Stability of singly hydrated silanone on silicon quantum dot surfaces: density functional simulations.

A key to understanding the optical characteristics of silicon quantum dots is the role of surface bonded species that introduce states to the band-gap. In particular, oxygen bonded in a silanone configuration is thought to be a source of shifts in emission during oxidation. We report the results of density-functional calculations examining the properties of such surface structures. We find single hydration of a simple, neutral silanone molecule leads to a barrierless conversion into a di-hydroxyl structure, and that similar processes are weakly activated on larger systems. However, we show that charging has a significant impact upon stability, with the attachment of an electron greatly increasing the barrier for converting silanone to di-hydroxyl termination. The relatively stable, negatively-charged silanone structures are predicted to lead to large red-shifts in the onset of optical absorption.

The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in bakers' yeast.

An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375,000-400,000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzes a reaction between 4'-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase, to reform CoA. This sequence of reactions, referred to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by which the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment segment is referred to as the 10,000-15,000 M(r) subunit. The 5'-ADP-4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthetase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10,000-15,000 M(r) subunit. Thus, the 10,000-15,000 M(r) subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.

Differential release of tumor necrosis factor and IL-6 from adrenal zona glomerulosa cells in vitro.

In previous studies, rat adrenal zona glomerulosa (ZG) cells were demonstrated to release interleukin-6 (IL-6). In the current study, cultures of ZG cells and bioassays for tumor necrosis factor (TNF) and IL-6 were used to determine if ZG cells release TNF and to define more fully the factors that regulate ZG IL-6 release. ZG cells released IL-6 and TNF, and this release was stimulated by lipopolysaccharide, interleukin-1 alpha, interleukin-1 beta, a protein kinase C activator, and a calcium ionophore without affecting intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) content. In contrast, adrenocorticotropic hormone (ACTH) increased the intracellular cAMP content, increased basal and secretagogue-stimulated IL-6 release but decreased basal and secretagogue-stimulated TNF release. The effects of ACTH on IL-6 and TNF release may be mediated by increases in intracellular cAMP because ACTH and dibutyryl cAMP modified IL-6 and TNF release in an identical manner. Therefore, IL-6 and TNF release from ZG cells can be differentially regulated. Because IL-6 and TNF modify adrenal steroid release, the adrenal production of these cytokines may have a role in the stress response.

Pyrularia thionin increases arachidonate liberation and prolactin and growth hormone release from anterior pituitary cells.

Pyrularia thionin is a 47 amino acid peptide isolated from the nuts of Pyrularia pubera. This peptide does not have intrinsic phospholipase A2 activity, but it increases the liberation of arachidonate from several tissues. Exposure of anterior pituitary cells to this toxin increases the liberation of arachidonate, increases the cellular levels of lysophospholipids, and decreases cellular phospholipids. Thus, phospholipase A2 is involved in the liberation of arachidonate stimulated by this peptide. Because this toxin also increases stearate liberation from the pituitary cells, either diacylglycerol lipase, phospholipase A1 or lysophospholipase may be directly or indirectly activated by this toxin. In addition to increasing fatty acid liberation, Pyrularia thionin increases the release of prolactin and growth hormone from anterior pituitary cells over the identical concentration ranges that this toxin liberates the fatty acids. Pyrularia thionin increased arachidonate liberation and prolactin release from perifused pituitary cells within 2 min, and following withdrawal of the toxin, arachidonate liberation and prolactin release returned to near basal levels within 6 min. Dopamine, a physiological inhibitor of prolactin release that closes calcium channels, decreased prolactin release stimulated by Pyrularia thionin. However, dopamine had no effect on the arachidonate liberation stimulated by this peptide. Similarly, D-600, an organic calcium channel blocker, decreased the prolactin and growth hormone release stimulated by the toxin without affecting the toxin-stimulated arachidonate liberation. Therefore, Pyrularia thionin increases arachidonate liberation through the rapid activation of phospholipase A2 by a mechanism that is not dependent on calcium uptake via D-600-inhibitable calcium channels. In contrast, the prolactin and growth hormone release stimulated by this toxin requires calcium uptake via D-600 inhibitable calcium channels.

