Cultures Of Anterior Pituitary Cells Research Articles

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys-NH 2. The N-terminal sequence [1–28] shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10 −10M (GH, PRL, ACTH) or 10 −9M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.

Phosphorylation of a group of proteins related to the physiological, multihormonal regulations of the various cell types in the anterior pituitary gland.

We previously identified a group of cytoplasmic phosphoproteins whose phosphorylation could be related to the multihormonal regulation of PRL in the homogeneous tumor-derived GH cell lines (set of proteins 1-11) and in heterogeneous normal anterior pituitary cells in culture (set of proteins 1-15). In normal cells, a mixture of hypothalamic hormones induced, like the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, stronger phosphorylation changes than TRH alone. Proteins of the set 1-15 are therefore likely to be present also in the nonmammotrophic anterior pituitary cell types, where their phosphorylation can be regulated by the hormones specific of the cell type considered. This interpretation is confirmed by the presence of the same proteins in cells of the corticotroph-like AtT-20/D16 cell line and the regulation of their phosphorylation by CRF. The same phosphoproteins were also detected in non dissociated anterior pituitary tissue from ovariectomized rats. Their phosphorylation was regulated by various hormones and other extracellular agents in a way similar to their regulation in anterior pituitary cells in culture. A 5-day estradiol implant pretreatment of the ovariectomized rats, which stimulates the anterior pituitary gland both directly and indirectly, resulted in a very high level of basal phosphorylation of proteins 1-15. Only very little further stimulation was achieved by the addition of exogenous hypothalamic hormones, indicating that the actual physiological regulatory pathways are the same as those unraveled in the various cell culture model systems. In conclusion, phosphorylation of proteins 1-15 1) can be related to the multihormonal regulation of the various anterior pituitary cell types in culture and 2) corresponds to intracellular molecular mechanisms actually involved in the physiological regulations of pituitary functions in the intact anterior pituitary gland.

Radioimmunoassay of inhibin in various mammals

A sensitive radioimmunoassay (RIA) for the determination of inhibin in peripheral plasma and tissue homogenates of different species has been developed using antisera to partially purified bovine follicular fluid (bFF) inhibin and 125I-labelled bFF 32 kDa inhibin. Antisera were produced by immunization of rabbits with partially purified bFF inhibin prepared by immunoaffinity chromatography. Increasing doses of a high titre antiserum could neutralize the suppressing effect of bFF, porcine follicular fluid and rat ovarian homogenate on FSH secretion from rat anterior pituitary cells in culture. Sensitivity of the assay was 3.1 ng International Research Standard of porcine inhibin per tube. Parallel inhibition curves were obtained for inhibin preparations from female and male animals of ten species, i.e. cattle, goats, sheep, cats, dogs, monkeys, pigs, horses, rats and man. Inhibin subunits and related proteins cross-reacted minimally with the antiserum used in the study. Plasma concentrations of inhibin in adult male and female rats were measured by the RIA before and at various times after gonadectomy. Inhibin levels in peripheral plasma before gonadectomy were significantly higher in adult female than in adult male rats. Inhibin levels decreased abruptly after gonadectomy in both sexes and they correlated negatively with plasma concentrations of FSH. This inhibin RIA will facilitate studies of the physiology of inhibin in various species of animals.

Burmese Russell's viper venom causes hormone release from rat pituitary cells in vitro

Acute and chronic hypopituitarism is associated with severe envenoming by the Burmese Russell's viper. We have demonstrated that in vitro, Burmese Russell's viper venom (0.1-10 micrograms/ml) causes a dose-dependent release of GH, TSH and ACTH from dispersed rat anterior pituitary cells in culture. At 10 micrograms/ml, venom causes a significant increase in the release of GH (344%, P less than 0.001), TSH (168%, P less than 0.005) and ACTH (greater than 700%, P less than 0.001). We have also shown that the component (or components) responsible for this stimulatory effect is stable to heat (60 degrees C, 1 h) and mild trypsinization. Repeated addition of venom (1 microgram/ml) to pituitary cells in a perifusion column system demonstrated attenuation of GH release. This reduced response was not due to depletion of the GH pool since the pituitary cells were subsequently able to respond to both GH-releasing factor (GRF) stimulation and KCl depolarization. Somatostatin in a dose which abolished GRF-stimulated GH release failed to affect venom-stimulated GH release, implying that venom acts in a cyclic AMP-independent manner. We conclude that Burmese Russell's viper venom has direct effects on pituitary hormone release in vitro. Whether these effects contribute to its known actions in vivo on the function of the pituitary remains to be established.

