in basal PRL release. The effect was dose-related between 10 and 30 nmol/l serotonin, but responsiveness declined towards basal levels with higher concentrations. When studied as a function of incubation time, basal release of PRL was significantly increased up to 1 h but decreased thereafter. Serotonin also enhanced the release of prolactin induced by 30 nmol/l thyrotropin-releasing hormone (TRH), at all doses tested. A serotonin concentration of as little as 30 nmol/l was already effective. A significant response was seen at 15 min and further increases occurred during the following incubation periods. Serotonin (approximately EC50 4.6 X 10(-8) mol/l) was less potent than TRH (EC50 about 1.2 X 10(-8) mol/l) to increase basal PRL release. On the other hand, the indole amine appeared to act with similar potency in stimulating PRL release both basal and TRH-induced. In addition, the combined effect of the releasing agents was found to be additive. These results suggest that serotonin and TRH could act through separate mechanisms. Methysergide, a serotoninergic blocking agent, had no effect on the in vitro PRL release either basal or TRH-induced, but it completely blocked that evoked by serotonin suggesting that serotonin may interact with specific receptors on the lactotropes. These findings clearly demonstrate that serotonin may stimulate the release of PRL by acting directly at the pituitary gland level.     

Arachidonate stimulates prolactin release in vitro: a role for the fatty acid and its metabolites as intracellular regulator(s) in mammotrophs

We investigated the involvement of arachidonate in the PRL secretory process using three experimental systems: hemipituitary glands incubated in vitro, cultured pituitary cells, and dispersed anterior pituitary cells perifused in columns. Arachidonate (100 microM) significantly (P less than 0.05) stimulated PRL release in the former system and stimulated PRL secretion in a dose-related manner in cultured cells. In hemipituitary glands, indomethacin, a cyclooxygenase inhibitor, potentiated the arachidonate-mediated stimulation, whereas nordihydroguaiaretic acid or BW755c abolished it. The latter two agents, but not indomethacin, abolished the effect of phospholipase A2 on PRL release in vitro. BW755c also inhibited the stimulatory effect of TRH on PRL release in both experimental systems. Conversely, the stimulation of PRL release by phorbol myristate acetate (PMA), although significantly reduced, was not abolished by either nordihydroguaiaretic acid or BW755c. Quinacrine, a phospholipase A2 inhibitor, also abolished the stimulatory effect of phospholipase A2 or TRH on PRL release. In cultured cells, quinacrine inhibits basal PRL release, but does not affect PRL release induced by arachidonate or (Bu)2 cAMP. These results more firmly establish a role for arachidonate as an intracellular mediator of PRL release and suggest the involvement of an arachidonate metabolic pathway(s) (lipoxygenase and epoxygenase) other than prostaglandin or thromboxane formation. The effect of PMA on PRL release may be attributable only in part to an increase in the production of arachidonate metabolites, and most of PMA's effect on PRL release may relate to its activation of protein kinase C.     

Control of prolactin release induced by suckling

In the present study, the role of dopamine and TRH in suckling-induced PRL release was investigated. Bupropion, a dopamine reuptake blocker, increased hypophysial stalk dopamine levels and inhibited suckling-induced PRL release. A short period of suckling, thought to induce a transient decrease in hypothalamic dopamine release, led to higher PRL levels following an iv injection of TRH than in rats which had not nursed their young for a short period after 4- to 6-h separation. These results, in combination with previous data, suggest that a decrease in hypothalamic dopamine release is important for suckling-induced PRL release. Increased PRL release may be in part due to an augmented hypothalamic release of TRH. Since serotonergic mechanisms seem involved in TRH release, lactating rats were treated with drugs acting on serotonergic pathways. Parachlorophenylalanine and pizotifen did not alter suckling-induced PRL release. Methysergide, a serotonin receptor blocker, prevented this PRL release when administered ip but not when injected into the lateral brain ventricle. Since methysergide is converted peripherally into metabolite(s) with dopamine agonistic activity, its effect on suckling-induced PRL release may be due to this action, rather than to its action on serotonin receptors. Thus, these data do not indicate that serotonergic mechanisms are important for suckling-induced PRL release. Passive immunization against TRH inhibited suckling-induced PRL release, indicating that TRH is a hypophysiotropic mediator of this PRL release.     

