Uncharged nuclear receptors for estrogen in breast cancers.

This study was undertaken to define the incidence and concentration of uncharged nuclear estrogen receptors (RN) in human breast cancer. The concentrations of RN and cytoplasmic uncharged receptor were determined on sucrose gradients following a 4-hr incubation at 4 degrees with 1.6 nM 17 beta-[3H]estradiol in 139 tumor specimens from 137 patients. RN was extracted from washed nuclear pellets in buffer containing 0.4 M KCl. The receptor molecule extracted had a high affinity for 17 beta-[3H]estradiol (Kd = 0.9 to 7.6 nM) and was specific for estrogen. The possibility of artifact due cytoplasmic contamination of the nuclear fraction or high-ionic-strength-induced exchange of charged nuclear receptors was rendered unlikely by validation experiments performed with pooled tumor tissue. Significant amounts of cytoplasmic uncharged receptor (greater than 7 fmol/mg protein) were found in 63.3% of the tumors. Similar significant amounts of RN were found in 29.5% of the tumors. Significant amounts of RN in the presence of undetectable cytoplasmic uncharged receptor were found in 2.9% of the tumors. The percentage of tumors that contain significant amounts of RN is approximately the same percentage of estrogen receptor-positive tumors that do not respond to ablative therapy.

Comparison of two isozymes of carbonic anhydrase in the rat anterior pituitary gland and pituitary tumors.

Two isozymes of carbonic anhydrase (EC 4.2.1.1.) were compared in the anterior pituitary gland of non-tumor-bearing rats and in the hormone-secreting pituitary tumors. In contrast to the pituitary gland, which contained 60 to 70% of the total carbonic anhydrase in the particulate subcellular fraction, three hormone-secreting pituitary tumors were devoid of the particulate (Triton X-100-solubilized)enzyme activity. Another pituitary tumor, 7315a, contained particulate carbonic anhydrase, but the activity was only 45% of the activity of normal pituitary gland. During the development of the rat brain, the particulate (Triton X-100-solubilized) carbonic anhydrase activity was undetectable in prepartions up to 21 days of age (body weight, 47 g). After that age, the carbonic anhydrase activity in the particulate fraction increased rapidly and reached the adult level at 37 days (body weight, 120 g), while the activity in the soluble fraction increased gradually after birth and then reached a plateau at 30 days (body weight, 81 g). These data show that the isozyme pattern of carbonic anhydrase in pituitary tumor tissue resembles the pattern in fetal cells more than the pattern in adult tissue.

Effects of iodine deficiency and high-fat diet on N-nitrosomethylurea-induced mammary cancers in rats.

The effects of an altered content of dietary iodine and fat on the development of N-nitrosomethylurea-induced mammary tumors in rats were studied and correlated with thyroid and pituitary function studies. In three separate experiments, animals fed a semisynthetic diet containing 11.8% fat had an earlier time of tumor appearance and greater tumor burden than did controls maintained on a diet containing 4.6% fat. These diet-associated changes were markedly inhibited by ovariectomy, indicating that the tumor growth was hormone responsive. We examined the possibility that the diet with increased fat content enhanced tumor growth through alterations in prolactin metabolism but could find no consistent elevation in serum prolactin and no increase in pituitary prolactin synthesis in vitro. Our data further showed that rats on an iodine-deficient form of the high-fat diet had no greater tumor growth than did animals receiving an iodine-supplemented form of the same diet. We conclude from these results that iodine deficiency does not promote mammary tumorigenesis. An incidental finding of great interest was that ovariectomy led to a highly significant depression of thyroid-stimulating hormone production in vitro. This suggests that estrogens may directly influence thyroid-stimulating hormone synthesis in vivo and thus contribute to the sex-related differences in thyroid physiology.

Demonstration and isolation of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C.

