Permeabilized Pituitary Cells Research Articles

Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion.

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca2+]i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca2+]i by ionomycin and activation of voltage-sensitive Ca2+ channels by Bay K 8644 or high K+, as well as Ca2+-induced LH release in permeabilized pituitary cells. The [Ca2+]i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca2+-dependent step in exocytosis without affecting Ca2+ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alphaT3-1 gonadotrophs treated with GnRH, K+, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

Role of guanine nucleotide-binding proteins, Gi alpha 3 and Gs alpha, in dopamine and thyrotropin-releasing hormone signal transduction: evidence for competition and commonality.

It is clear that dopamine (DA) at high concentrations (> 100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (< 10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of Gi alpha 3 and Gs alpha in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 micrograms/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of Gi alpha 3 or Gs alpha. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol DA/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61.4 +/- 7.6% of basal values; mean +/- S.E.M.) while 10 nmol DA/l augmented (120.0 +/- 7.0%) PRL release in five separate experiments. Treatment of cells with anti-Gi alpha 3 attenuated the inhibitory effect of high dose DA (87.3 +/- 14.5%). However, elimination of Gi alpha 3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121.0 +/- 5.2%). Interestingly, immunoneutralization of Gs alpha resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69.7 +/- 7.3%) while combined treatment of anti-Gi alpha 3 and anti-Gs alpha abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70.7 +/- 12.5% and 10 nmol/l, 87.5 +/- 21.4%). These data reveal that ligand-activated DA receptors can interact with both Gi alpha 3 and Gs alpha. Elimination of the stimulatory component (Gs alpha) favors the DA receptor activation of the inhibitory pathway (Gi alpha 3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/l as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188.0 +/- 31.0% of basal values under control conditions. Unexpectedly, immunoneutralization of Gs alpha completely blocked the ability of TRH to induce PRL release (101.8 +/- 12.0%). This neutralizing effect was specific to Gs alpha in that blockade of Gi alpha 3 activity had no significant effect on TRH-stimulated PRL release (166.2 +/- 13.1%). These data are the first to support a direct role of Gs alpha in TRH signal transduction within PRL-secreting cells.

Growth hormone releasing hexapeptide (GHRP-6) activates the inositol (1,4,5)-trisphosphate/diacylglycerol pathway in rat anterior pituitary cells.

Growth hormone-releasing hexapeptide (GHRP-6) is known to stimulate secretion of growth hormone (GH) in vivo and in vitro in a variety of species. However, the cellular effects of GHRP-6 remain largely unknown. We have tested the influence of GHRP-6 on the inositol phospholipid second messenger system in cultured anterior pituitary cells. Cultured pituitary cells responded upon challenge with GHRP-6 with a dose-dependent release of GH. Moreover, incubation of GHRP-6 with pituitary cell cultures labelled with myo-[3H]inositol resulted in a dose-dependent rise in [3H]inositol phosphates. Brief stimulation of pituitary cells with GHRP-6 increased phosphorylation of MBP4-14, a specific protein kinase C substrate, when incubated with the cytosol- or plasma membrane fraction from the stimulated cells. Furthermore, introduction of MBP4-14 into the cytosol in digitonin permeabilized pituitary cells caused increased phosphorylation of this substrate. GHRP-6 induced a rise in intracellular Ca2+ in individual somatotrophs loaded with the Ca2+ indicator, Fura-2. Preincubation (3 min) with somatostatin (SRIF) diminished the Ca2+ spike elicited by GHRP-6, while no effect of SRIF was observed when added simultaneously with GHRP-6. These results indicate that GHRP-6-stimulated GH-secretion involves the diacylglycerol/inositol(1,4,5)trisphosphate pathway with a resulting rise in cytosolic Ca2+.

Paradoxical effects of dopamine (DA): Gi alpha 3 mediates DA inhibition of PRL release while masking its PRL-releasing activity.