Effects of somatostatin and urotensin II on tilapia pituitary prolactin release and interactions between somatostatin, osmotic pressure Ca++, and adenosine 3',5'-monophosphate in prolactin release in vitro

Both somatostatin (SRIF) and urotensin II, a dodecapeptide from the teleost caudal neurosecretory system, inhibit PRL release from the organ-cultured rostral pars distalis of the tilapia, Sarotherodon mossambicus, in a dose-related manner. The inhibitory action of SRIF on PRL release was completely prevented by the presence of the calcium ionophore A23187. PRL release was also blocked when Ca++ was excluded from the incubation medium, even in the presence of the ionophore. Both dibutyryl cAMP (dbcAMP) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, alone or in combination, stimulated PRL release during incubation in high osmotic pressure medium. The effect of dbcAMP appeared to be dose related. Together, dbcAMP and 3-isobutyl-1-methylxanthine were also effective in preventing the inhibition of PRL release by SRIF. These results are consistent with the notion that Ca++, and possibly cAMP, may be important mediators of PRL secretion, and it is likely that SRIF may inhibit PRL release by blocking a Ca++- or cAMP-mediated mechanism.     

Gender-biased activity of the novel prolactin releasing peptides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects

The prolactin- (PRL) releasing activities of the newly described PRL-releasing peptides (PrRPs) were compared to that of thyrotropin-releasing hormone (TRH) in dispersed, rat anterior pituitary cell cultures. A dose-related stimulation of PRL release by TRH was observed in cells harvested from both intact male and random cycle female pituitary donors. The minimum effective dose of TRH ranged from 1 to 10 nM. Neither PrRP-20 nor PrRP-31 significantly altered PRL secretion in cells from male donors even at doses as high as 1 microM. In cells harvested from females, only the highest doses of PrRP-20 and PrRP-31 tested (0.1 and 1.0 microM) significantly stimulated PRL secretion. The PRL-releasing action of TRH was observed already at 15 min of incubation, whereas those of PrRP-20 and PrRP-31 appeared only after 1 and 2 h of incubation, and the magnitude of PRL release in the presence of 1 microM PrRPs was significantly less than that of a similar dose of TRH. These data do not suggest a physiologically relevant role for the PrRPs in the neuroendocrine regulation of PRL secretion in intact male and nonlactating, random-cycle female rats.     

PERIFUSION STUDIES OF BROMOCRIPTINE-TREATED AND UNTREATED MACROPROLACTINOMAS: EFFECTS OF DOPAMINE, BROMOCRIPTINE AND TRH

There have been no detailed in-vitro studies of PRL secretion by human macroprolactinoma cells exposed to bromocriptine (BC) to within a few days of surgical removal. We have studied cells from four such tumours (serum PRL 7.05-247 U/l) and six untreated tumours (serum PRL 4-80.35 U/l) using a perifusion technique. The BC-treated tumours had shown tumour shrinkage and were treated until 40-96 h before surgery, but in one patient serum PRL had not suppressed below 15 U/l despite chronic treatment. Pretreatment serum PRL responses to TRH were blunted in all 10 patients. During perifusion with dopamine (DA, 5 mumol/l) untreated prolactinomas had a higher PRL secretion rate (19.3 +/- 2.7 microU/mg tissue/min, mean +/- SEM) than BC-treated (3.9 +/- 0.7, P = 0.005). When DA was removed, PRL secretion from untreated tumours increased to 129.7 +/- 18.7 microU/mg/min, but in three of the BC-treated, little increase occurred. In the fourth (from the patient whose serum PRL had not fully suppressed) PRL secretion increased from 4.4 to 25.6 microU/mg/min after DA withdrawal, and DA and BC dose-related inhibition of PRL was similar to that observed in untreated tumours. TRH (10 ng/ml), without DA, provoked increased PRL release from both untreated (266% basal secretion, n = 3) and BC-treated (298%, n = 3) tumours; this effect was completely inhibited by DA (5 mumol/l). The absence of hormones other than PRL following potassium (55 mmol/l) excluded contaminating normal pituitary. We conclude: (1) The effects of BC on prolactinoma PRL secretion may persist for at least 4 days; (2) partial in-vivo BC resistance can be due to factors other than DA receptor malfunction; (3) the apparent discrepancy between in-vivo and in-vitro TRH responses was consistent with the presence of increased hypothalamic DA tone in vivo; and (4) BC may have differential effects on TRH and DA-controlled PRL pools in the tumourous lactotroph.     