The corss-linking reagent p,p'-difluoro-m,m'-dinitrodiphenylsulfone has been used to fix in the tetramer form the various species of hemoglobin present in mixtures of hemoglobin A and hemoglobin C and of hemoglobin S and hemoglobin C. Following reaction, the presence of the hybrid hemoglobins alpha 2 A beta A beta C and alpha 2 A beta S beta C in these hemoglobin mixtures was demonstrated electrophoretically and the hybrids were isolated by ion-exchange chromatography. The identity of the alpha 2 A beta A beta C hybrid was further verified by peptide analysis. The success in cross-linking alpha 2 A beta 2 C, alpha 2 A beta A beta C, and alpha 2 A beta S beta C with p,p'-difluoro-m,m'-dinitrodiphenylsulfone shows that the distance between the alpha chain amino terminals in solution for these hemoglobin species is the same as in normal hemoglobin.

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity.

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.

The role of immunopeptides in the regulation of anterior pituitary hormone release.

The anterior pituitary lobe secretes hormones that regulate the functioning of the immune system which, in turn, produces thymic hormones and interleukin proteins capable of altering neuroendocrine responsiveness. Interleukin-1 is released during inflammation and activates the hypothalamic-pituitary-adrenal axis, which subsequently diminishes the immune response. Interleukin-6 (IL-6) stimulates prolactin and growth hormone release in vitro from anterior pituitary cells which, in turn, are capable of producing IL-6. The possible production of IL-6 by the anterior pituitary in situ suggests an autocrine and/or paracrine role for this cytokine in the regulation of hormone release.

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour.

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.

Regulation of the intracellular calcium concentration in MMQ pituitary cells by dopamine and protein kinase C.

The MMQ pituitary cell line, which expresses a membranal dopamine receptor, was used to examine the individual contributions of dopamine and protein kinase C (PKC) to control of the intracellular calcium concentration. The calcium concentrations, monitored with the fluorescent dye Indo-1, increased in response to elevated K+, BAY K8644, and maitotoxin, implicating the presence of voltage-dependent calcium channels. Dopamine had no detectable independent effect, but significantly inhibited the rise in intracellular calcium mediated by activation of voltage-dependent calcium channels; this dopaminergic action was prevented by haloperidol. Acute pharmacological activation of PKC for 60 s inhibited the stimulatory effects of these calcium channel activators, and this acute inhibitory action was abolished by prior depletion of PKC. In contrast, however, PKC depletion did not alter the calcium response to BAY K8644 or maitotoxin. Thus, MMQ cells appear to have voltage-dependent calcium channels which, at rest, are either at low density or in a closed state. The rise in intracellular calcium resulting from stimulation of the channels is under inhibitory control by an apparent D-2 dopamine receptor. When pharmacologically activated, phorbol diester-sensitive PKC limits the rise in the cellular calcium level associated with calcium uptake. In the absence of pharmacological activation, however, this enzyme system does not appear to play a role in the cellular calcium response to BAY K8644 or maitotoxin.

Impaired calcium mobilisation in the 7315a prolactin-secreting pituitary tumour.

The 7315a tumour secretes prolactin, but is refractory to enhancement of prolactin release by thyrotrophin-releasing hormone (TRH). In order to investigate further this refractoriness of the 7315a tumour cell, we compared cells from the tumour and from the normal pituitary with regard to TRH-enhanced fractional 45Ca2+ efflux and inositol phosphate production. TRH caused a large efflux of calcium from normal pituitary cells, but only mildly enhanced calcium efflux from the tumour cells. In contrast, TRH enhanced total inositol phosphate generation in both groups of cells to a similar degree. We therefore conclude that prolactin release from 7315a tumour cells is refractory to TRH due, at least in part, to impaired mobilisation of intracellular calcium by inositol phosphates.

Angiotensin peptides stimulate phosphoinositide breakdown and prolactin release in anterior pituitary cells in culture.

We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5-fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release.

Adrenocorticotropin increases interleukin-6 release from rat adrenal zona glomerulosa cells.