Transmembrane signals mediating adrenocorticotropin release from mouse anterior pituitary cells

The effect of arginine vasopressin (AVP) and corticotropin releasing factor (CRF) an adrenocorticotropin (ACTH) secretion, phosphatidylinositol breakdown and cAMP accumulation was examined in primary cultures of mouse anterior pituitary cells. AVP and CRF added alone stimulated ACTH secretion in a dose-dependent manner. At 10 −8 M concentration of peptide, AVP and CRF stimulated ACTH secretion 2.8- and 4.6-fold, respectively. AVP and CRF added in combination at equal doses gave an additive effect. CRF enhanced cAMP accumulation, but AVP had no effect on basal or CRF-induced cAMP accumulation. Both forskolin (10 −5 M) and 8-bromo-cAMP (10 −3 M) increased ACTH secretion in these cells by 2.8- and 1.7-fold, respectively. AVP induced the breakdown of phosphoinositides, and CRF alone, or in combination with AVP did not modify this effect. Phorbol 12-myristate 13-acetate (10 −7 M) dioctanoylglycerol (10 -4 M) and phospholipase C (100 mU/ml) also stimulated ACTH secretion in these cells by 4.2-, 2.4-, and 3.7-fold, respectively. Depletion of intracellular and extracellular Ca 2+ decreased ACTH secretion, but had no significant effect on CRF-induced cAMP accumulation. However, AVP-induced phosphoinositide breakdown was dependent on extracellular Ca 2+. These results indicate that CRF stimulates ACTH secretion via the cAMP-dependent pathway and AVP via the phosphoinositide breakdown-phospholipase C pathway. In the presence of AVP and CRF, both pathways appear to operate independently to produce an additive effect on ACTH secretion.

Nitroimidazole Derivatives Inhibit Anterior Pituitary Cell Function Apparently by a Direct Effect on the Catalytic Subunit of the Adenylate Cyclase Holoenzyme*

Nitroimidazole derivatives dose-dependently decreased basal and CRF-stimulated ACTH release, basal and GRF-stimulated rat GH release, and basal rat PRL release in primary cultures of rat anterior pituitary cells. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with the nitroimidazole derivatives. Miconazole, econazole, isoconazole, clotrimazole, and bifonazole had similar or more pronounced effects on anterior pituitary function compared to ketoconazole, whereas metronidazole and etomidate were less effective. The positive correlation between the number of phenylated side-chains or phenolic rings of the imidazole molecule and the efficacy to inhibit activity on pituitary hormone secretion suggests a structure-activity relationship of these compounds. The effects of the nitroimidazole derivatives on anterior pituitary hormone release and biosynthesis were mediated by cAMP. Thus, basal and CRF-, cholera toxin-, and forskolin-stimulated adenylate cyclase activities in rat anterior pituitary cell membranes determined by cAMP formation were suppressed by the nitroimidazole derivatives. Pertussis toxin did not diminish the nitroimidazole derivative effect on cAMP formation. The adenylate cyclase inhibitory effect of these substances was independent of the presence of GTP in the assay system, underlining a direct effect on the catalytic subunit. In addition, basal and forskolin-stimulated cAMP generation in membranes of S49 lymphoma cyc-variants, which lack a functional Gs protein, was efficiently suppressed (by up to 90%) by the nitroimidazole derivatives. In conclusion, ketoconazole and other nitroimidazole derivatives inhibit anterior pituitary hormone synthesis and secretion apparently by a direct effect on the catalytic subunit of the adenylate cyclase system.

Presence of calcitonin-like peptide in rat milk: possible physiological role in regulation of neonatal prolactin secretion.