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.

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.

Induction of interleukin-6 release by interleukin-1 in rat anterior pituitary cells in vitro: evidence for an eicosanoid-dependent mechanism.

We have reported previously that a subpopulation(s) of anterior pituitary cells released IL-6 and that this release was stimulated by interleukin-1 (IL-1) through a non-cAMP-dependent mechanism. We now report that IL-1 induces IL-6 release from anterior pituitary cells in an eicosanoid-dependent manner. Dispersed rat anterior pituitary cells were briefly prelabeled (2-3 h) with [3H]arachidonic acid (AA) to esterify the fatty acid within the lipid pool. Incubation of these prelabeled cells with 25 ng/ml IL-1 beta caused an increase only within 1-2 min in the amount of free [3H]AA detected in the extracts of the cells. During 15- to 30-min incubations, IL-1 beta (25 ng/ml) caused an increased accumulation of [3H]AA in the incubation medium which reached levels similar to those induced by 100 nM TRH. Perifused anterior pituitary cells responded to IL-1 beta (25 ng/ml) with a rapid (less than 2 min), biphasic, and reversible efflux of [3H]AA. The [3H]AA appears to have been derived from choline phospholipids, as formation of [3H]glycerophosphorylcholine was substantially increased by exposure of [3H]choline-prelabeled cells to either IL-1 alpha (171%) or IL-1 beta (236%); in addition, the complete deacylation of phosphatidylcholine suggests that other fatty acid species are liberated as a consequence of IL-1 receptor activation and, thus, may also contribute to the actions of IL-1 alpha and IL-1 beta. However, the levels of [3H]phosphorylcholine and [3H]choline were unchanged as well as those of catabolites of other lipid species. These data suggested an involvement of phospholipase-A2 (PLA2) in mediating the IL-1 induction of IL-6 release. Subsequently, we used inhibitors of the PLA2, cyclooxygenase, and lipoxygenase enzymes to investigate a possible role for the generation of AA and its subsequent enzymatic conversion in the signal transduction pathway activated by IL-1. The PLA2 inhibitor aristolochic acid (10 microM) blocked IL-1 beta-induced IL-6 release and the release of IL-6 caused by Pyrularia pubera thionin (5 micrograms/ml), a stimulator of PLA2 activity. The cyclooxygenase inhibitor indomethacin (10 microM) did not inhibit IL-1 beta-induced IL-6 release. In contrast, the general lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) and the more specific 5-lipoxygenase inhibitors AA861 and RHC5901 (both 10 microM) reduced basal and blocked IL-1 beta-induced IL-6-release.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-1 stimulates interleukin-6 release from rat anterior pituitary cells in vitro.

We have reported previously that anterior pituitary cells released interleukin-6 (IL-6) and that this release was stimulated by lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or agents that increased intracellular cAMP concentrations. We now report that IL-1 stimulates IL-6 release from anterior pituitary cells in vitro. IL-1 alpha and IL-1 beta (0.04-25 ng/ml) significantly increased IL-6 release 3- to 4-fold in a concentration-related manner during 6-h incubations; however, there was no change in extracellular or intracellular cAMP concentrations. IL-1 alpha and IL-1 beta (10 ng/ml), vasoactive intestinal peptide (VIP, 500 nM), prostaglandin E2 (PGE2, 1 microM), and LPS (1 ng/ml) stimulated IL-6 release to a similar degree. In the presence of VIP and PGE2, IL-1 alpha and IL-1 beta increased IL-6 release without any apparent further change in extracellular or intracellular cAMP. Conversely, LPS did not increase cAMP concentrations, and IL-1 did not significantly increase IL-6 release in the presence of LPS. The preexposure of anterior pituitary cells to 1 microM PMA caused the apparent down-regulation of protein kinase C activity because 100 nM PMA was no longer effective to stimulate IL-6 release; however, the ability of IL-1 alpha, IL-1 beta, PGE2, or LPS to stimulate IL-6 release was not altered. In addition, IL-1 alpha and IL-1 beta stimulated IL-6 release in the presence of maximally stimulative concentrations of PMA. The synthetic glucocorticoid dexamethasone (10 nM) significantly inhibited IL-6 release induced by IL-1 alpha, IL-1 beta, or LPS. The separation of anterior pituitary cells on unit gravity BSA gradients generated fractions of IL-6-producing cells that were inducible by LPS and IL-1 beta and separate from the PRL-, ACTH-, GH-, or LH-producing cell fractions. These data suggest that IL-1 stimulates IL-6 release from a subpopulation of anterior pituitary cells via a glucocorticoid-sensitive and non-cAMP-mediated pathway that is different from those pathways used by VIP, PGE2, and PMA.

