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Biomedical subjects

R M MacLeod

Publications and source records attributed to R M MacLeod.

At least 37 records · Page 2Linked to original sources
PubMed1990

The paracrine role of angiotensin in gonadotrophin-releasing hormone-stimulated prolactin release in rats.

It is well known that lactotrophs are in close proximity to gonadotrophs in the lateral region of the pituitary gland, and thus there is interest in interactions between these two types of cell. The present study was undertaken to investigate the role of angiotensin II (AII) in gonadotrophin-releasing hormone (GnRH)-induced prolactin release, and to examine the effect of oestradiol on the paracrine interaction among anterior pituitary cells of young male rats. Over a 3-day period, one group of rats was injected twice with polyoestradiol phosphate (0.5 microgram/g body weight; PEP-treated group), and a second with saline (control group). Their anterior pituitary glands were enzymatically dispersed and, subsequently, the cells were allowed to reaggregate for 48 h. A 20-min perifusion with 100 nmol GnRH/l increased (P less than 0.01) prolactin release from these anterior pituitary cell aggregates. The integrated value for prolactin release was 9.1 +/- 2.9 ng/10(7) cells. In the PEP-treated group, basal release of prolactin was greater than that in the control group (P less than 0.01). However, during exposure to GnRH, the integrated amount of prolactin release by the PEP-treated group (12.5 +/- 4.8 ng/10(7) cells) was not significantly different from that of the control group, although in each individual experiment the GnRH-stimulated prolactin release from the PEP-treated cells was higher than that from the cells that had not been exposed to PEP. The release of angiotensin I (AI) from these perifused pituitary aggregates was significantly (P less than 0.01) increased by GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed1989

Thymic stromal elements contain an anterior pituitary hormone-stimulating activity.

Conditioned medium from thymic reticular monolayers displayed time-dependent accumulations of a concentration-responsive pituitary hormone-releasing activity that has been named thymic neuroendocrine-releasing factor (TNRF). Dopamine blocked and somatostatin (SRIF) attenuated TNRF-induced prolactin (PRL) release. Conversely, SRIF had no effect on TNRF-induced growth hormone (GH) release. TNRF potentiated thyrotropin-releasing hormone (TRH)-stimulated PRL release and was additive to the effects of GH-releasing hormone (GHRH) on GH release. Anterior pituitary cells perifused with TNRF responded with immediate, sustained and reversible increases in hormone release. Partial purification revealed this activity to be greater than 10,000 in molecular weight. These data suggest that the thymus may affect pituitary function.

Animals↗
PubMed1989

Estradiol attenuates prolactin secretion and phosphoinositide hydrolysis in MMQ cells.

We previously isolated a clonal cell line, designated MMQ, which only secretes prolactin (PRL) and whose secretory process is nonresponsive to thyrotropin releasing hormone (TRH) and angiotensin II (AII). In the present study, we injected MMQ cells into rats to determine whether the tumor cells would become responsive to secretagogues when subsequently propagated in vitro. We also investigated what effects in vivo administration of 17 beta-estradiol would have on secretagogue-induced PRL release and on intracellular biochemical mechanisms in these cells. MMQ cells were implanted subcutaneously in the backs of female rats. One group was injected with 100 micrograms polyestradiol phosphate (PEP) every 5 days, a second with saline. The inoculants grew into solid tumors within 3 weeks. The day after the tumors were removed and enzymatically dispersed, the cells, now designated MMQt cells, were perifused in vitro. Basal PRL released by MMQt cells was approximately 1 ng/min/10(7) cells and perifusions with 100 nM TRH or AII for 5 min significantly increased PRL release above baseline (integrated areas: 1.8 +/- 0.4 and 5.2 +/- 1.3 ng/10(7) cell, respectively; P less than 0.01). Two ng/ml maitotoxin (MTX), a calcium channel activator, increased PRL release (38.2 +/- 6.7 ng/10(7) cells; P less than 0.01). In PEP-treated perifused MMQt cells, basal in vitro PRL release was not different from that observed in the control group, but the responses to TRH, AII and MTX were greatly attenuated (TRH: 0.6 +/- 0.1, AII: 1.3 +/- 0.2 and MTX: 9.2 +/- 2.5 ng/10(7) cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1989

Effect of 17 beta-estradiol on phosphoinositide metabolism and prolactin secretion in anterior pituitary cells.