Previous results have shown that salmon calcitonin (sCT), a peptide in rat brain and pituitary gland, inhibits basal and TRH-stimulated PRL release and reduces PRL mRNA levels in isolated anterior pituitary cells of adult rats in culture. Rat milk contains a variety of neuropeptides and hormones, some of which are absorbed in bioactive form to exert endocrine influences in the developing offspring. The present studies were undertaken to investigate whether a CT-like peptide is present in rat milk. Circulating PRL levels in neonatal rats are low, and there is an abrupt increase in the basal secretion of this hormone at weaning. A second objective was to examine whether CT plays a role in the regulation of PRL secretion in neonatal animals. A sensitive and specific RIA for sCT was developed and used to assay rat milk on various days of lactation for sCT-like immunoreactivity. sCT-like activity was present in the water-soluble (infranatant) fraction of milk throughout lactation in concentrations as high as 1589 pg/ml. There were no statistically significant differences in immunoreactive levels of the peptide in milk samples from different days of lactation. sCT-like immunoreactivity in rat milk infranatant coeluted with synthetic sCT on reverse phase HPLC, and these HPLC fractions inhibited basal PRL release when added to cultures of anterior pituitary cells. This inhibition of PRL release by the sCT-immunoreactive HPLC fractions was comparable to that exerted by equivalent concentrations of synthetic sCT. Newborn rats were injected sc with 10 microliters normal rabbit serum or anti-sCT serum from the day of birth until postpartum day 10. The rats were killed on day 11, and their sera were analyzed for PRL. Anti-sCT-injected rats showed a significant increase in serum PRL levels compared to those in untreated or normal serum-treated rats. These results demonstrate that a CT-like peptide, which is a potent inhibitor of PRL release, is present in rat milk throughout lactation, and that passive immunization with a highly specific anti-sCT serum leads to an increase in serum PRL levels in neonatal rats. CT, possibly of milk origin, may be a physiologically relevant PRL-inhibiting factor during the neonatal period.

Stimulation by atrial natriuretic factor of cyclic GMP production in cultured anterior and intermediate pituitary tissues: evidence for a major contribution of proliferating nonendocrine cells

Primary cultures of anterior and intermediate pituitary tissues were monitored immunocytochemically for the presence of endocrine and nonendocrine cells and simultaneously tested for their ability to produce cyclic GMP in response to atrial natriuretic factor (ANF). Cells cultured for 3 days and 6 days, in which nonendocrine (vimentin-positive) cells were found to rapidly overgrow the endocrine cells, showed a dramatic elevation in cyclic GMP production stimulated by ANF, with maximum stimulation 300–700% that seen in 1-day cultured cells. Also, ANF-induced accumulation of cyclic GMP in an enriched population of vimentin-positive cells appeared to closely match that triggered in a 3-day culture of anterior pituitary cells, emphasizing the major role played by nonendocrine cells and their ability to synthesize cyclic GMP. In contrast, in the homogeneous population of tumor corticotrophs AtT-20, there was a close relationship between cyclic GMP formation and cell density. It thus appears that contamination of primary cultures of anterior and intermediate pituitary tissues by proliferating nonendocrine cells (mainly fibroblasts), in which ANF-induced accumulation of cyclic GMP may be confused with that of the very secretory cells, leads to overestimation and masking of guanylate cyclase activity of endocrine cells.

Selective Suppression of Follicle-Stimulating Hormone Secretion in Anterior Pituitary Cells by the Gonadal Steroid 3α-Hydroxy-4-Pregnen-20-One*

The effect of 3 alpha-hydroxy-4-pregnen-20-one (3HP), a Sertoli cell steroid, on the secretion of gonadotropins from rat anterior pituitary cells in culture was examined and subsequently compared with the action of other gonadal steroids and steroids structurally related to 3HP. Pituitary cells from randomly cycling, sexually mature female rats were isolated and maintained in culture 72 h before use. On the day of treatment, medium was changed, steroids (10(-16)-10(-4) M) and/or LHRH (10(-8) M) were added, and cells were allowed to incubate for a further 24 h. Medium was then examined for gonadotropin content by RIA. 3HP treatment of anterior pituitary cells resulted in a significant reduction of both basal and LHRH-induced FSH secretion, while LH secretion was unaffected. The lowest effective dose of 3HP (10(-16) M) significantly decreased basal FSH secretion to 65.6% of control levels. The lowest effective dose of 3HP that significantly inhibited (by 31%) LHRH-induced FSH secretion was 10(-14) M 3HP. Maximum suppression by 3HP of basal FSH secretion occurred between 10(-10)-10(-8) M, and maximum suppression of LHRH-induced secretion occurred at 10(-12) M. None of the other gonadal steroids tested (progesterone, testosterone, 5 alpha-dihydrotestosterone, 17 beta-estradiol, 20 alpha-hydroxy-4-pregnen-3-one, and 5 alpha-pregnane-3,20-dione) had a similar selective effect on FSH secretion; progesterone, testosterone, and 17 beta-estradiol actually resulted in increased FSH release, and 5 alpha-pregnane-3,20-dione resulted in significant increase in basal LH. A number of metabolites and structural variations of 3HP were examined in this in vitro system at concentrations of 10(-12)-10(-6) M, and none exhibited a similar selective FSH-suppressing activity as 3HP. The data suggest that the selective FSH-suppressing effect of 3HP seen previously in vivo and here in vitro is due to 3HP itself and not the result of a metabolite of this molecule.