Effects of angiotensin II and natriuretic peptides of the eel on prolactin and growth hormone release in the tilapia,Oreochromis mossambicus

The effects of angiotensin II (ANG II) and natriuretic peptides (NPs) of the eel (ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; and VNP, ventricular natriuretic peptide) on prolactin (PRL(188) and PRL(177)) and growth hormone (GH) release from the organ-cultured tilapia pituitary were examined. Eel ANG II at concentrations greater than 1 nM stimulated the release of PRL(188) and PRL(177) in a dose-related manner during the first hour of incubation. Significant stimulation by 100 nM ANG II on PRL(177) release was observed until 4h of incubation, and on PRL(188) release until 12 h. No effect of ANG II was seen on GH release. None of the NPs altered the release of PRLs at any time point. On the other hand, eel VNP at concentrations greater than 1 nM stimulated GH release in a dose-related manner after 4 h, and significant stimulation was observed until 48 h. Eel CNP was less effective than eel VNP; significant stimulation of GH release was observed at 1 and 10 nM during 24-48 h of incubation. No significant effect of eel ANP on GH release was seen at any concentration. ANG II had no effect on GH release at any time point. There was no change in mRNA levels of PRLs or GH in the pituitaries incubated with ANG II for 8 h or those incubated with the NPs for 48 h. These results indicate rapid and short-lasting stimulation by ANG II on PRL release and slow and long-lasting stimulation by VNP and CNP on GH release from the tilapia pituitary.     

Effect of 17 beta-estradiol on phosphoinositide metabolism and prolactin secretion in anterior pituitary cells

The present study was undertaken to investigate the effect of 17 beta-estradiol (E2) administration on in vitro prolactin (PRL) release and intracellular phosphoinositide metabolism. The incorporation of [3H]inositol (Ins) into phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphophate [PtdIns(4,5)P2], and the generation of inositol phosphate (InsPx) following thyrotropin-releasing hormone (TRH) stimulation were studied in primary cultures of anterior pituitary cells obtained from ovariectomized rats. Administration of polyestradiol phosphate (PEP) to ovariectomized rats produced a significant increase (p less than 0.05) in serum PRL levels. This treatment also enhanced significantly (p less than 0.01) the in vitro release of PRL in a progressive manner during 24, 48 and 72 h of culture of dispersed anterior pituitary cells. The radioisotopic labeling by [3H]Ins of all species of phosphoinositides was progressive throughout 72 h of culture, and a good correlation was observed between intracellular phosphoinositide synthesis and PRL release from these cells. PEP treatment enhanced significantly (p less than 0.05-0.01) [3H]Ins incorporation into PtdIns and PtdIns(4)P after 48 and 72 h of culture, although it did not alter [3H]Ins incorporation into PtdIns(4,5)P2. Furthermore, this treatment caused a small, but significant increase (p less than 0.01) in InsPx generation following TRH stimulation. However, the increased [3H]Ins incorporation into phosphoinositide and InsPx generation that we observed after TRH stimulation was significantly (p less than 0.01) less than the increased amount of in vitro PRL release following PEP treatment. There was no significant correlation between the percentage increases in PRL release and phosphoinositide metabolism following the same treatment. These data suggest that phosphoinositide metabolism is enhanced in the anterior pituitary cells of ovariectomized rats by treatment with PEP, but this system does not appear to be tightly coupled or causally related to the much greater production of PRL release.     