The present study was undertaken to investigate the effect of 17 beta-estradiol (E2) administration on in vitro prolactin (PRL) release and intracellular phosphoinositide metabolism. The incorporation of [3H]inositol (Ins) into phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphophate [PtdIns(4,5)P2], and the generation of inositol phosphate (InsPx) following thyrotropin-releasing hormone (TRH) stimulation were studied in primary cultures of anterior pituitary cells obtained from ovariectomized rats. Administration of polyestradiol phosphate (PEP) to ovariectomized rats produced a significant increase (p less than 0.05) in serum PRL levels. This treatment also enhanced significantly (p less than 0.01) the in vitro release of PRL in a progressive manner during 24, 48 and 72 h of culture of dispersed anterior pituitary cells. The radioisotopic labeling by [3H]Ins of all species of phosphoinositides was progressive throughout 72 h of culture, and a good correlation was observed between intracellular phosphoinositide synthesis and PRL release from these cells. PEP treatment enhanced significantly (p less than 0.05-0.01) [3H]Ins incorporation into PtdIns and PtdIns(4)P after 48 and 72 h of culture, although it did not alter [3H]Ins incorporation into PtdIns(4,5)P2. Furthermore, this treatment caused a small, but significant increase (p less than 0.01) in InsPx generation following TRH stimulation. However, the increased [3H]Ins incorporation into phosphoinositide and InsPx generation that we observed after TRH stimulation was significantly (p less than 0.01) less than the increased amount of in vitro PRL release following PEP treatment. There was no significant correlation between the percentage increases in PRL release and phosphoinositide metabolism following the same treatment. These data suggest that phosphoinositide metabolism is enhanced in the anterior pituitary cells of ovariectomized rats by treatment with PEP, but this system does not appear to be tightly coupled or causally related to the much greater production of PRL release.

Animals↗
PubMed1989

Interleukin-6 stimulates anterior pituitary hormone release in vitro.

Interleukin-6 (IL-6), a cytokine produced by inflammatory reactions, was found to stimulate PRL, GH and LH release from anterior pituitary cells at concentrations similar to those which affected lymphocyte mitogenesis. Perifused pituitary cells responded to IL-6 with prompt increases in hormone release that declined rapidly following cessation of exposure. Dopamine (DA) attenuated IL6-induced PRL release. In addition, IL-6 potentiated both GHRF- and TRH-induced hormone release without an affect on intracellular cAMP. These data demonstrate a new biological activity for IL-6 and provide evidence for immune system regulation of anterior pituitary hormone release.

Animals↗
PubMed1989

Evidence that phorbol diester-sensitive protein kinase-C(s) may not be directly involved in secretagogue-stimulated prolactin release and arachidonate liberation.

This report presents findings pertaining to the role of protein kinase-Cs in the release of PRL and liberation of arachidonate from PRL-secreting cells. In our experiments, protein kinase-C activators increased PRL release and arachidonate liberation from anterior pituitary cells and from the PRL-secreting cell line MMQ. In cells depleted of pituitary protein kinase-Cs by chronic exposure to protein kinase-C activators, such as phorbol dibutyrate or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, TRH, angiotensin-II, and neurotensin each increased PRL release and [3H]arachidonate liberation in a normal manner. In addition, the PRL-releasing activities of protein kinase-C activators and those of TRH appeared to be synergistic, an unexpected effect if these substances were functioning through the same intracellular pathways. It, therefore, appears that phorbol diester-sensitive protein kinase-Cs may not be involved in the increased secretion of PRL or liberation of arachidonate that is caused by TRH, angiotensin-II, or neurotensin.

Angiotensin II↗
PubMed1988

Dopaminergic reduction of intracellular calcium: the role of calcium influx.

The effects of dopamine (DA) on 45Ca2+ ion movement and prolactin release in dispersed female rat anterior pituitary cells were studied to elucidate the mechanism for DA reduction of intracellular calcium levels. In 45Ca2+ prelabeled cells, DA inhibited fractional calcium efflux and prolactin release simultaneously and continuously in a concentration-dependent manner (IC50 20 nM DA). We then studied unidirectional calcium influx and observed haloperidol-reversible, concentration-dependent DA suppression of calcium influx into unlabeled cells. These data complement and extend reported fluorescent dye studies and suggest that dopamine primarily inhibits calcium influx, thereby reducing intracellular calcium levels, which leads to suppression of prolactin release and is manifest secondarily as a reduction in fractional 45Ca2+ efflux.

Animals↗
PubMed1988

Angiotensin II increases pituitary cell prolactin release and arachidonate liberation.

The effects of angiotensin II (AII) on prolactin release and arachidonate liberation were studied in anterior pituitary cells preincubated with [3H]arachidonate to label the cellular phospholipids. AII increased prolactin release and [3H]arachidonate liberation over similar concentration ranges with the dynamics of these two events proving identical. Dopamine attenuated both prolactin release and [3H]arachidonate liberation. The diacylglycerol lipase inhibitor RHC80267 decreased AII-stimulated prolactin release and arachidonate liberation. Further evidence that AII-induced release of arachidonate is mediated by a diacylglycerol lipase is suggested by the finding that AII increased [14C]stearate liberation from cells prelabeled with the fatty acid. Although arachidonate itself may have some role in prolactin secretion, it is likely that arachidonate metabolites are more directly involved because BW755c and AA861, inhibitors of arachidonate metabolite formation, increased AII-stimulated arachidonate liberation, but decreased prolactin release.