Role of calcium in dopaminergic regulation of TRH- and angiotensin II-stimulated prolactin release.

The contributions of intracellular and extracellular calcium to thyrotropin-releasing hormone (TRH)- and angiotensin II (ANG II)-stimulated prolactin (PRL) release and the role of calcium in dopaminergic inhibition of these events were examined because of unresolved controversies in these areas. Dispersed normal female rat anterior pituitary cells were used to evaluate radiocalcium fluxes and the intracellular calcium concentration ([Ca]i). Both peptides increased PRL release, fractional 45Ca2+ efflux, and [Ca]i in a spike and plateau pattern, and neither increased 45Ca2+ uptake. In a low-calcium buffer, TRH and ANG II stimulated less than 5% of the normal PRL response, yet efflux was at least 50% of normal and [Ca]i was 20-40% of normal. Dopamine reduced TRH-stimulated PRL release by greater than 90% and abolished the plateau, yet the calcium responses to TRH were at least 50% of normal. Although dopamine prevented the plateau component of peptide-stimulated [Ca]i, the plateau phase of efflux persisted. Thus TRH and ANG II may control at least two cell-associated calcium pools, one readily depleted and the other highly resistant to depletion, without evidence for stimulation of calcium uptake. Dopamine inhibits PRL release stimulated by these peptides, with a relatively greater influence on the plateau component, through mechanisms only minimally related to calcium flux. Dopamine may slightly increase the extrusion of calcium mobilized by these peptides and thus may limit the anticipated increase in [Ca]i.

Characterization of a new animal model of chronic hyperprolactinemia.

Animal models of chronic prolactin (PRL) excess have included rats bearing transplantable pituitary tumors that have produced other hypophyseal hormones in addition to PRL. We report characterization of a new model, the Buffalo rat implanted with the MMQ tumor, a line developed from the 7315a line. Rats implanted with the MMQ tumor have serum PRL levels that increase with time and correlate with the estimated volume of the subcutaneous tumor. When rats are killed 4 weeks after implantation, serum PRL levels are strikingly higher in tumor-bearing rats compared with controls (females, 2,723 +/- 266 v 192 +/- 46 ng/mL, P less than .0001; males, 1,637 +/- 213 v 99 +/- 11 ng/mL, P less than .0001). Serum PRL levels measured by the Nb2 lymphoma assay were higher than immunoassay measurements in both tumor-bearing and control Buffalo rats. Sephadex chromatography of serum from tumor-bearing rats showed that most of the PRL immunoreactivity co-eluted with 125I-rPRL. Neither serum growth hormone (GH) nor luteinizing hormone (LH) levels were different from controls in tumor-bearing rats. Female MMQ-bearing rats had lower estradiol levels. At death, the wet weights of adrenal glands, kidneys, and gonads were not affected by the presence of tumor. In contrast, tumor-bearing rats had increased spleen weight and histological evidence of white pulp hyperplasia. The Buffalo rat implanted with the PRL-only MMQ tumor is a promising new tool for the study of chronic hyperprolactinemia.