Physiological concentrations of ouabain rapidly inhibit prolactin release from the tilapia pituitary

Ouabain, a cardiac glycoside and inhibitor of Na(+), K(+)-ATPase, is now believed to be a steroid hormone in mammals. We have recently identified ouabain immunoreactivity in the plasma of the tilapia, a euryhaline teleost. Changes in plasma concentrations of immunoreactive ouabain (20-40 pM) in response to salinity change were well correlated with the changes in plasma osmolality and cortisol. Our previous studies have shown that cortisol rapidly inhibits prolactin (PRL) release from the tilapia pituitary by suppressing intracellular Ca(2+) ([Ca(2+)]i) and cAMP. In the present study, low doses of ouabain (10-1000 pM) inhibited PRL release dose-dependently during 2-24 h of incubation. There was no effect on growth hormone (GH) release, except for a significant increase at 1000 pM during 8-24 h of incubation. Significant dose-related increases in PRL release were observed at higher doses of ouabain (100-1000 nM), whereas significant inhibition was seen in GH release at 1000 nM during 2-24h of incubation. Ouabain at 1-100 pM had no effect on Na(+), K(+)-ATPase activity of the pituitary homogenate. The enzyme activity was inhibited by higher concentrations of ouabain, 10% at 1 nM, 15% at 10 nM, 28% at 100 nM, and 45% at 1000 nM. Ouabain also attenuated stimulation of PRL release by the Ca(2+) ionophore, A23187, and by a combination of dibutyryl cAMP and a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthin. Intracellular Ca(2+) concentrations were monitored in the dispersed PRL cells with the Ca(2+)-sensitive dye, fura-2. Ouabain at 1 nM reversibly reduced [Ca(2+)]i within seconds, whereas 1 microM ouabain increased [Ca(2+)]i. A rapid reduction in [Ca(2+)]i was also observed when PRL cells were exposed to 1 microM cortisol, whereas there was no consistent effect at 1 nM. These results suggest that ouabain at physiological concentrations rapidly inhibits PRL release from the tilapia pituitary by suppressing intracellular Ca(2+) and cAMP metabolism. The stimulation of PRL release by high concentrations of ouabain (100-1000 nM) may result from an increase in [Ca(2+)]i, and subsequent depolarization due to the inhibition of Na(+), K(+)-ATPase activity.     

15-Lipoxygenase products stimulate prolactin secretion from a cloned strain of rat pituitary cells

Arachidonic acid and its lipoxygenase products may contribute to the process of prolactin (PRL) release. In the present study we investigate the role of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on PRL secretion from GH3 cells. The incubation of GH3 cells with the lipoxygenase product 15-HETE significantly increased PRL release in a concentration-dependent manner. Nordihydroguaiaretic acid (NDGA), which reduces the production of arachidonate metabolites via the lipoxygenase pathway, also reduced basal and TRH or arachidonic-acid-stimulated-PRL release. The inhibitory effect of NDGA on PRL release could be overcome by the addition of 15-HETE. The time course curve of PRL release from cells challenged by 15-HETE had the same profile as that of cells stimulated by TRH. The stimulating effect of 15-HPETE (ED50 = 0.7 x 10(-9) M), which is the direct precursor of 15-HETE, on PRL release was greater than TRH or 15-HETE (ED50 = 6.5 x 10(-9) M). Furthermore 15-HPETE and 15-HETE seemed to affect the release of newly synthesized PRL. These data indicate that 15-HETE and 15-HPETE could be important intracellular components in the control of PRL secretion and may account for at least a part of arachidonate-induced PRL release from GH3.     

Influence of starvation on hormonal control of hypophyseal secretion in rats

The reduction of hypophyseal hormone secretion during starvation is not completely understood. A previous study showed that the concomitant reduction of plasma TSH and T3 may be related to an increased sensitivity of the thyrotrope cell to T3. This suggests that regulation of hypophyseal secretion by peripheral hormones may be altered in starved rats. As GH and PRL secretion are under the control of thyroid and steroid hormones, the aim of the present study was to investigate the modification of feed-back control by T3 or E2 on hypophyseal secretion during starvation. For this purpose, pituitary GH, PRL and TSH contents and their plasma responses to TRH injection were measured in euthyroid, thyroidectomized (Tx), T3-supplemented Tx and E2-treated male Wistar rats before and after a 3-day starvation. TRH (0.25 micrograms/100 g) was injected iv through a chronically-implanted catheter. Our results show that GH content and GH plasma response to TRH are dramatically increased in T3-treated Tx starved rats, suggesting that starvation also increases the effectiveness of T3 influence on somatotrope cell secretion. By contrast, effects of T3 on PRL secretion remain unchanged during starvation. Furthermore, starvation in E2-treated rats is associated with a marked rise in the PRL and GH responsiveness to TRH without any significant change of hormonal pituitary content. This suggests that, in starved rats, E2 increases the effects of TRH on lactotrope and somatotrope secretion. No significant effect on TSH secretion could be demonstrated. Thus, starvation seems to act differentially on the feed-back mechanisms controlling the hormonal secretion of the three adenohypophyseal target cells to TRH.     