Angiotensin II↗
PubMed1988

A possible role for lipoxygenase and epoxygenase arachidonate metabolites in prolactin release from pituitary cells.

We studied the effects of selected leukotrienes and hydroxyeicosatetraenoic acids (HETEs) on prolactin release from primary cultures of female rats anterior pituitary cells. Leukotrienes B4, C4, and D4 had no effect on basal prolactin release; however, they did enhance prolactin release that was stimulated by 1 or 5 nM thyrotropin-releasing hormone (TRH). Leukotriene C4 also enhanced prolactin release that was induced by phorbol myristate acetate (a protein kinase C activator) by maitotoxin (a calcium uptake stimulator), and by angiotensin II. 5-HETE, 12-HETE, and 15-HETE stimulated basal prolactin release at high concentrations (1 microM and greater), and 5-HETE and 12-HETE enhanced TRH- and angiotensin II-induced prolactin release at lower (nanomolar) concentrations as well. In order to determine the role of endogenous arachidonate metabolites in prolactin release, pituitary cell cultures were exposed to selected inhibitors of the 5-lipoxygenase enzyme, which metabolizes arachidonate to leukotrienes and 5-HETE, and to those of the epoxygenase enzyme, which metabolizes arachidonate to epoxyeicosatrienoic acids. These inhibitors decreased basal and secretagogue-induced prolactin release. In additional experiments, it was determined that TRH enhances the liberation from pituitary cells of arachidonate metabolites with high-performance liquid chromatography elution profiles similar to those of leukotriene C4 and omega-OH-leukotriene B4 (a metabolite of leukotriene B4) and the HETEs. Therefore, the production of leukotrienes, HETEs, and epoxyeicosatrienoic acids may be necessary for the normal release of prolactin.

Animals↗
PubMed1988

Gonadotropin-releasing hormone-stimulated phosphoinositide hydrolysis in the anterior pituitary. Modulation by protein kinase C but not by cyclic nucleotides.

Gonadotropin-releasing hormone (GnRH) produces a rapid and concentration-dependent hydrolysis of polyphosphoinositides in rat anterior pituitary cells in culture. Evaluation of the action of the decapeptide by measurement of [3H]-inositol phosphates and of prelabeled phosphoinositides demonstrated an effect on phosphatidylinositol-4,5-bis-phosphate and phosphatidylinositol-4-phosphate earlier than on phosphatidylinositol. The receptor antagonist [D-pGlu1,D-Phe2,D-Trp3,6]-luteinizing hormone-releasing hormone blocked the effect of GnRH on [3H]-inositol phosphate production. Protein kinase C activators attenuated GnRH-induced phosphoinositide hydrolysis, while neither cyclic AMP analogs nor cyclic GMP analogs were effective. These results indicate that phosphoinositide hydrolysis represents an important postreceptor transducing mechanism for GnRH action at the gonadotroph and that protein kinase C (but not cyclic nucleotides) may exert a negative feedback control on GnRH receptor-coupling mechanisms.

Animals↗
PubMed1988

Characterization of the MMQ cell, a prolactin-secreting clonal cell line that is responsive to dopamine.

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.

Calcium↗
PubMed1988

The dynamics of arachidonic acid liberation and prolactin release: a comparison of thyrotropin-releasing hormone, angiotensin II, and neurotensin stimulation in perifused rat anterior pituitary cells.

The dynamics of arachidonic acid (AA) liberation and PRL release were highly correlated in perifused rat anterior pituitary cells during stimulation by three different neuropeptides: TRH, angiotensin II (AII), and neurotensin (NT). After preincubation of these cells with 1 microCi [3H]AA, a 20-min perifusion with AII (100 nM), TRH (100 nM), or NT (1 microM) elicited a sharp initial increase in PRL release and [3H]AA efflux, which rapidly subsided (within 6 min) to less elevated levels of PRL release and AA liberation. The plateau responses were sustained throughout the remainder of the 20-min treatment period; after the cessation of neuropeptide perifusion, the responses rapidly returned to basal levels. AII and TRH elicited a greater initial stimulation of PRL release and AA liberation, whereas NT resulted in less pronounced initial responses and a greater plateau of sustained PRL release and AA liberation. Dopamine (DA; 500 nM) or calcium-depleted medium (containing 60 microM EGTA) evenly attenuated the stimulation of PRL release throughout exposure to the neuropeptides; however, the initial stimulation of AA efflux by AII and TRH was relatively resistant to inhibition by DA or calcium-depleted medium. In contrast, the stimulation of AA liberation by NT was abolished by DA or calcium-dependent medium. These results establish that the time course of AA liberation is complimentary to that of PRL release during stimulation by AII, TRH, and NT and support a possible role for AA liberation and metabolism as one of the mechanisms that participates in the regulation of PRL release. A lesser ability of NT to elicit functional and biochemical responses to intracellular calcium mobilization is postulated as an explanation for the observed differences among AII, TRH, and NT effects on PRL release and AA liberation.