In an attempt to elucidate the mechanism(s) underlying the paradoxical (inhibitory vs. stimulatory) effects of DA on PRL release, we treated permeabilized pituitary cells with antibodies directed against the carboxyl-terminus of the alpha-subunit of the guanine nucleotide binding protein, Gi3. Immunoneutralization of Gi alpha 3 completely blocked the inhibitory effect of 1000 nM DA on PRL release, as assessed by reverse hemolytic plaque assays. In contrast, DA at a 100-fold lower concentration (10 nM) had no effect on PRL release under control conditions, but elicited a stimulatory response in the presence of anti-Gi alpha 3. Examination of the frequency distribution of plaque sizes indicated that suppression and augmentation of PRL secretion by DA was not attributable to distinct subpopulations of lactotropes. Taken together, these data suggest that all pituitary lactotropes have the potential to respond to the inhibitory and stimulatory activities of DA. However, the stimulatory action of this monoamine is normally masked by tonic activation of Gi alpha 3 which couples DA to its inhibitory pathway.

Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain

GTP-binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab-related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca 2+-dependent or Ca 2+-independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL(30–47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca 2+-stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 μM peptide. The peptide (100 μM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence-specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5′-[ γ-thio]triphosphate (GTPγS) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that the two inhibitory sites of action of GTPγS can be distinguished by their dependence on GTPγS concentration and their sensitivity to pertussis toxin. Ca 2+-stimulated exocytosis was half-maximally inhibited by 6.8 μM GTPγS, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTPγS inhibition of Ca 2+ -stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to secod messenger generation.

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Protein kinase C is now recognized to comprise a family of closely related subspecies (PKCs). When cultured rat pituitary cells were permeabilized by digitonin for 5 min in the absence of Ca2+, endogenous PKC activity was decreased by 72%. PKC depletion was also achieved by prior treatment (24 hr) with high concentrations of phorbol 12-myristate 13-acetate (PMA). When purified activated brain PKCs were added for 30 min to PMA-pretreated, digitonin-permeabilized cells, only alpha- and beta- but not gamma-type PKC stimulated luteinizing hormone release. Since PKC was implicated as a mediator of gonadotropin secretion, gonadotropin-releasing hormone might utilize alpha- and beta-type PKCs for stimulation of gonadotropin secretion; alpha- and beta-type PKCs might participate also in other exocytotic responses in diverse biological systems in which PKC was implicated.

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: Possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2- 3H] inositol were permeabilized by ATP 4−, exposed to guanine nucleotides and resealed by Mg 2+. Addition of guanosine 5′-0-(3-thio triphosphate) (GTPγS) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [ 3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5′-0-(2-thiodiphospate) (GDPβS) or by neomycin. Surprisingly, addition of GDPβS (100–600 uM) on its own resulted in a dose-related increase in [ 3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP>GTPγ S>ATP>CTP. The stimulatory effect of UTP, ATP and CTP, but not GTPγS or GDPβS, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTPγS decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.

Gonadotropin-Releasing Hormone- Induced Rise in Cytosolic Free Ca2+Levels: Mobilization of Cellular and Extracellular Ca2+Pools and Relationship to Gonadotropin Secretion

Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (approximately 8 s) mobilization of an ionomycin-sensitive intracellular Ca2+ pool. A second component of Ca2+ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca2+ concentration ([Ca2+]i). Thereafter, influx of Ca2+ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca2+]i during the second phase of GnRH action. Addition of inositol 1,4,5-trisphosphate (IP3) to permeabilized pituitary cells resulted in a Ca2+ transient, released from a nonmitochondrial pool, which maintained ambient free Ca2+ concentration around 170 nM in an ATP-dependent mechanism. Successive stimulations of the cells with IP3 produced an attenuated response. Elevation of the gonadotroph [Ca2+]i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca2+ channels by the dihydropyridine Ca2+-agonist [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nM) response being reached by 10(-8) M of the drug, both [Ca2+]i elevation and GnRH-induced LH release were inhibited similarly (40-50%) by the dihydropyridine Ca2+-antagonist nifedipine. The results indicate that peak [Ca2+]i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP3 formation.(ABSTRACT TRUNCATED AT 250 WORDS)