Protein kinase C modulates cell swelling-induced Ca2+ influx and prolactin secretion in GH4C1 cells.

In GH4C1 rat pituitary cells, cell swelling stimulates prolactin (PRL) secretion by increasing Ca2+ influx through nifedipine-sensitive Ca2+ channels; however, the mechanism by which cell swelling opens Ca2+ channels is still unclear. To evaluate the role of protein kinase C (PKC) in this phenomenon, we studied the effect of down-regulating PKC by 12-h pretreatment with phorbol ester or by treatment with H-7, a protein kinase C inhibitor. Cell swelling induced by either 27% medium hyposmolarity or 80 mM isotonic urea caused a prompt rise in both [Ca2+]i and PRL secretion in otherwise untreated control GH4C1 cells. Removal of medium Ca2+ enhanced the osmotically induced cell swelling but prevented the increase in [Ca2+]i and PRL secretion. Both PKC down-regulation and H-7 suppressed the cell swelling-induced increases in [Ca2+]i concentration and PRL secretion, although they enhanced the induced cell volume expansion. Our data indicate that in GH4C1 cells PKC plays an important positive modulating role in the osmotic opening of plasmalemma Ca2+ channels, a critical component of the early transduction chain by which cell swelling causes PRL secretion in tumor-derived clonal pituitary cells.     

Medium hyperosmolarity depresses thyrotropin-releasing hormone-induced Ca2+ influx and prolactin secretion in GH4C1 cells.

We studied the influence of graded degrees of hyperosmolarity on the dynamics of the thyrotropin-releasing hormone (TRH)-induced rise in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion in GH4C1 cells. TRH caused two phases of increase in [Ca2+]i that were differentially altered by hyperosmolarity: 100% hyperosmolarity (600 mOsm) depressed only 20% of an initial high-amplitude [Ca2+]i burst (first phase) dependent on Ca2+ mobilized from intracellular pools, but it abolished a sustained low-amplitude second phase dependent on extracellular Ca2+ influx. Low degrees of hyperosmolarity suppressed PRL secretion due to Ca2+ influx while high degrees suppressed secretion due to mobilized Ca2+. These data suggest that in GH4C1 cells hypertonic inhibition of secretion may result from both blocking Ca2+ influx and mechanisms unrelated to [Ca2+]i.     

pH homeostasis in pituitary GH4C1 cells: basal intracellular pH is regulated by cytosolic free Ca2+ concentration.

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.     

Pretreatment with 1,25-dihydroxycholecalciferol enhances thyrotropin-releasing hormone- and inositol 1,4,5-trisphosphate-induced release of sequestered Ca2+ in permeabilized GH4C1 pituitary cells.

In GH4C1 cells 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] has been shown to enhance the TRH- and bombesin-induced increase in intracellular Ca2+ ([Ca2+]i). The aim of the present study was to investigate whether this increase in [Ca2+]i could be due to enhanced release of sequestered Ca2+ in cells pretreated with 1,25-(OH)2D3. In digitonin-permeabilized cells, the addition of 10 microM inositol 1,4,5-trisphosphate (IP3) rapidly increased free Ca2+ ([Ca2+]) to 50 +/- 10 nM (mean +/- SE) in cells pretreated with 1 nM 1,25-(OH)2D3 for 24 h, compared with 25 +/- 5 in control cells (P < 0.05). Furthermore, stimulating permeabilized cells with TRH increased [Ca2+]. The increase in control cells was 20 +/- 2, compared with 55 +/- 11 in cells pretreated with 1,25-(OH)2D3 (P < 0.05). Repeated additions of IP3 resulted in an attenuation of the response of [Ca2+] in both control cells and cells pretreated with 1,25-(OH)2D3. However, only the first addition of IP3 resulted in an enhanced increase in [Ca2+] in cells pretreated with 1,25-(OH)2D3 compared with control cells. If the cells were stimulated first with TRH and then with IP3, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Furthermore, if cells were stimulated with IP3 and then with TRH, no difference in the [Ca2+] response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Stimulating the permeabilized cells with thapsigargin resulted in an increase in [Ca2+]. However, no difference in the response was observed between control cells and cells pretreated with 1,25-(OH)2D3. Addition of GTP or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) had no effect on [Ca2+]. The results suggest that 1,25-(OH)2D3 has a modulatory effect on an IP3-sensitive intracellular Ca2+ pool in GH4C1 cells.     

