Search

Bovine anterior pituitary cells phosphorylate prolactin (PRL). We describe the phosphorylation of endogenous and exogenous bPRL in highly enriched subcellular fractions of bovine anterior pituitary using [gamma-32P]-ATP. 32P-labeling of endogenous and exogenous bPRL occurred in all subcellular membrane fractions, but most significantly in the fraction enriched for secretory granules. Zn2+ (0.8 mM), Cu2+ (0.8 mM), and Mn2+ (9.8 mM) increased bPRL phosphorylation by 268, 214, and 154%, respectively, relative to basal phosphorylation with no added cations. Neither Mg2+ (10 mM) nor Ca2+ (0.9 mM) increased bPRL phosphorylation above basal levels. Phosphorylation was dependent on the concentration of Zn2+ with an apparent Km of 570 microM. bPRL phosphorylation occurred over a wide pH range of 5.9-8.3, with the greatest activity at pH of 6.7 or greater. Phosphorylation of bPRL was time-dependent. The apparent Kms of the bPRL kinase for exogenous bPRL and ATP were 15.3 and 267 microM, respectively. bPRL incorporation of 32P was unaffected by the presence of calcium and calmodulin, cAMP, phosphotidylserine and diolein, or spermine. From these results we conclude that in vitro phosphorylation of bPRL occurs under physiological conditions that would be found in pituitary cells.

In the anterior pituitary, cGMP is produced in response to a number of stimuli, but intracellular events distal to cGMP production are obscure. Since cGMP-dependent protein kinase (PKG) is a major effector of cGMP actions in other tissues we have determined whether PKG and its specific substrates might be present and responsive to external signals in the ovine anterior pituitary. Photoaffinity labelling with [32P]cGMP revealed a specific 78 kDa protein in ovine anterior pituitary that comigrated with purified bovine lung PKG-I. PKG in protein extracts from anterior pituitary or cultured anterior pituitary cells was enriched by DEAE ion-exchange chromatography and assayed for activity. Both tissue and cultured cells had a relatively high PKG activity by comparison with aortic smooth muscle (known high activity) and brain (known low activity). Subcellular distribution studies showed that in anterior pituitary, aortic and brain, PKG activity was present in both cytosol and triton-extracted membrane fractions, while in platelets the activity was associated with only the membrane fraction. To determine if this PKG might be responsive to extracellular signals an activity ratio assay was used. Incubation of cultured cells with atrial natriuretic peptide (ANP) and sodium nitroprusside, activators of membrane and cytosolic guanylate cyclases respectively, increased the activity of PKG. To determine events distal to PKG activation, a search for potential substrates of PKG was performed. Few substrates were detectable upon addition of purified PKG to tissue lysates due to the high background activity of endogenous protein kinases in the anterior pituitary. However, 19 substrates of PKG were detected in heat-stable and 14 in acid-soluble protein extracts of the anterior pituitary, in which background phosphorylation was almost abolished. After partial purification through Q-Sepharose ion-exchange chromatography some of these proteins were preferentially phosphorylated by addition of PKG-I, while the others were additionally substrates of exogenous cAMP-dependent protein kinase (PKA) or Ca2+ and phospholipid-dependent protein kinase (PKC). A 132-kDa substrate showed an identical phosphopeptide map to a PKG substrate previously described in vascular smooth muscle and platelets. These data demonstrate for the first time the presence of functional PKG activity and multiple PKG substrates in the anterior pituitary where they may play a role in mediating the intracellular actions of cGMP.

In order to explore the role of certain GTP binding proteins in the rat anterior pituitary, we have analyzed the subcellular distribution of the proteins rho and rab. They were found in both membrane and cytosolic fractions. Rab1 and rab2 were localized in both Golgi and endoplasmic reticulum (ER) membranes, while rab4 and rab6 were found in fractions enriched with Golgi and plasma membranes, implicating these proteins in the control of vesicular intracellular trafficking as described in other systems. Rab3 was localized like a fraction of synaptophysin, suggesting a role for rab3 in the targeting of "synaptic-like' microvesicles. We have identified three substrates of C. botulinum exoenzyme C3. A 26-kDa substrate with an isoelectric point (pI) of 5.2, probably rhoB, was localized in the lightest fractions such as rab3 and synaptophysin proteins. Two other 23-24 kDa substrates with pI of 5.5-5.8, probably rhoA and/or rhoC, were found in both fractions enriched with ER and secretory granules. Rho proteins have been implicated in the control of actin polymerization. Their localization in anterior pituitary suggests that rhoB could control the association of synaptic-like microvesicles and plasma membrane, and that rhoA/rhoC could play a role in secretory granule exocytosis; these two pathways being involved in cytoskeleton protein reorganisation in response to extracellular signals.