Effects of tri-iodothyronine, cortisol and transcriptional inhibitors on vitamin D3-enhanced thyrotrophin secretion by rat pituitary cells in vitro

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to selectively enhance agonist-induced TSH release in the rat thyrotroph in vitro. The interaction of 1,25-(OH)2D3 with tri-iodo-thyronine (T3) and cortisol was studied in primary cultures of dispersed anterior pituitary cells. TRH (1 nmol/l)-induced TSH release over 1 h was enhanced by 70% (P less than 0.01) following exposure to 10 nmol 1,25-(OH)2D3/l for 24 h. Pretreatment with T3 (1 pmol/l-1 mumol/l) for 24 h caused a dose-dependent inhibition of TRH-induced TSH release. Net TRH-induced TSH release was inhibited by 85% at T3 concentrations of 3 nmol/l or greater. Co-incubation with 1,25-(OH)2D3 resulted in enhanced TRH-induced TSH release at all T3 concentrations tested (P less than 0.001). The increment of TRH-induced TSH release resulting from 1,25-(OH)2D3 pretreatment was equivalent in the presence or absence of maximal inhibitory T3 concentrations. At 1 nmol T3/l, there was a two- to threefold relative increase in 1,25-(OH)2D3-enhanced TRH-induced TSH release. Incubation with cortisol (100 pmol/l-100 nmol/l) had no effect on basal or TRH-induced TSH release, nor did it alter 1,25-(OH)2D3-enhanced TRH-induced TSH release when added 24 h before, or at the time of addition of 1,25-(OH)2D3. Actinomycin D and alpha-amanitin abolished 1,25-(OH)2D3-enhanced TSH secretion. These data demonstrate that the action of 1,25-(OH)2D3 in the thyrotroph required new RNA transcription, and was not affected by cortisol. In the presence of T3, the response of the thyrotroph to TRH induced by 1,25-(OH)2D3 was increased. We have shown that 1,25-(OH)2D3 has significant effects on the action of TRH and T3 in vitro. These findings support the proposal that 1,25-(OH)2D3 may modulate TSH secretion in vivo.

Differentiation of Striated Muscle Fibers in Monolayer Cultures of Rat Anterior Pituitary: (rat pituitary/cell culture/differentiation/striated muscle).

Striated muscle fibers appeared in monolayer cultures of rat anterior pituitary cells maintained in αMinimum Essential Medium (αMEM). As muscle differentiation in cultures of pituitary cells under ordinary conditions has not hitherto been reported, an in vitro study was undertaken to determine what factor(s) is responsible for this myogenesis. When dispersed anterior pituitary cells were culrured in three different media, αMEM, Medium 199 and Dulbecco's Modified Eagle Medium (DMEM), only αMEM induced a high incidence of striated muscles. The nature of the serum (fetal calf, calf and horse) and its concentration (1-10%) did not affect myogenesis. In monolayers in αMEM, the sequence of differentiation of striated muscle was as follows: 1) Elongated cells, resembling myoblasts appeared; 2) these cells fused; and finally 3) cross striations appeared. Rhythmic contraction was most intense in striated muscle fibers, but it was also obsrved in myotubes without cross striations and even in myogenic cells before fusion. The possible origin of muscles in these pituitary cultures is discussed.