The permeant molecule urea stimulates prolactin secretion in GH4C1 cells by inducing Ca2+ influx through dihydropyridine-sensitive Ca2+ channels

Isotonic urea in medium with a normal 1.2 mM Ca²⁺ concentration induced a striking rise in both cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and prolactin (PRL) secretion, each of whose peaks were proportional to the concentration of urea between 5 and 120 mM. There was a significant linear relationship between the peaks of induced [Ca²⁺]_i and PRL secretion (r = 0.99, P < 0.001). The increase in both [Ca²⁺]_i and PRL secretion was completely abolished by removal of medium Ca²⁺ or by 2 μM nifedipine. Hypertonie urea was ineffective in inducing either an increase in [Ca²⁺]_i or PRL secretion. These data support the hypothesis that plasma membrane expansion is a potent non-toxic inducer of hormone secretion and that in GH₄C₁ cells an increase in [Ca²⁺]_i produced by enhanced influx of extracellular Ca²⁺ through dihydropyridine-sensitive Ca²⁺ channels plays an important role in this phenomenon.     

Dual actions of 1,25-dihydroxycholecalciferol on intracellular Ca2+ in GH4C1 cells: evidence for effects on voltage-operated Ca2+ channels and Na+/Ca2+ exchange

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] amplifies the TRH-induced spike phase of increase in cytosolic free calcium ([Ca2+]i). In the present report we describe the results of investigations on the mechanisms of action of 1,25-(OH)2D3 on Ca2+ homeostasis in these cells. Pretreatment with 1 nM 1,25-(OH)2D3 for at least 24 h caused no change in basal uptake of 45Ca2+ compared with that in control cells or in 45Ca2+ uptake induced by the calcium channel agonist Bay K 8644. However, when the cells were depolarized with 50 mM K+, 1,25-(OH)2D3-treated cells showed an up to 90% enhancement of uptake (3-120 min) of 45Ca2+. An enhanced increase in [Ca2+]i was also observed in fura-2-loaded cells. The effect was specific and dose dependent for 1,25-(OH)2D3. The calcium channel antagonists nimodipine and verapamil inhibited completely the enhancing action of 1,25-(OH)2D3 as did the protein synthesis inhibitor cycloheximide. No enhanced uptake of 45Ca2+ into intracellular stores was detected when cells were incubated with 1,25-(OH)2D3. Na+/Ca2+ exchange was determined by measuring exchange of extracellular 45Ca2+ for intracellular Na+. Na+/Ca2+ exchange was dependent on intracellular Na+, was inactive when Li+ replaced Na+, was insensitive to calcium channel antagonists, and showed electrogenic properties. In cells incubated with 1,25-(OH)2D3 for at least 24 h, Na+/Ca2+ exchange was enhanced up to 54% compared with that in control cells. Enhanced exchange was dose dependent and specific for 1,25-(OH)2D3. Ca2+ channel antagonists were without effect while dichlorobenzamil inhibited partially the 1,25-(OH)2D3 enhancement of Na+/Ca2+ exchange. Cycloheximide abolished completely the action of 1,25-(OH)2D3 on Na+/Ca2+ exchange. We conclude that in GH4C1 cells, 1,25-(OH)2D3 enhances membrane calcium transport by modulating voltage-operated Ca2+ channels and activating Na+/Ca2+ exchange by mechanisms requiring new protein synthesis.     

Medium hyperosmolarity inhibits prolactin secretion induced by depolarizing K+ in GH4C1 cells by blocking Ca2+ influx.