The LHRH receptor in alpha T3-1 gonadotrope cells was shown to bring about a marked and sustained activation of MAP kinase. This response was prevented by protein kinase C inhibition or down-regulation and could be partially mimicked by phorbol ester. Additional evidence for inhibition of this response by pertussis toxin and partial mimicry by mastoparan (in a pertussis toxin-sensitive manner) provides the first evidence for Gi/Go-mediated signal transduction by the LHRH receptor. This dual mechanism of MAP kinase activation appears to be exceptional amongst the G protein-linked receptors that have been investigated.

The negative feedback control of thyrotropin production by the anterior pituitary involves local 5'-deiodination of L-thyroxine (L-T4) to the active thyroid hormone 3,3',5-triiodo-L-thyronine (T3) by two 5'-deiodinase isozymes which are distinctly regulated by thyroid hormone. T3 rapidly increased steady-state mRNA levels and activity of type I iodothyronine-5'-deiodinase (5'DI) in rat anterior pituitary and in reaggregate cultures of anterior pituitaries. Type II 5'-deiodinase activity determined in parallel with 3,3',5-triiodo-L-thyronine (rT3) as substrate was markedly lower than that of 5'DI. 5'DI mRNA levels and activity were higher in anterior pituitaries of female compared to male rats. Neither gender differences nor T3 stimulation of 5'DI activity were found in the posterior part. These data demonstrate a T3 dependent expression of 5'DI in euthyroid anterior pituitary and suggest that this isozyme serves a major function within the complex network of thyroid hormone homeostasis.

The protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu) induced the release of both luteinizing hormone (LH) and growth hormone (GH) from proestrous rat anterior pituitary pieces in vitro. Phorbol 12,13-dibutyrate-induced LH, but not GH release was readily inhibited by the phospholipase A2 (PLA2) inhibitors, quinacrine, aristolochic acid, ONO-RS-082 and chloracysine. Furthermore, PDBu induced release of [3H]arachidonic acid ([3H]AA) from pre-labelled anterior pituitary tissue that was prevented in the presence of quinacrine, aristolochic acid and ONO-RS-082 but not the diglyceride lipase inhibitor RHC 80267. The effect of PDBu was completely inhibited by staurosporine and the selective PKC inhibitor Ro 31-8220 but only partially by low concentrations of H7; consistent with the involvement of both H7-sensitive and H7-resistant forms of PKC in the activation of PLA2 by PDBu. The protein synthesis inhibitor cycloheximide inhibited the release of both [3H]AA and LH that had been induced by PDBu, whereas LH release induced by the PLA2 activator mellitin was cycloheximide-insensitive. These results suggest that PKC activators may induce LH but not GH release from anterior pituitary tissue by a mechanism involving activation of a PLA2, brought about by a process which is reliant on protein synthesis.

Carboxypeptidase H (CP-H) has been characterized in anterior and posterior lobes of bovine pituitary with regard to subcellular distribution, enzymatic properties, nature of membrane association, number of forms and C-terminal processing. Anterior lobe contained both soluble and membrane forms of CP-H with similar enzymatic properties after extraction at pH 5.5. The soluble forms represented about 70% of the total activity. Two soluble and two membrane forms, of 53 and 56 kDa, were demonstrated by immunoblotting and after purification. The amount of the 56 kDa soluble form increased with the salt content of the extraction buffer. Endoglycosidase digestions showed that the difference in size was not due to differential N-linked glycosylation and also showed that CP-H oligosaccharides do not become resistant to endoglycosidase H. CP-H in posterior lobe was also composed from about 70% soluble and 30% membrane forms. Only 53 kDa CP-H was detected in soluble and membrane fractions of posterior lobe. All forms of CP-H from both lobes reacted with antiserum directed to the C-terminus. These results do not support previous observations that soluble and membrane forms of CP-H can be distinguished by size and C-terminal processing.

The purpose of this study was to investigate the involvement of protein kinase C in growth hormone-releasing factor (GRF) action by directly measuring the effect of GRF on protein kinase C activity in purified male rat somatotrophs. Somatotrophs were incubated with GRF (10(-7) M) for 0.33, 1, 3, 10, 30 and 90 min. Protein kinase C present in soluble and particulate fractions was partially purified using DEAE-cellulose chromatography, and protein kinase C activity was assayed. In control experiments, to insure protein kinase C activity could be activated, two known protein kinase C activators, phorbol 12-myristate 13-acetate (PMA) and dioctanoyl-rac-glycerol (diC8) were added for 3 min. Protein kinase C activity is present in somatotrophs. Under basal conditions the majority of the enzyme activity is located in the cytosol (approximately 90%). The protein kinase C activators caused a significant translocation of protein kinase C activity from soluble to particulate fractions at 3 min. GRF did not cause a translocation of protein kinase C activity even though GH release was significantly increased by 3 min. GRF did not significantly alter the specific activity of protein kinase C in the soluble or particulate fractions, except for a small (approximately 10%) increase in soluble activity at 90 min. We conclude that protein kinase C is present in the somatotrophs of the anterior pituitary. Protein kinase C, however, does not mediate the action of GRF and its role in signal transduction in somatotrophs awaits elucidation.