THE INTERACTION OF TRH AND DOPAMINERGIC MECHANISMS IN THE REGULATION OF STIMULATED PROLACTIN RELEASE IN MAN

The manner by which dopaminergic and TRH mechanisms interact to control PRL release is not known. Whilst dopamine receptor antagonists and TRH both release PRL, it is not known if the PRL released by these two mechanisms reflects similar aspects of physiological control, or if PRL responses to these mechanisms of release can be dissociated. We addressed this question by studying the PRL responses to maximal stimulatory dose of TRH and domperidone (a DA receptor antagonist), which were administered sequentially, simultaneously or separately on different occasions. Six normal volunteers undertook three sets of studies: (1) standard PRL stimulation tests to 400 micrograms TRH, 5 mg domperidone or simultaneous TRH/domperidone administration, (2) domperidone bolus-infusion study in which either 5 mg domperidone or 400 micrograms TRH was administered i.v. at 120 min during a 240 min infusion of domperidone (50 micrograms/min) which was preceded by a 5 mg i.v. bolus of the drug, and (3) TRH bolus-infusion study in which domperidone or TRH was administered i.v. at 120 min during a 240 min infusion of TRH (0.4 micrograms/min) which was preceded by a 400 micrograms i.v. bolus of the drug. In Study 1, simultaneous TRH/domperidone administration induced an incremental rise in PRL (5195 +/- 940 mIU/l) which was significantly greater (P less than 0.0005) than with either domperidone (3730 +/- 825 mIU/l) or TRH (1335 +/- 300 mIU/l) alone. In study 2, TRH administration at 120 min resulted in a significant rise (P less than 0.01) in PRL (delta PRL 960 +/- 232 mIU/l) whilst the second dose of domperidone did not, thus suggesting that the initial bolus and subsequent infusion had resulted in complete DA receptor blockade. In Study 3, domperidone administered at 120 min induced a marked rise in PRL (delta PRL 3609 +/- 963 mIU/l). In contrast, the corresponding TRH stimulus resulted in a small rise (delta PRL 142 +/- 32 mIU/l) suggesting that the PRL release induced by the initial bolus and subsequent infusion had been near maximal. Thus, TRH is able to induce significant PRL release in the presence of maximal DA receptor blockade, and domperidone, in the presence of maximal TRH stimulation, is also capable of inducing significant PRL release. These observations together with the ability of TRH/domperidone to induce a greater PRL response than either agent alone, suggest that each stimulus has a specific releasing action on a fraction of intracellular PRL which is not accessible to the other.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pseudopregnant rats

The effectiveness of TRH in releasing PRL after transient dopamine (DA) blockade was investigated in female rats between days 3 and 11 of pseudopregnancy (PSP). At 0930 h on the morning of the experiment, each animal was injected with the DA antagonist domperidone (0.01 mg/rat, iv) or vehicle (acetic acid in saline); 5 min later, the DA agonist 2-bromo-alpha-ergocryptine maleate (CB-154; 0.5 mg/rat, iv) was administered. Sixty minutes later, TRH (1.0 micrograms/rat, iv) was administered. Blood samples were withdrawn via indwelling catheters before, 5, 20, 40, and 70 min after domperidone or vehicle administration, and 5 and 10 min after TRH administration. On day 3 of PSP, TRH-induced PRL release was significantly enhanced by the domperidone-CB154 treatment compared to that in vehicle-treated control rats. By day 9 of PSP, the effectiveness of TRH in stimulating PRL release after domperidone treatment was decreased by 50% compared to that on day 3 of PSP. This reduction in PRL response to TRH was not due to decreased progesterone levels, as no difference was observed in plasma progesterone between days 3 and 9. Rats that were given domperidone on day 11 of PSP did not exhibit a significant increase in sensitivity to TRH; however, the effectiveness of TRH was enhanced by domperidone on day 11 of PSP in animals that were hysterectomized on day 2 of PSP. Since DA receptor blockage increased the sensitivity to a putative PRL-releasing factor (TRH) and this mechanism was eliminated around the time that the PRL surges of PSP disappear, we suggest that this pituitary mechanism is a critical component of the PRL release mechanism during the surges of PSP. Further, the observed loss of the mechanism between days 9 and 11 of PSP may be due to the direct influence at the anterior pituitary of a uterine PRL inhibitory factor which has been recently described.     