Medium hyperosmolarity between 300 (normal medium osmolarity) and 600 mOsm inhibited in a concentration-correlated fashion (r greater than 0.97, p less than 0.001) the rise in intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced in GH4C1 cells by depolarizing 30 mM K+. [Ca2+]i concentration and PRL secretion were tightly related between 300 and 600 mOsm (r = 0.976, p less than 0.001); 50% inhibition of both occurred at 450 mOsm. Medium hyperosmolarity slowed the rate of Ca2+ influx. At 600 mOsm the rise in both [Ca2+]i and PRL secretion was abolished but PRL secretion induced by 1 microM phorbol 12-myristate 13-acetate was not significantly reduced. Our data suggest that inhibition of Ca2+ influx may be the primary mechanism by which extracellular hyperosmolarity inhibits PRL secretion induced by high medium K+ in GH4C1 cells. Depression of the Ca2+ intracellular transduction system may play a pathophysiological role in vivo in conditions such as dehydration and hypertonic coma.     

Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+

Vasoactive intestinal peptide (VIP) and peptide (P) with N-terminal histidine and C-terminal isoleucine (PHI) stimulated prolactin (PRL) secretion from GH4C1 cells equipotent with ED50 values of 30-50 nM. In a parafusion system optimized to give high time resolution both VIP and PHI increased PRL secretion with a delay of about 60 s and subsequent to the activation of the adenylate cyclase. Thyroliberin (TRH) increased PRL secretion within 4 s. The dose-response curves for VIP- and PHI-stimulated cAMP accumulation were superimposable on those for PRL secretion. At submaximal concentrations the effects of VIP and PHI on both cAMP accumulation and PRL secretion were additive, whereas the effects were not additive at concentrations giving maximal effects. VIP and PHI increased [Ca2+]i measured by quin-2 in a different way than TRH, without inducing changes in the electrophysiological membrane properties of the GH4C1 cells. We conclude that both VIP and PHI stimulate PRL secretion via a cAMP-dependent process involving an increase in [Ca2+]i.     

Alpha-adrenergic inhibition of thyrotropin-releasing hormone-induced prolactin secretion in GH4C1 cells is associated with a depressed rise in intracellular Ca2+.

alpha-Adrenergic receptors are present on the plasma membrane of normal anterior pituitary cells and alpha-adrenergic agonists may play a role in the secretion of corticotropin (ACTH) and thyrotropin (TSH). However, alpha-adrenergic involvement in prolactin (PRL) secretion is uncertain. We have therefore examined this question in the PRL-secreting clonal rat pituitary tumor-derived GH4C1 cells. Norepinephrine (NE), an alpha-adrenergic agonist, had no effect on basal PRL secretion but abolished thyrotropin-releasing hormone (TRH)-induced PRL secretion in a dose-dependent manner (EC50 100 nM). NE also significantly suppressed the TRH-stimulated rise in [Ca2+]i. Phentolamine (PA), a non-selective alpha-adrenergic antagonist, reversed the inhibitory effect of NE on both the TRH-stimulated PRL secretion and [Ca2+]i rise. NE did not inhibit the rise in PRL secretion or [Ca2+]i induced by depolarizing 30 mM K+, 30% hyposmolarity or BAY K-8644, a specific L-type Ca2+ channel agonist. The inhibitory effect of NE on TRH-induced PRL and [Ca2+]i changes was also present when Ca2+ influx was prevented by removing medium Ca2+ or by blocking L-type Ca2+ channels with 2 microM nifedipine. The TRH-stimulated first-phase rise in [Ca2+]i in GH4C1 cells is believed to result primarily from release of sequestered Ca2+ from an intracellular pool through the activation of inositol 1,4,5-trisphosphate (IP3) and this [Ca2+]i spike stimulates PRL secretion. Our data thus suggest that GH4C1 cells have alpha-adrenergic receptors and that alpha-adrenergic agonists either suppress IP3 generation or block IP3 release of sequestered intracellular Ca2+.     