The present study examines the effect of reduction of protein kinase C (PKC) activity, as induced by either phorbol ester (PMA) down-regulation or staurosporine inhibition, on the secretion of ACTH from cultured anterior pituitary (AP) cells. Short-term (3 h) exposure of cells to 5 nM PMA resulted in almost complete desensitization to both PMA and vasopressin (AVP), while there was only a minor incidence on the effect of corticotropin-releasing factor (CRF). In contrast, long-term (12-24 h) exposure of cells to PMA, as well as pretreatment with staurosporine, dramatically reduced the stimulatory influence of CRF. This was shown not to be due to a decline in ACTH cells' stores, nor to the toxicity of phorbol ester or to a negative autofeedback of ACTH. Pretreatment of corticotrophs with PMA failed to dampen the CRF-induced cyclic AMP formation, while it caused a decline in the effects of forskolin and 8-bromoadenosine cyclic AMP. Stimulated ACTH secretion subsequent to either veratridine- or high K(+)-induced cell depolarization was likewise decreased. We conclude that in corticotrophs the stimulatory action of not only AVP, but also of that of CRF on ACTH secretion strongly relies on PKC activity. In the case of CRF, however, this may not be a primary consequence of receptor occupation, as evidence suggests an indirect relationship which may involve PKC regulation of Ca2+ channels and/or the ion's intracellular messenger function.

The Ca2+-phospholipid-dependent protein kinase, initially described by Takai et al. (J. Biol. Chem. 254, 3692-3695, 1979), has been identified in the anterior pituitary gland of the rat and sheep. The enzyme is essentially undetectable in initial cell extracts but marked activity is manifest following DEAE chromatography, suggesting the potential presence of an endogenous inhibitor of this enzyme. Two forms of this protein kinase exist in both sheep and rat anterior pituitary gland, both of which are similarly dependent upon Ca2+, phosphatidyl serine and diacylglycerol. Several endogenous substrates for this protein kinase have been observed in both the pars distalis and pars tuberalis of the sheep adenohypophysis.

A calmodulin-activated protein kinase has been identified in bovine anterior pituitary membranes. This enzyme phosphorylated one endogenous substrate of subunit molecular weight 53,000 in the membranes. Phosphorylation of this protein was rapid, was half-maximal at 2.5 microM calcium in the presence of saturating concentrations of calmodulin (CaM), and was inhibited by trifluoperazine and thioridazine. A second protein was phosphorylated by an endogenous protein kinase in anterior pituitary membranes. Phosphorylation of this 42,000 Mr protein was reduced by calcium, was independent of exogenously added CaM, and was increased by trifluoperazine or thioridazine. The 42,000 Mr protein may be the alpha-subunit of pyruvate dehydrogenase. Calcium-dependent protein phosphorylation was also observed in intact cells; the largest increases were seen in proteins of Mr 42,000, 21,000 and 17,000.

Phospholipid-dependent, Ca2+-activated protein kinase (C-kinase) was recently shown to be expressed in rat pituitary. The enzyme is activated by Ca2+ and phosphatidylserine (PS). Diacylglycerol (DG), which is liberated during phosphoinositide turnover, and the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) activate pituitary C-kinase in the presence of PS, even at resting levels of intracellular Ca2+ (10(-7) M), and increase the apparent affinity of the enzyme for Ca2+. While micromolar concentration of Ca2+ had no effect on the apparent affinity of the enzyme for PS (Km approximately 15 micrograms/ml), elevation of Ca2+ to the millimolar range produced a sharp increase in the apparent affinity for PS (Km approximately 5 micrograms/ml). Elevation of PS (up to 500 micrograms/ml) could not replace Ca2+ in supporting maximal enzyme activity even in the presence of DG. Cytosolic pituitary C-kinase (70% of total enzyme activity) is recovered in an inactive state and can be activated without further purification. The particulate enzyme (30%) is recovered in a cofactors-insensitive form but can be activated after detergent-solubilization and anion exchange chromatography. Endogenous redistribution of soluble pituitary C-kinase to the membrane does not convert it to its proteolytic product which is insensitive to Ca2+, PS and DG. Pituitary C-kinase characterized here most likely plays a key role in signal transduction mechanisms involved in pituitary functions.