Interleukin-6 (IL-6) is produced by adrenal zona glomerulosa cells; its release is stimulated by several secretagogues, including IL-1 alpha, IL-1 beta, and angiotensin II. The present study reports that ACTH (0.1-100 nM) increased the release of IL-6 from primary cultures of rat adrenal cells in a concentration-dependent manner. This increase was accompanied by an increase in cAMP content in cell extracts and in the incubation medium. The dynamics of IL-6 release from the adrenal cells also were investigated using a perifusion system; approximately 50 min were required for the effects of IL-1 alpha, IL-1 beta, and ACTH on IL-6 release to become apparent. Following withdrawal of the secretagogues, IL-6 release returned to basal levels within 90-120 min. In some experiments, the adrenal zona glomerulosa was separated from the zona fasciculata/reticularis to determine the origin of secretagogue-stimulated IL-6 release. PGE2 and forskolin increased IL-6 release from both cell types, but maximal release from zona glomerulosa cells was more than 10-fold greater than that from zona fasciculata/reticularis cells. ACTH (0.1-100 nM) increased intracellular cAMP levels in cells from both cell types in a concentration-dependent manner, but increased IL-6 release only from zona glomerulosa cells. Dexamethasone, an inhibitor of IL-6 production in several tissues, had no effect on either basal or stimulated IL-6 production in the adrenal. Because IL-1 beta is produced primarily by tissues of the immune system, whereas ACTH is a classical endocrine hormone, we investigated the effect of interaction of these proteins on IL-6 release from the adrenal. Together, IL-1 beta and ACTH stimulation of IL-6 release was greater than the sum of the effects of each substance separately; however, IL-1 beta did not potentiate the effect of ACTH on cAMP levels. Similarly, IL-1 beta potentiated IL-6 release stimulated by forskolin and (Bu)2cAMP. Thus, the adrenal may be an important convergence point between the immune and endocrine systems, and because IL-6 release is regulated by IL-1 alpha, IL-1 beta, ACTH, and angiotensin II, and this cytokine stimulates corticosterone release, IL-6 may play an important paracrine role in integrating the signals derived from these systems.

Thyrotropin-releasing hormone and lysine-bradykinin stimulate arachidonate liberation from rat anterior pituitary cells through different mechanisms.

TRH and lysine-bradykinin (Lys-bradykinin) increase PRL release and arachidonate liberation from anterior pituitary cells. We investigated whether the arachidonate liberation stimulated by TRH and Lys-bradykinin originates in pituitary lactotropes and whether these events are accomplished through similar mechanisms. Lys-bradykinin and TRH rapidly (0.5 min) increased the intracellular [3H]arachidonate content of rat anterior pituitary cells. Lys-bradykinin also increased [3H]arachidonate liberation and PRL release from lactotrope-enriched pituitary cells, but not from a pituitary cell preparation with a diminished number of lactotropes. In contrast, TRH increased [3H]arachidonate liberation from both lactotrope-enriched and lactotrope-diminished preparations; this increased [3H]arachidonate liberation stimulated by TRH in the lactotrope-diminished cells may originate in the thyrotropes. The effects of TRH and Lys-bradykinin on [3H]arachidonate and [14C]stearate liberation in perfused pituitary cells also were determined. Both secretagogues increased arachidonate and stearate liberation in a biphasic manner, characterized by a transient spike, followed by a lower magnitude wave of fatty acid release. The spike phase produced by Lys-bradykinin was more pronounced than that produced by TRH. The calcium dependence of TRH- and Lys-bradykinin-stimulated arachidonate liberation also was investigated. Cobalt and the low calcium medium containing ionomycin were used to block the secretagogue-induced increase in intracellular calcium concentrations. These conditions blocked TRH-stimulated arachidonate liberation, but only marginally decreased Lys-bradykinin-stimulated arachidonate liberation, indicating that the two peptides act through different mechanisms. Therefore, TRH stimulation of arachidonate liberation is linked to an increase in intracellular calcium. In contrast, Lys-bradykinin increases arachidonate liberation through a calcium-independent intracellular mediator. This calcium-independent increase in arachidonate liberation may involve the bradykinin receptor being coupled directly to a phospholipase, a G-protein that provides a link between the bradykinin receptor and the phospholipases that liberate arachidonate, or bradykinin-induced activation of a protein kinase-C that activates the phospholipases and subsequently liberates arachidonate.

The biphasic regulation of prolactin secretion by dopamine agonist-antagonists.