Thyrotropin-releasing hormone rapidly stimulates a biphasic secretion of prolactin and growth hormone in GH4C1 rat pituitary tumor cells

The characteristics of TRH-induced acute PRL and GH secretion were studied in GH4C1 cells, a clonal rat anterior pituitary tumor cell line which secretes PRL and GH. The experiments were carried out both in a flow system in which microcarrier (Cytodex)-attached cells were perifused at a constant rate and in a conventional static culture system. In both systems, cells responded to TRH in a qualitatively similar manner. TRH significantly stimulated PRL and GH secretion within 5 sec without a detectable lag period. The secretion rate was highest during the initial 1 min, declined sharply thereafter despite the continuous presence of TRH, and plateaued at a lower level. The maximum dose of TRH caused 250-700% of basal secretion during the early period (approximately 8 min; first phase) and about 150% of basal secretion thereafter (second phase). The sustained lower secretion (second phase) was maintained as long as cells were exposed to TRH (up to 2.5 h), and the secretion rate returned to the basal level within 30 min of removal of TRH from the medium. The half-maximal doses for the first and second phase secretion were 2-3 and 0.5-1 nM, respectively, in both the perifusion and static culture systems. Over a 2-day period, TRH stimulated PRL synthesis and inhibited GH synthesis. The dose-response curves for these long term effects on hormone synthesis were similar to the dose-response curves for the first phase of release. [N3-methyl-His2]TRH gave similar results, but was more potent than TRH. [N3-methyl-His2]TRH stimulated first phase release with an ED50 of 0.4-0.8 nM, second phase release with an ED50 of 0.1-0.2 nM, and hormone synthesis with an ED50 of 0.7-0.8 nM. Preincubation of the cells with Ca+2-free medium significantly depressed both first and second phase secretion. Preexposure of the cells to cycloheximide (10 micrograms/ml) had little effect on the first phase of secretion, but reduced second phase secretion. The acute effects of TRH on GH and PRL were identical, except that the secretory response tended to be greater for PRL. We conclude that 1) TRH causes hormone secretion very rapidly in a biphasic manner; 2) the first phase of secretion consists primarily of the release of stored hormone, whereas the second phase includes the release of newly synthesized hormone; 3) the dose-response curve of second phase secretion is shifted to the left compared with that of first phase secretion; and 4) both phases of secretion are at least partially dependent on extracellular Ca+2.     

Comparison of patterns of prolactin release in GH4C1 cells and primary pituitary cultures

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA, an activator of C-kinase), the cation ionophore A23187, forskolin (an activator of adenylate cyclase) and thyrotropin-releasing hormone (TRH) on prolactin release from anterior pituitary cells in primary culture were investigated and compared to the effects of these same agents on prolactin release from GH4C1 cells. In both GH4C1 cells and primary pituitary cultures, 100 nM TRH increased prolactin release 3- to 5-fold within 4 min after the stimulation started. This peak response was followed by a fall to a sustained increased rate of release approximately 1.5-fold above the basal rate. The decline after the early peak was slower in primary cultures than in GH4C1 cells. Addition of 20 microM A23187 to primary cultures caused a rapid 2- to 4-fold increase in release that fell to basal values within 12 min after the stimulation started. In GH4C1 cells, A23187 caused a rise in prolactin release of less than 2-fold that was sustained longer than the rise seen in primary cultures. Perifusion of either type of cells with 50 nM TPA caused a rapid 2- to 2.5-fold increase in release that also was sustained for 30 min or more in both types of cells. Perifusion with combined TPA and A23187 caused a 3- to 5-fold increase in rate of release from each cell type that declined rapidly to a 2-fold sustained release in primary cultures, and that declined more slowly in GH4C1 cells. Forskolin, 1 microM, had only a small effect by itself, but potentiated the effect of TPA or combined TPA and A23187 in both types of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lidocaine inhibits prolactin secretion in GH4C1 cells by blocking calcium influx.