Ether as an Anesthetic for Decapitation in the Rat: Gonadotropin Secretion by Subsequently Established Anterior Pituitary Cell Cultures

Historically, for the establishment of dispersed anterior pituitary cell cultures, rodents have been killed by decapitation without anesthesia. Because decapitation fails to induce immediate unconsciousness, the American Veterinary Medical Association Panel on Euthanasia has recently recommended that rodents should not be decapitated without previous anesthesia or sedation. Investigators are therefore confronted with the dilemma of wishing to euthanize rodents humanely yet not wishing to potentially compromise experimental data through the use of anesthetics. We present our observations on the effects of diethyl ether anesthesia administered prior to decapitation on the gonadotropin secretory characteristics exhibited in vitro by cultured rat anterior pituitary cells. Neither light nor complete (surgical level) ether anesthesia had any statistically significant effect on either luteinizing hormone or follicle-stimulating hormone responsiveness or cell content of luteinizing hormone and follicle-stimulating hormone or DNA content. The data indicate that ether anesthesia would appear to be acceptable for those studies involving subsequent in vitro luteinizing hormone and follicle-stimulating hormone secretion.

Effects of hypothyroidism, tri-iodothyronine and glucocorticoids on growth hormone responses to growth hormone-releasing hormone and His-d-Trp-Ala-Trp-d-Phe-Lys-NH2

The aim of this study was to investigate the role of thyroid hormones and glucocorticoids on GH secretion. Secretion of GH in response to GH-releasing hormone (GHRH) (5 micrograms/kg) was markedly (P less than 0.001) decreased in hypothyroid rats in vivo (peak GH responses to GHRH, 635 +/- 88 micrograms/l in euthyroid rats vs 46 +/- 15 micrograms/l in hypothyroid rats). Following treatment with tri-iodothyronine (T3; 20 micrograms/day s.c. daily for 2 weeks) or cortisol (100 micrograms/day s.c. for 2 weeks) or T3 plus cortisol, a marked (P less than 0.01) increase in GH responses to GHRH was observed in hypothyroid rats (peak GH responses, 326 +/- 29 micrograms/l after T3 vs 133 +/- 19 micrograms/l after cortisol vs 283 +/- 35 micrograms/l after cortisol plus T3). In contrast, none of these treatments modified GH responses to GHRH in euthyroid animals. Hypothyroidism was also associated with impaired GH responses to the GH secretagogue, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). Secretion of GH in response to GHRP-6 in vivo was reduced (P less than 0.01) in hypothyroid rats (peak GH responses, 508 +/- 177 micrograms/l in euthyroid rats vs 203 +/- 15 micrograms/l in hypothyroid rats). In-vitro studies carried out using monolayer cultures of rat anterior pituitary cells derived from euthyroid and hypothyroid rats showed a marked impairment of somatotroph responsiveness to both GHRP-6 and somatostatin in cultures derived from hypothyroid rats. In summary, our data suggest that thyroid hormones and glucocorticoids influence GH secretion by modulating somatotroph responsiveness to different GH secretagogues.

Calcitonin inhibition of growth hormone-releasing hormone-induced GH secretion in normal men

Calcitonin has been shown to modulate pituitary hormone secretion in a variety of ways. In this study we examined the effects of a salmon calcitonin infusion on GHRH-induced GH secretion in 5 normal men. In addition, in vitro experiments were performed using primary cultures of rat anterior pituitary cells in order to examine whether there is a direct pituitary effect of CT. Infusion of CT significantly blunted the GH response to GHRH in all subjects without affecting basal GH secretion or plasma calcium levels. Infusion of CT was accompanied by significant increases in ACTH, beta-endorphin, cortisol and free fatty acid levels, and by a significant decrease in serum insulin levels. The addition of CT to primary cultures of rat pituitary cells did not alter basal or stimulated secretion of GH or ACTH. These results indicate that: 1) CT blunts the GH response to GHRH; 2) CT infusion results in the stimulation of the hypothalamic-pituitary-adrenal axis, and 3) this effect is probably exerted at the hypothalamic level, since no direct activity of CT was documented in vitro on either GH or ACTH secretion.

Platelet-activating factor stimulates prolactin release from dispersed rat anterior pituitary cells in vitro.