Complete amino acid sequence analysis of a peptide isolated from the thymus that enhances release of growth hormone and prolactin.

We have previously reported that thymosin fraction 5 (TF5), a partially purified calf thymus preparation, contains a peptide(s) that can enhance the production of GH and/or PRL from rat anterior pituitary cells in vitro. Using reverse phase HPLC, we have now isolated and chemically characterized from TF5 a peptide possessing this activity. This peptide, termed MB-35, is a highly charged basic molecule of 35 amino acid residues and a mol wt of 3756. A computer-assisted search of published protein sequences has revealed that this peptide has a 100% homology with a region of the histone H2A. Biological studies using rat pituitary cells have revealed that MB-35 is active alone or in combination with GH-releasing factor (GRF) or TRH and can increase the production of GH and/or PRL beyond that achievable with GH-releasing factor and TRH alone. The observation that histone H2A, the parent molecule, is without activity is of keen interest, since it suggests that nucleoproteins may have heretofore unknown physiological activities, perhaps related to cell cycle and/or other events associated with DNA activation events.

Dopamine receptor and adrenoceptor agonists inhibit prolactin release from MMQ cells.

MMQ cells, a prolactin-secreting cell line possessing dopamine receptors, were exposed to the calcium channel activator maitotoxin and dopamine or adrenoceptor agonists or antagonists. Dopamine (500 nM) or the dopamine agonists lisuride (50 nM), terguride (50 nM), and N-0437 (50 nM) decreased maitotoxin-stimulated prolactin release from perifused MMQ cells. In this system, sulpiride (100 nM), a dopamine D2 antagonist, reversed the prolactin inhibition produced by lisuride (20 nM). In static incubations of MMQ cells, lisuride (10-500 nM) inhibited maitotoxin-stimulated prolactin release in a concentration-dependent manner; this inhibition was attenuated in a concentration-related manner by sulpiride (100-500 nM). In addition, sulpiride reversed dopamine (50-500 nM), lisuride (10-500 nM), and terguride (50-500 nM) inhibition of forskolin-stimulated cAMP generation. The alpha 2-adrenergic agonist clonidine inhibited maitotoxin-stimulated prolactin release from perifused MMQ cells; this inhibition was abolished by idazoxan, an alpha 2-adrenergic antagonist. In contrast, serotonin or the serotonin antagonist methysergide had no effect on prolactin release from MMQ cells. These data indicate that activation of dopamine D2 receptors and alpha 2-adrenoceptors by classically defined pharmacological agents inhibits prolactin release and cellular cAMP levels in MMQ cells. Therefore, MMQ cells may provide a valuable model for the development of pharmacological agents and assist in the identification of the mechanisms involved in the dopaminergic inhibition of prolactin release.

Hypercalciuria in a new rat model of hyperprolactinemia.

Hypercalciuria has been reported in rats with mild hyperprolactinemia due to implantation of anterior pituitary glands under the kidney capsule and in rats bearing transplantable tumors that secrete large amounts of prolactin (PRL) and growth hormone (GH). We studied Buffalo rats implanted subcutaneously with the new MMQ pituitary tumor line that secretes only PRL. Urinary calcium excretion increased as the tumors grew. Three weeks after tumor implantation in female rats, the urinary calcium excretion was 1.102 +/- 0.092 mg/100 g body weight (BW).24 hours compared with controls, 0.296 +/- 0.079, P less than .0005. Male tumor-bearing rats also had increased urinary calcium excretion compared with male controls. In tumor-bearing rats the urinary calcium excretion factored for urinary sodium excretion, dietary calcium intake, or urinary creatinine excretion was elevated. Urinary calcium excretion was correlated with serum PRL levels and with estimated tumor volume. Serum calcium, immunoassayable parathyroid hormone, and urinary cyclic adenosine monophosphate (cAMP) excretion were normal in the tumor-bearing rats. There was some evidence of loss of bone calcium in rats bearing the MMQ tumor, and serum levels of calcitonin were decreased. These results are similar to those found in anterior pituitary-grafted hypercalciuric rats. It is unlikely that parathyroid hormone (PTH) abnormalities are responsible for the hypercalciuria in the MMQ-bearing rats. The pituitary gland may have an effect on the distal renal tubule to decrease calcium reabsorption.