The mechanism of the inhibitory effect of local anesthetics on hormone secretion was studied in the GH4C1 line of rat pituitary tumor-derived cells. Lidocaine between 0.1 and 5 mM exerted significant dose-dependent inhibition on the increment in cytosol Ca2+ concentration ([Ca2+]i) and prolactin (PRL) secretion induced by 30 mM K+. For both effects the IC50 was 0.25 mM and maximal inhibition occurred at 5 mM. A normal response returned within 20 min after removal of lidocaine from the incubation medium. 1 microM tetrodotoxin had no effect on the 30 mM K+ induced [Ca2+]i transient or PRL secretion, indicating that Na+ channels are not involved in the inhibitory effect of lidocaine. Lidocaine similarly inhibited the [Ca2+]i increment and PRL secretion induced by 30% medium hyposmolarity and 1 microM Bay K 8644. Lidocaine was much less effective in inhibiting secretion induced by 1 microM phorbol 12-myristate 13-acetate (TPA) or 5 microM forskolin. 5 mM procaine produced effects similar to those of lidocaine. Our data suggest that in GH4C1 cells local anesthetics depress secretagogue-induced PRL secretion primarily by blocking Ca2+ influx, probably through L-type Ca2+ channels.     

The dopaminergic regulation of anterior pituitary 45Ca2+ homeostasis and prolactin secretion

A role for the regulation of cellular Ca2+ homeostasis in the dopaminergic control of prolactin secretion was investigated in rat anterior pituitary glands. Withdrawal of dopamine stimulated the uptake of 45Ca2+ into hemipituitary tissue by 48% after 3 min. Radioisotope desaturation from tissue prelabelled with 45Ca2+ was significantly retarded in the presence of dopamine. Withdrawal of dopamine rapidly stimulated 45Ca2+ efflux from prelabelled tissue by 79% and was accompanied by a three- to fourfold rise in prolactin secretion. The 45Ca2+ efflux response to dopamine withdrawal was reduced in tissue prelabelled in the presence of dopamine. Agonist displacement with metoclopramide mimicked the effect of dopamine withdrawal on 45Ca2+ efflux and prolactin secretion. These observations demonstrate that the stimulation of prolactin release by dopamine withdrawal is accompanied by a redistribution of cellular Ca2+ and support the hypothesis that dopamine inhibits secretion by decreasing Ca2+ influx in the mammotroph cell.     

Effects of trifluoperazine on prolactin release and cyclic AMP formation and degradation in GH4C1 pituitary cells

In GH4C1 cells, the calmodulin antagonist trifluoperazine (TFP) showed a dose-dependent, biphasic effect on the basal release of PRL. An inhibition of PRL release was observed with 15-50 mumol/l TFP, whereas a concentration of 100 mumol/l and above had a stimulatory effect. The increase in basal hormone release evoked by TRH (1 mumol/l) and high extracellular concentration of K+ (50 mmol/l) was eliminated by 30 mumol/l TFP. The stimulatory effect of 100 mumol/l TFP on basal hormone release was not affected by addition of TRH (1 mumol/l) or K+ (50 mmol/l). The Ca2+ antagonists Co2+ (5 mmol/l) and verapamil (100 mumol/l), and the Ca2+ chelator EgTA (4 mmol/l) abolished the stimulatory effect of TRH (1 mumol/l) and of K+ (50 mmol/l) on PRL release, whereas only Co2+ inhibited the stimulation caused by 100 mumol/l TFP. TFP (75 mumol/l) caused a transient increase in the concentration of cellular cAMP. Incubation of intact GH4C1 cells with TFP (75 mumol/l), had an inhibitory effect on both the low and the high affinity form of cAMP phosphodiesterase. Basal as well as TRH-stimulated adenyl cyclase activity were inhibited by TFP, and this effect was counteracted by addition of calmodulin.     

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.     