The biologically active phospholipid (platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated PRL release from dispersed rat anterior pituitary cells in culture. PAF-induced PRL release was dose dependent, with threshold stimulation at 1 nM and maximal stimulation at 100 nM. Stimulation occurred as early as 1 min of incubation and persisted for 2 h. The action of PAF on PRL release is consistent with a receptor-mediated mechanism based on the observations that the action of PAF is blocked by dopamine agonists and the PAF receptor antagonists L 652731 and SRI 63072. The structural analogs 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, which lack the biological activity of PAF, are not able to stimulate PRL release over the dose range 0.2-2 microM. In addition, the PAF precursor lyso PAF and diacyl-sn-glycero-3-phosphocholine (phosphatidylcholine) were ineffective in stimulating PRL release. PAF induced the secretion of PRL and GH but not that of LH or TSH from hemipituitaries in short term incubations. PAF did not effect PRL release from GH3 cells. In conclusion, these data indicate that PAF stimulates PRL release from primary cultures of rat anterior pituitary cells in a dose-related, rapid, and specific manner.

Differential Modulation of D1 and D2 Dopamine‐Sensitive Adenylate Cyclases by 17β‐Estradiol in Cultured Striatal Neurons and Anterior Pituitary Cells

Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17 alpha-estradiol, dexamethasone, nor progesterone used at the same concentration (10(-9) M) was effective. Furthermore, the modulation of DA-sensitive adenylate cyclases by the steroid required long-term exposure of living cells to 17 beta-estradiol since neither 17 beta-estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA-sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17 beta-estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17 beta-estradiol on DA target cells in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular adenosine triphosphate activates phospholipase C and mobilizes intracellular calcium in primary cultures of sheep anterior pituitary cells

In primary cultures of sheep anterior pituitary cells extracellular ATP (ED50 0.4–0.8 μM) stimulated efflux of 45Ca2+ from a slow-turnover intracellular pool. ADP was also effective whereas AMP and adenosine were not. The ATP effect was not due to cell permeabilization as 100 μM ATP did not elicit efflux of 2-deoxy[3H]glucose metabolites. This 45Ca2+ mobilization may be mediated by inositol trisphosphate, since ATP (ED50 1 μM) stimulated inositol phosphate generation. These results demonstrate P2-purinoceptors in sheep anterior pituitary cells which are coupled to phospholipase C activation and intracellular Ca2+ mobilization, and raise the possibility of a regulatory role for extracellular ATP in the anterior pituitary.

Effect of 17 β-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.

Iso Stimulation of GH and cAMP: Comparison of β-Adrenergic- to GRF-Stimulated GH Release and cAMP Accumulation in Monolayer Cultures of Anterior Pituitary Cells in vitro

Growth hormone (GH) release and cAMP content were measured in monolayer cultures of anterior pituitary cells after beta-adrenergic and GH-releasing factor (GRF) receptor activation. Isoproterenol (Iso, ED50-20 nM) was less potent than GRF (ED50-20 pM) in stimulating GH release. Iso caused a rapid stimulation of GH release that was maximal after 15 min and declined thereafter, while GRF caused a more gradual increase in GH secretion that was maximal after 30 min and remained elevated after 3 h. Both Iso- and GRF-stimulated GH release were preceded by an increase in cAMP content in the pituitary cells. Further, the addition of 3-isobutyl-1-methylxanthine (IBMX) to the medium enhanced the GH-stimulatory and cAMP-accumulating effects of both secretagogues. Experiments performed with native catecholamines and synthetic catecholamine agonists and antagonists indicated that the GH-stimulatory effect of Iso was mediated by a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors. Additionally, experiments performed with cultured GH3 tumor cells, found that incubation with GRF, Iso, vasoactive intestinal polypeptide, forskolin, or cholera toxin caused an increase in cAMP content in the cells. However, compared to the responses observed in primary pituitary cultures the GH secretory response to these agents was comparatively small. Together, these studies suggest that a mixed population of beta 1-adrenergic and beta 2-adrenergic receptors may act, at least in part, on somatotrophs in the anterior pituitary to stimulate GH release. Although both GRF and beta 2-adrenergic receptor agents affect GH release through a common second messenger system, their differing pharmacokinetic properties suggest distinct intracellular mechanisms.

Intrapituitary Cell-Cell Communication Regulates ACTH Secretion

Exposure of rat anterior pituitary cells in culture to a cytotoxin specific for CRF-target cells is followed by increased basal ACTH secretion from the remaining cells three days after the last exposure to the cytotoxin. When CRF-target-depleted populations are co-cultured with unexposed pituitary cells ACTH secretion is more than double that predicted in the absence of cell-to-cell interaction. These data provide evidence for an intrapituitary cell-to-cell signalling system modulating ACTH secretion.