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.

Interactions of dopamine and neurotensin on calcium fluxes and prolactin release in normal rat pituitary cells.

We studied the role of calcium in dopaminergic control of the neuroendocrine effects of neurotensin. In primary cultures of dispersed normal female rat anterior pituitary cells the interactions of dopamine and neurotensin were examined with reference to the rate of PRL release, the magnitude of 45Ca2+ uptake, the rate of fractional 45Ca2+ efflux, and the dynamic response of the intracellular calcium concentration (Cai) monitored with the fluorescent dye, Indo-1. Neurotensin stimulated calcium uptake and also mobilized a pool of intracellular calcium to increase Cai in a sustained plateau-like pattern. The response of PRL release and fractional efflux to neurotensin, however, each displayed typical spike and plateau profiles. In the presence of dopamine the stimulation of PRL release and calcium uptake due to neurotensin were abolished, and the rise in Cai was barely detectable, but neurotensin-stimulated fractional efflux persisted almost unchanged. These data suggest that dopamine may modulate Cai by inhibiting calcium uptake and possibly also by enhancing cellular calcium extrusion under stimulated conditions. Further, the increased inositol trisphosphate production reportedly stimulated by neurotensin apparently does not generate a spike-like response of intracellular calcium, and stimulated hormone release may display a spike and plateau pattern solely with a plateau Cai profile.

A comparison of the concentration-dependent actions of thyrotropin-releasing hormone, angiotensin II, bradykinin, and Lys-bradykinin on cytosolic free calcium dynamics in rat anterior pituitary cells: selective effects of dopamine.

Using Indo-1 as a fluorescent probe, we studied the dynamics and the underlying mechanisms of the response of cytosolic free calcium ([Ca2+]i) to different concentrations of four prolactin secretagogues, thyrotropin-releasing hormone, angiotensin II, bradykinin, and lys-bradykinin in rat anterior pituitary cells. Low concentrations (1-100 pM) of these peptides caused a sustained increase in [Ca2+]i, whereas high concentrations (up to 100 nM) caused a large transient elevation of [Ca2+]i that was followed by a lower sustained plateau. Experiments with protein kinase C-depleted cells suggested that phorbol diester-sensitive protein kinase C was not involved in the transition of [Ca2+]i from spike to plateau seen with high concentrations of secretagogue. Specific concentrations of secretagogue mobilized different pools of [Ca2+]i, as indicated by experiments with Ca2(+)-depleted medium. Low concentrations of secretagogue induced a Ca2+ response that was abolished by Ca2(+)-depleted medium, whereas high concentrations generated a [Ca2+]i response that was refractory to Ca2(+)-depleted medium. Dopamine (100 nM) abolished the [Ca2+]i plateau response to all four agents at low concentrations and selectively reduced the plateau component of the responses elicited at high concentrations of secretagogue. If the plateau component is represented by utilization of either extracellular Ca2+ or a cell-associated EGTA-accessible pool(s) of Ca2+, then dopamine modulates one or both of these calcium sources.

Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami.