Isolation and characterization of a homologue of mammalian prolactin-releasing peptide from the tilapia brain and its effect on prolactin release from the tilapia pituitary

In the tilapia (Oreochromis mossambicus), as in many teleosts, prolactin (PRL) plays a major role in osmoregulation in freshwater. Recently, PRL-releasing peptides (PrRPs) have been characterized in mammals. Independently, a novel C-terminal RF (arginine-phenylalanine) amide peptide (Carrasius RF amide; C-RFa), which is structurally related to mammalian PrRPs, has been isolated from the brain of the Japanese crucian carp. The putative PrRP was purified from an acid extract of tilapia brain by affinity chromatography with antibody against synthetic C-RFa and HPLC on a reverse-phase ODS-120 column. The tilapia PrRP cDNA was subsequently cloned by polymerase chain reaction. The cDNA consists of 619 bp encoding a preprohormone of 117 amino acids. Sequence comparison of the isolated peptide and the preprohormone revealed that tilapia PrRP contains 20 amino acids and is identical to C-RFa. Incubation of the tilapia pituitary with synthetic C-RFa (100 nM) significantly stimulated the release of two forms of tilapia PRL (PRL188 and PRL177). However, the effect of C-RFa was less pronounced than the marked increase in PRL release in response to hyposmotic medium. The ability of C-RFa to stimulate PRL release appears to be specific, since C-RFa failed to stimulate growth hormone release from the pituitary in organ culture. In contrast, rat and human PrRPs had no effect on PRL release. C-RFa was equipotent with chicken GnRH in stimulating PRL release in the pituitary preincubated with estradiol 17beta. Circulating levels of PRL were significantly increased 1 h after intraperitoneal injection of 0.1 microg/g of C-RFa in female tilapia in freshwater but not in males. These results suggest that C-RFa is physiologically involved in the control of PRL secretion in tilapia.     

Effects of repeated stimuli on prolactin release in vitro from normal and adenomatous rat lactotrophs

It is now well known that dopamine (DA) plays a major role in the inhibitory control of prolactin (PRL); however, the mechanisms that are physiologically involved in the stimulation of PRL release are still under investigation. Indeed, although suppression of DA inhibitory tonus, administration of thyrotropin-releasing hormone (TRH) or vasoactive intestinal peptide (VIP) are all known PRL releasers, it is not clear whether they interact during physiological periods of PRL release such as suckling and estrus. No clear indications exist, furthermore, on whether they all act upon a same pituitary pool that may become depleted following repeated exposure to stimuli. Refractoriness to a single or a repeated stimulus has been reported to occur in prolactinoma-bearing or normal humans, respectively, the mechanism of which is still matter for discussion. Our present studies performed by perifusing normal or adenomatous rat lactotrophs attached to Cytodex I microcarrier beads was undertaken to try and answer some of these questions. The experimental period consisted in perifusing the cells for 1 h with Dulbecco's modified Eagle's medium (DMEM) containing DA 10(-5) M, then for 2 h with either DMEM, DMEM and TRH 10(-8) M, DMEM and VIP 10(-7) M, then again with DA in DMEM for 1 h, and finally with DMEM, DMEM and TRH, or DMEM and VIP. Three experiments of various combinations were performed. Lower PRL levels were observed under DA, while two periods (first and second) of PRL release followed the suppression of DA infusion with or without the addition of either one of the two peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