Difference in calcium requirements for forskolin-induced release of prolactin from normal pituitary cells and GH4C1 cells in culture

We investigated the role of Ca++ in cAMP-stimulated PRL release from 1) primary cultures of male rat pituitary glands, 2) primary cultures of estrogen-induced pituitary tumors from Fischer rats, and 3) the pituitary tumor cell line GH4C1. Forskolin, an activator of adenylate cyclase, increased intracellular cAMP concentrations in GH4C1 cells at least 20-fold within 15 min. This increase occurred in the presence or absence of added extracellular Ca++ or in the presence of D600 or Co++. Forskolin increased PRL release from the three types of cells. The three systems differed in the Ca++ sensitivity of forskolin-induced release, but showed little difference in the Ca++ sensitivity of K+-induced release. This was shown in two ways. The cells were incubated either 1) in a medium without added Ca++ or 2) in the presence of a Ca++ channel inhibitor, D600. In normal cells, K+- and forskolin-induced release were equally inhibited when extracellular Ca++ was removed or D600 was added. In GH4C1 cells, Ca++ removal or D600 addition (100 microM) completely blocked K+-induced release, but had little effect on forskolin-induced release. The response of Fischer tumor cells was intermediate between those of normal and GH4C1 cells. 45Ca++ uptake by GH4C1 cells was not affected by forskolin, whereas the release of 45Ca++ from preloaded cells was increased slightly only 30 min after the addition of forskolin in three of four experiments. The difference in Ca++ requirements between normal and GH4C1 cells for forskolin stimulation may be due to the release of cellular Ca++ stores by cAMP. These stores may not be as large in normal cells as they are in GH4C1 cells, and therefore the requirement for extracellular Ca++ occurs. Alternatively, GH4C1 cells may release PRL by a mechanism different from that which normal cells use.     

Comparison of the regulation of carboxypeptidase E and prolactin in GH4C1 cells, a rat pituitary cell line

The treatment of GH4C1 cells, a prolactin-producing rat anterior pituitary cell line, with estradiol (1 nM), insulin (300 nM) and epidermal growth factor (10 nM) has been previously shown to substantially increase both the intracellular level of prolactin, as well as the number of secretory granules. In this study, we examined the effect of this treatment on levels of carboxypeptidase E (CPE), a prohormone-processing enzyme. GH4C1 cells contain CPE mRNA and enzymatic activity. The secretion of both prolactin and CPE activity from GH4C1 cells is stimulated 10-fold by 50 mM KCl and 2- to 3-fold by 100 nM thyrotropin-releasing hormone, suggesting that these two proteins are contained in secretory granules. Treatment of GH4C1 cells with estradiol, insulin, and epidermal growth factor causes an increase in the intracellular level of CPE to approximately 2-fold of control values. This change is much smaller than the change in the level of prolactin: intracellular prolactin is increased 140-fold by the treatment. Kinetic analysis of the CPE activity indicates that the treatment does not alter the Km of substrate hydrolysis, with the change in activity the result of an increase in apparent Vmax. Northern blot analysis indicates that the level of CPE mRNA is not influenced (less than 10%) by the treatment, whereas the level of prolactin mRNA is increased 9-fold. These results indicate that CPE is not coordinately regulated with prolactin in the GH4C1 cell line, although some regulation of CPE activity does occur.     

12-O-tetradecanoyl-phorbol-13-acetate decreases influx of extracellular Ca2+ induced by depolarization in GH4C1 cells: effects of pretreatment with 1,25-dihydroxycholecalciferol.