Interleukin-6 (IL-6) is an inflammatory cytokine that stimulates T-cell activation and B-cell differentiation. We recently reported that picomolar concentrations of IL-6 stimulated PRL, GH, and LH release in vitro. These data suggested that IL-6 may function as a hypothalamic releasing factor for anterior pituitary hormones. Medial basal hypothalami (MBH) were incubated for 60-90 min in Krebs-Ringer bicarbonate buffer supplemented with 0.025% BSA, and the conditioned medium was assayed for IL-6 concentrations by the 7TD1 cell growth factor assay. It was found that MBH released IL-6 in vitro. Although depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, somatostatin release from the MBH was increased significantly. The bacterial endotoxin lipopolysaccharide (LPS; 1-100 ng/ml) induced significant increases in IL-6 release from the MBH. The presence of IL-6 in the hypothalamus suggested a possible role for this cytokine in the regulation of neuropeptide release; however, the release of somatostatin was not affected by 20 ng/ml IL-6. Comparison studies of neural and neuroendocrine tissues revealed that the anterior and posterior pituitaries released larger amounts of bioactive IL-6 than the MBH or parietal cortex during a 4-h incubation; induction of IL-6 release by endotoxin occurred only in the anterior pituitary and hypothalamus. IL-6 mRNA was detectable in the MBH and anterior pituitary tissue after a 4-h incubation; however, no IL-6 mRNA was detectable in freshly isolated tissues. LPS (100 ng/ml) and (Bu)2cAMP (1 mM) increased IL-6 mRNA accumulation in and IL-6 release from the MBH and anterior pituitary. These data suggest that the MBH synthesizes and releases IL-6 via a nonneuronal source in vitro.

Physiological and biochemical effects of bradykinin and lys-bradykinin in pituitary cells.

The presence of kallikrein activity, bradykinin (BK) and lys-bradykinin (LBK) in the pituitary gland suggests a possible physiological role of kinins therein. We demonstrated that BK and LBK increased prolactin (PRL), but not growth hormone release, from rat anterior pituitary cells cultured in vitro. Such stimulatory effect on PRL secretion appears to involve B2-type BK receptors, as suggested by the antagonizing effect of B6572 (a B2-type BK receptor antagonist) on PRL release. The BK-induced increase in PRL release is associated with an enhanced [3H]arachidonate (AA) efflux, an elevated cytosolic free calcium concentration [(Ca2+]i), and increased inositol phosphate (InsPx) production. Bradykinin and LBK stimulated [3H]AA liberation, [Ca2+]i elevation and PRL release at lower concentrations than those necessary to stimulate InsPx production. Therefore, AA release and [Ca2+]i elevation may be more important to PRL release than is InsPx production. Dopamine (DA) inhibited BK- or LBK-stimulated PRL release and slightly attenuated the stimulated [Ca2+]i response, but had no effect on stimulated [3H]AA efflux and InsPx generation. This study suggests that BK and LBK may have either an autocrine or a paracrine role in regulating PRL secretion, and are subject to modulation by DA.

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.

Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phosphate production.

Calcitonin is present in both the hypothalamus and pituitary of the rat, and normal rat anterior pituitary cells express calcitonin receptors. Calcitonin has been reported to inhibit or to stimulate PRL release from rat anterior pituitary cells. We have investigated the effects of salmon calcitonin on basal and stimulated PRL release from rat anterior pituitary cells and have studied the effects of this peptide on the intracellular biochemical pathways involved in PRL release. Salmon calcitonin had no significant effect on basal PRL release, but inhibited (P less than 0.01) TRH-stimulated PRL release without affecting PRL release promoted by angiotensin II, neurotensin, phorbol myristate acetate (a protein kinase C activator), or maitotoxin (a calcium channel activator). Salmon calcitonin had no effect on the increase in PRL release and intracellular cAMP concentration after exposure of pituitary cells to vasoactive intestinal peptide or forskolin. Salmon calcitonin significantly decreased (P less than 0.01) the TRH-stimulated rise in inositol phosphates without affecting the angiotensin II-stimulated increase in inositol phosphates. Similarly, salmon calcitonin decreased the TRH-stimulated increase in cytosolic calcium and arachidonate liberation by pituitary cells. We conclude that salmon calcitonin selectively decreases TRH-stimulated PRL release by a mechanism that involves a decrease in inositol phosphate production, as well as a subsequent reduction in cytosolic calcium levels and in arachidonate liberation.