The in vitro secretion of PRL by the pituitary gland is inhibited by low concentrations of dopamine. Dopamine receptor-blocking agents, haloperidol and pimozide, in concentrations between 10-100 nM, neutralize virtually completely the dopamine-mediated inhibition of PRL secretion. At these concentrations, the dopamine antagonists alone are without effect on PRL secretion. Higher concentrations of these agents, however, have a powerful action to inhibit PRL secretion. Perphenazine was without direct effect on the in vitro secretion of PRL. These observations may be of general importance to the study of dopamine action in other organ systems.

5-Hydroxyeicosatetraenoic acid increases prolactin release from rat anterior pituitary cells.

The enzymatic breakdown of phospholipids to form arachidonic acid and its subsequent conversion to metabolites produced via the lipoxygenase pathway in anterior pituitary cells may contribute to the process of PRL release. The incubation of primary cultures of pituitary cells from female rats with the lipoxygenase product 5-hydroxyeicosatetraenoic acid (5-HETE; 5-100 microM) significantly increased PRL release in a concentration-dependent manner. The release of PRL induced by 45 microM 5-HETE was completely blocked by 1 microM dopamine. Penfluridol, an agent that binds to and inactivates several Ca+2-binding proteins, including calmodulin, decreased (P less than 0.01) basal and 5-HETE-stimulated PRL release. Similarly, 50 microM D-600, a Ca+2 channel antagonist, significantly (P less than 0.01) reduced basal and 5-HETE-induced PRL release. BW755c or RHC 80267, both of which reduce the production of arachidonic acid metabolites, including 5-HETE, significantly reduced basal PRL release. The inhibitory effects of BW755c and RHC 80267 on PRL release, however, could be overcome by the addition of 5-HETE. In conclusion, 5-HETE or similar lipoxygenase metabolites may be important cellular components in the process of PRL release, and the inhibitory action of dopamine on PRL would seem to be mediated at some step after stimulation by these metabolites.

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.

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.

Adenosine 3',5'-monophosphate (cAMP) and calcium-calmodulin interrelation in the control of prolactin secretion: evidence for dopamine inhibition of cAMP accumulation and prolactin release after calcium mobilization.

Calcium (Ca2+) ionophore A23187 increased the intracellular cAMP content and PRL release in normal rat anterior pituitary cells. Cotreatment with dopamine reduced both control and A23187-stimulated cAMP accumulation and PRL release. The dopamine antagonist spiperone restored the response of cAMP to ionophoric stimulation after pretreatment with dopamine in the greatest concentration used. Penfluridol, a compound with Ca2+-calmodulin-blocking properties, decreased control and A23187-stimulated cAMP content and PRL release. W7, a selective calmodulin-blocking agent, reduced basal cAMP and PRL release, whereas W5, a W7 analog with only 20% of its calmodulin-blocking ability, did not affect cAMP or PRL secretion. These data indicate that the Ca2+-calmodulin and cAMP systems are interrelated in the regulation of PRL secretion. They are also consistent with the hypothesis that the inhibition of PRL release by dopamine occurs after Ca2+ is mobilized and when or before it stimulates adenylate cyclase activity.

Association of 45Ca2+ mobilization with stimulation of growth hormone (GH) release by GH-releasing factor in dispersed normal male rat pituitary cells.

Dispersed normal male rat anterior pituitary cells were prelabeled with 45Ca2+ and perifused to study the influence of GH-releasing factor (GRF) on fractional calcium efflux and GH release. The cells were exposed for 2 min to 0, 0.03, 0.1, 0.3, 1.0, or 10.0 nM GRF in separate perifusion columns, and the response to each concentration was determined by integration of the area under the curve. Concentrations of 0.1 nM GRF and higher produced a simultaneous and significant stimulation of calcium efflux and GH release. The increase in calcium efflux was proportional to GRF concentration and was maximally responsive at 1 nM GRF. The value for the entire integrated response of GH release increased continuously with GRF concentration, but GH released rapidly (0-4 min) in response to GRF achieved a maximal response at 1 nM GRF and was significantly correlated with calcium efflux. Somatostatin (100 nM) abolished the stimulation of GH release and calcium efflux due to 10 nM GRF. We conclude that GRF receptor activation is intimately associated with calcium mobilization, although the relative dependence upon intracellular or extracellular calcium sources has yet to be defined. This interaction occurs at a GRF concentration about 10 times lower than that observed to cause a measurable increase in intracellular biochemical messengers such as cAMP, phosphatidylinositol, or arachidonate. We postulate that GRF-stimulated calcium mobilization is a rapid and very sensitive event contributing to GRF-stimulated GH release.