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol (1,25(OH)2D3) causes amplification of both the TRH-induced spike phase in cytosolic free calcium [( Ca2+]i) and the increase in [Ca2+]i induced by depolarization with K+. In the present study we investigated the actions of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Ca2(+)-homeostasis in GH4C1 cells pretreated with 1,25(OH)2D3 for 24 h. In control and 1,25(OH)2D3-pretreated cells, incubation with TPA (0.1-300 nM) for 15 min in the presence of 45Ca2+ did not affect the basal uptake of 45Ca2+. However, if the cells were treated with 50 mM K+, TPA induced a time- and concentration-dependent decrease in depolarization-induced net 45Ca2+ uptake. A maximal decrease of 30-50% was observed with 100-300 nM TPA, 1,25(OH)2D3 pretreated cells being more responsive to the action of TPA than control cells. sn-1-Oleoyl-2-acetyl-glycerol, which mimics the action of TPA on protein kinase C (PKC), did not alter depolarization-induced uptake of 45Ca2+. Two agents which inhibit PKC activity, polymyxin B and K252A, did not prevent the effect of TPA on depolarization-induced uptake of 45Ca2+, whereas staurosporin totally inhibited the action of TPA. In Fura-2 loaded cells pretreated with 1,25(OH)2D3, incubation with 200 nM TPA for 9 min decreased the depolarization-induced spike and plateau phases of change in [Ca2+]i; only the spike phase was decreased in control cells. TPA did not affect basal [Ca2+]i in either group. Treatment with TPA for only 3 min decreased the TRH-induced spike in [Ca2+]i only in 1,25(OH)2D3 pretreated cells; however, after a 5-min treatment with TPA, the TRH-induced spike in [Ca2+]i was decreased in both control and 1,25(OH)2D3 pretreated cells. TPA did not affect the spike in [Ca2+] induced by 50 nM ionomycin. Na+/Ca2+ exchange was not altered by TPA, nor did TPA enhance efflux of 45Ca2+ from cells preloaded with 45Ca2+ for 2.5 h. We conclude that, in GH4C1 cells, TPA modulates plasma membrane calcium flux, probably via an inhibitory action on voltage-operated Ca2+ channels. This inhibitory action may be independent of activation of PKC, and 1,25(OH)2D3 pretreated cells are more responsive to the actions of TPA than are control cells. These results are consistent with our previous findings that 1,25(OH)2D3 enhances voltage-dependent Ca2+ channel activity in GH4C1 cells.     

Role of extracellular calcium and calmodulin in prolactin secretion induced by hyposmolarity, thyrotropin-releasing hormone, and high K+ in GH4C1 cells

The mechanism by which 30% medium hyposmolarity induces PRL secretion by GH4C1 cells was compared with that induced by 100 nmol/l TRH or 30 mmol/l K+. Removing medium Ca2+, blocking Ca2+ channels with 50 mumol/l verapamil, or inhibiting calmodulin activation with 20 mumol/l trifluoperazine, 10 mumol/l chlorpromazine or 10 mumol/l pimozide almost completely blocked hyposmolarity-induced secretion. The smooth muscle relaxant, W-7, which is believed relatively specific in inhibiting the Ca2(+)-calmodulin interaction, depressed hyposmolarity-induced PRL secretion in a dose-dependent manner (r = -0.991, p less than 0.01). The above drugs also blocked or decreased high K(+)-induced secretion, but had much less effect on TRH-induced secretion. Secretion induced by TRH, hyposmolarity, or high K+ was optimal at pH 7.3-7.65 and was significantly depressed at pH 6.0 or 8.0, indicating that release of hormone induced by all 3 stimuli is due to an active cell process requiring a physiologic extracellular pH and is not produced by nonspecific cell toxicity. The data suggest hyposmolarity and high K+ may share some similarities in their mechanism of stimulating secretion, which is different from that of TRH.     

Kisspeptin-1 directly stimulates LH and GH secretion from goldfish pituitary cells in a Ca(2+)-dependent manner

It has been established that kisspeptin regulates reproduction via stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion, which then induces pituitary luteinizing hormone (LH) release. Kisspeptin also directly stimulates pituitary hormone release in some mammals. However, in goldfish, whether kisspeptin directly affects pituitary hormone release is controversial. In this study, synthetic goldfish kisspeptin-1((1-10)) (gKiss1) enhances LH and growth hormone (GH) release from primary cultures of goldfish pituitary cells in column perifusion. gKiss1 stimulation of LH and GH secretion were still manifested in the presence of the two native goldfish GnRHs, salmon (s)GnRH (goldfish GnRH-3) and chicken (c)GnRH-II (goldfish GnRH-2), but were attenuated by two voltage-sensitive calcium channel blockers, verapamil and nifedipine. gKiss-induced increases in intracellular Ca(2+) in Fura-2AM pre-loaded goldfish pars distalis cells were also inhibited by nifedipine. These results indicate that, in goldfish, (1) direct gKiss1 actions on pituitary LH and GH secretion exist, (2) these actions are independent of GnRH and (3) they involve Ca(2+) signalling.