Discrepant reports exist regarding the presence of glandular kallikrein or other trypsin-like serine proteases in the pituitary. The existence of pituitary kallikreins in latent forms could explain these discrepancies. I report that trypsin treatment of rat anterior pituitary homogenates activates two serine proteases which generate kinins from kininogen and selectively cleave chromogenic substrates for kallikreins. One protease (enzymatically and immunologically resembling glandular kallikrein) and activated 5-fold by trypsin and was 20 times more abundant in female than in male lobes due to hormonal regulation by ovarian estrogens. The second kallikrein (activated 20-fold by trypsin) was unaffected by estrogens. The results demonstrate that rat anterior pituitary kallikreins predominantly exist in latent forms requiring activation for detection. Additionally, glandular kallikrein is a major estrogen-induced protein in the rat anterior pituitary. No other member of this large protease family is known to be regulated by estrogens.

Changes in the activity of DNA polymerase and [3H]thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats. The activities of DNA polymerases alpha and beta, extracted in Tris--HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively alpha, while in the phosphate soluble fraction it is a mixture of alpha and beta. The administration of oestrogens to male rats increases [3H]thymidine incorporation and enhances the activity of DNA polymerases in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further increment of [3H]thymidine incorporation and of DNA polymerase activity in the Tris-soluble fraction. In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction. Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase alpha. This activity changes in parallel with [3H]thymidine incorporation into DNA which is an indication of cell proliferation in the gland. This is discussed with respect to a negative feedback mechanism between intracellular prolactin concentration and DNA synthesis in the APG.

Glandular kallikrein, a trypsin-like serine protease, and prolactin (PRL) are both estrogen-induced proteins in rat anterior pituitary lactotrophs. The estrogen agonist and antagonist effects of tamoxifen (TAM, a triphenylethylene antiestrogen) and chlorotrianisene (TACE, a triphenylethylene estrogen) on anterior pituitary glandular kallikrein and PRL were examined to see if TAM and TACE differentially affect these estrogen response of lactotrophs after in vivo dosing of rats. TAM and TACE acted as partial agonists on PRL and uterine weight induction. In contrast, on glandular kallikrein induction TAM acted as a pure estrogen antagonist and TACE acted as an almost pure antagonist. The results document that both TAM and TACE exhibit protein-specific estrogen agonist and antagonist efficacies in lactotrophs, with the estrogen induction of glandular kallikrein being particularly sensitive to antagonism by TAM in vivo. The marked antiestrogen character of TACE was surprising since TACE has been classified and clinically used as an estrogen.

The effect of chronic estrogen treatment on the stimulation and dopamine inhibition of anterior pituitary (AP) adenylate cyclase (AC) activity was examined. Treatment of ovariectomized female rats with estradiol for 21 days resulted in a 450% increase in AP weight compared to ovariectomized controls. Stimulation of AC by guanine nucleotides (GN) (1 nM-0.1 mM) and vasoactive intestinal peptide (1 microM) was reduced by 50%. Stimulation of AC by fluoride ions was unchanged by estradiol treatment. Stimulation above basal by forskolin was reduced by variable amounts (23-50%), and depended on the concentration of forskolin used. Inhibition of AC mediated by D2-dopamine receptors was decreased by 45%. Estrogen treatment had no effect on the toxin-catalyzed incorporation of [32P]ADP into stimulatory and inhibitory GN regulatory proteins. These results indicate that the effect of estrogen on the anterior pituitary include modulation of stimulated, dopamine-inhibited and basal AC activity.

Subcutaneous injection of oestradiol-17beta enhanced the thymidine kinase activity in the anterior pituitary of immature male rats, but did not alter the thymidylate synthetase level. The kinase activity reached a sharp maximum 36 hours after injection of the steroid and then decreased to its original value. The estimated minimum active dose was 0.020 mug per rat. The 17alpha isomer was inactive. Cycloheximide and actinomycin D, according to injection schedule, prevented the rise in activity, suggesting a regulation of pituitary thymidine kinase at the level of protein biosynthesis. The induced enzyme exhibited the same Km and the same thermal inactivation profile as the constitutive enzyme. With the doses of oestradiol required for induction of the enzyme, no significant variations of serum and pituitary LH and FSH concentrations were detected. Based on these and previous results it is suggested that, in the male pituitary, the concentration ratio of circulating oestrogens over androgens controls not only the secretory function but also the production of specific enzymes.