The inhibition of phosphatidylinositol turnover: a possible postreceptor mechanism for the prolactin secretion-inhibiting effect of dopamine.

We studied the association between the inhibition of phosphatidylinositol (PI) turnover and the inhibition of PRL secretion in the presence of dopamine. The incorporation of radiolabeled phosphate into anterior pituitary gland PI as well as serum PRL levels were significantly (P less than 0.01) greater in female than in male rats. No significant sex-related difference was found in the incorporation by pituitary tissue of 32P into phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Dopamine decreased the incorporation of 32P into PI, but not into PC or PE, by female rat pituitary glands; this effect was reversed by two dopamine receptor-blocking agents, haloperidol and pimozide. After dopamine was removed from the incubation medium, basal 32P incorporation into PI was restored within 10 min. The administration of bromocriptine (500 micrograms/kg, ip, 4 h earlier) significantly reduced pituitary PI turnover. Conversely, in vivo injection of alpha-methyl-p-tyrosine (alpha MpT; 200 mg, ip, 2.5 h before death), an inhibitor of catecholamine biosynthesis, dramatically increased serum PRL levels. In vitro incorporation of 32P into PI, but not into PC or PE, increased (+130%) when these glands were incubated for 30 min with radiolabeled phosphate. The in vitro addition of 0.5 microM dopamine to glands from alpha MpT-treated rats counteracted the stimulation of 32P incorporation into PI produced by alph MpT treatment. In rats bearing the transplantable PRL-secreting tumor MtTW15, the hyperprolactinemia produced by the tumor stimulates hypothalamic turnover of dopamine, with a consequent inhibition of pituitary gland PRL secretion. 32P incorporation into PI, but not into PC or PE, was significantly (P less than 0.01) inhibited (-41%) in pituitary glands from these rats. The injection of alpha MpT (200 mg/kg, ip) or haloperidol (2 mg/kg, ip) 12 and 3 h before death into MtTW15 tumor-bearing rats abolished the inhibition of 32P incorporation into pituitary PI. Dopamine also decreased PI turnover in the 7315a PRL-secreting pituitary tumor. Our data indicate that the PI cycle may be an intracellular mechanism controlling PRL release in the rat and that the changes in its cleavage and turnover may be an early postreceptor event responsible for the inhibition of PRL secretion produced by factors such as dopamine.

Production of interleukin-6 by anterior pituitary cells is stimulated by increased intracellular adenosine 3',5'-monophosphate and vasoactive intestinal peptide.

Interleukin-6 (IL-6) is an inflammatory cytokine that is produced by a variety of cells and tissues. We recently demonstrated that IL-6 is produced by anterior pituitary cells in response to the bacterial endotoxin lipopolysaccharide and phorbol diester in vitro. Lipopolysaccharide (0.01-100 ng/ml) increased, whereas dexamethasone (0.1-100 nM) decreased, IL-6 production by anterior pituitary cells in vitro as measured by the 7TD1 cell growth factor assay. In addition, we now report that IL-6 production by anterior pituitary cells is stimulated by agents that elevate intracellular cAMP concentrations. Exposure of anterior pituitary cells to (Bu)2cAMP (0.01-10 mM), prostaglandin E2 (1.0-1000 nM), forskolin (50-1000 nM), or cholera toxin (0.25-250 ng/ml) for 6 h resulted in concentration-related increases in the production of IL-6, which, in the cases of forskolin and cholera toxin, correlated well with increased intracellular cAMP concentrations. Vasoactive intestinal peptide (1-1000 nM), which stimulates adenylate cyclase activity in the anterior pituitary, caused a concentration-related enhancement of IL-6 production that was unaffected in the presence of 10-100 nM somatostatin. In contrast, GH-releasing factor had no effect on IL-6 production. These data suggest that anterior pituitary cells produce IL-6 in response to increased intracellular cAMP, and that the neuropeptide vasoactive intestinal peptide may act to regulate IL-6 production.