The exaggerated prolactin response to thyrotropin-releasing hormone and metoclopramide in 1,2-dibromo-3-chloropropane-induced azoospermia

Twelve males with azoospermia secondary to exposure to the nematocide 1,2-dibromo-3-chloropropane were challenged to iv LRH (100 micrograms), TRH (200 micrograms), and metoclopramide (MET; 10 mg) administered 30 min apart. When compared to 24 male controls, both basal FSH and LH levels as well as peak gonadotropin responses to LRH were increased in the azoospermic group. The patients also had increased total estradiol (E2) and testosterone (T) as well as testosterone-binding globulins levels. Free T levels, however, were not significantly different from the controls. Basal PRL levels were similar in the two groups. However, the peak PRL responses to both TRH and MET were significantly increased in the azoospermia subjects (P < 0.001). In both groups, the PRL response to MET was greater than to TRH. In the individual control and azoospermic subjects, there was no correlation between the PRL response and E2, T, or the E2 to T ratio. However, a positive correlation did exist between testosterone-binding globulin levels and the PRL response to TRH and MET. Although the precise mechanism underlying the PRL hyperresponsiveness is unknown, it may be an estrogen-induced phenomenon.     

The role of calcium in prolactin release from the pituitary of a teleost fish in vitro

The release of PRL from the pituitary of a teleost fish, the tilapia (Oreochromis mossambicus), has been previously shown to be dependent on calcium. However, the source(s) and specific action(s) of calcium in the secretory process have not been identified. Also undefined are the mechanisms by which regulators of PRL cell function may alter calcium distribution. In the present investigation, the elevation of medium K+ concentration during static incubations to a depolarizing concentration (56 mM) produced no change in cumulative PRL release over control levels during the 18-20 h of incubation. During perifusion incubation, exposure to high K+ concentrations briefly stimulated (less than or equal to 10 min) and then depressed PRL release. In contrast, reduced medium osmotic pressure elicited a rapid elevation in PRL release that was sustained for 2 h or more. D600, a calcium entry blocker, at 10(-5) M diminished the K+-induced pulses of PRL release. The same concentration, however, did not alter the release of PRL evoked by reduced osmotic pressure. In contrast, CoCl2, which blocks a range of calcium-mediated processes in addition to calcium influx, suppressed PRL release during perifusion and static incubations in hyposmotic medium. These findings suggest that while PRL secretion from the tilapia pituitary is calcium dependent, calcium entry through voltage-regulated plasmalemma channels may not be a prerequisite to the actions of reduced osmotic pressure.     

Mechanism(s) by which the transient removal of dopamine regulation potentiates the prolactin-releasing action of thyrotropin-releasing hormone

The transient removal of dopamine (DA) selectively potentiated the prolactin (PRL) releasing action of thyrotropin-releasing hormone (TRH) but not vasoactive intestinal peptide (VIP). Consistent with these findings, the PRL-stimulating actions of agents which activated the Ca2+/protein kinase C second messenger pathway but not the adenylate cyclase system were also potentiated. In the current study we have extended these findings to determine the second messenger system mediating the potentiating action of the removal of DA. Dispersed anterior pituitary cells from E2-treated Sprague-Dawley rats were cultured on plastic coverslips. Cells tonically superfused with DA (500 nM were challenged with TRH (100 nM) 20 min after no additional treatment or a 10-min treatment with 8-Br-cyclic adenosine monophosphate (8-Br-cAMP), the Ca2+ ionophore A23187,12-O-tetradecanoyl-phorbol-13-acetate (TPA), TRH, or VIP. The potentiation of the TRH response was compared to the 4- to 5-fold potentiation observed following the removal of DA for 10 min 8-Br-cAMP at the concentration used (500 microM) was unable to alter the basal rate of PRL release, but, as VIP (500 nM), potentiated 2- to 3-fold the PRL-releasing action of TRH. A prior administration of TRH (100 nM) did not affect the responsiveness of the cells to a second challenge with TRH 20 min later. Both A23187 (20 microM) and TPA (5 or 50 nM) induced a sustained rise in the rate of PRL release. TPA-treated cells showed an increased responsiveness to TRH, whereas A23187-treated cells did not.(ABSTRACT TRUNCATED AT 250 WORDS)