Angiotensin II↗
PubMed1988

Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receptor.

We have examined the influences of dopamine and the D2 receptor agonist bromocriptine on phosphoinositide metabolism in primary cultures of rat anterior pituitary cells, monitoring changes in the levels of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Basal incorporation of [3H]inositol ([3H]Ins) into phosphoinositides was progressive, and radioisotopic equilibrium was attained in all three species within 48 h. The inclusion of dopamine or bromocriptine in the incubation medium promoted concentration-dependent reductions in the rate, but not the magnitude, of phosphoinositide radiolabeling. The onset of this effect was rapid; inhibition of [3H]Ins incorporation by dopamine (500 nM) and bromocriptine (100 nM) could be detected within 2 h. This treatment also produced a comparable reduction in the incorporation of [32P]orthophosphate into PtdIns(4,5)P2. In extended time-course studies, bromocriptine dramatically retarded the radiolabeling of PtdIns(4)P and PtdIns(4,5)P2, and apparent equilibria in these species were attained only after 96 h. We also assessed the ability of dopamine to modify the concentration-response characteristics of [3H]Ins-labeled inositol phosphate ([3H]InsPx) production by TRH, angiotensin II (AII), neurotensin (NTS), bombesin (BBS), and vasoactive intestinal polypeptide (VIP). Neither dopamine nor bromocriptine altered the rate or magnitude of TRH-, AII-, NTS-, or BBS-related InsPx generation. VIP was completely ineffective in stimulating InsPx generation. PRL release was significantly reduced in all dopamine-treated groups. That the InsPx concentration-response relationships for each of these peptides remained unimpaired by exposure to dopamine or bromocriptine extends our previous observation that the phosphoinositide-specific phospholipase-C is insensitive to dopaminergic tone. Consistent with our earlier findings, these data indicate that activation of the D2 dopamine receptor attenuates the activity of mechanisms associated with the serial phosphorylations of PtdIns and PtdIns(4)P, reactions that give rise to PtdIns(4)P and PtdIns(4,5)P2, respectively. It is our conclusion that dopamine, in addition to its other actions, attenuates the phosphorylation, rather than the hydrolysis, of anterior pituitary phosphoinositide. This attenuation appears to be mediated by an inhibitory coupling of the D2 receptor with the phosphoryltransferase activities that catalyze PtdIns(4)P and PtdIns(4,5)P2 formation.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Calmodulin modulates prolactin secretion in vitro: studies with calmodulin containing liposomes.

The control of prolactin secretion by Ca calmodulin and cyclic AMP was studied. Ca++ ionophore A23187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvement in these processes we used either selective calmodulin antagonist, the naphthalene sulphonamide derivative W7, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic AMP accumulation and prolactin secretion. Insertion of Ca calmodulin within the cells stimulated prolactin secretion without modifying cyclic AMP accumulation. W7 inhibited the Ca calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin participates to the process of prolactin release.

Animals↗
PubMed1987

Immunoglobulins of Lambert-Eaton myasthenic syndrome inhibit rat pituitary hormone release.

We speculated that IgG from patients with Lambert-Eaton myasthenic syndrome (LES) would inhibit calcium-dependent hormone secretion in rat anterior pituitary (AP) cells. Primary cultures of normal AP cells were continuously exposed to crude IgG from an LES patient or from healthy control subjects, and the incubation media were assayed for prolactin (PRL) or growth hormone (GH). The LES IgG caused a time- and concentration-dependent reduction of PRL and GH release compared with control IgG over 0.5 to 48 hours using concentrations of 0.01 to 4.0 mg/ml. The calcium-channel activator maitotoxin stimulated 45Ca2+ uptake and PRL release from AP cells under control conditions, and the LES IgG significantly reduced both actions of maitotoxin. Thus LES IgG appears to modulate an AP antigenic site, perhaps representing an integral component of voltage-gated calcium channels that may share properties with similar presynaptic elements at the neuromuscular junction.

Animals↗
PubMed1987

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.

Animals↗
PubMed1987

Attenuation of pituitary polyphosphoinositide metabolism by protein kinase C activation.

Phorbol myristate acetate (PMA) stimulates pituitary hormone release by activating protein kinase C (PKC). By doing so, PMA mimics the diacylglycerol (DAG) produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). The present study demonstrates that PMA and DAG augment prolactin release and attenuate the elevations of inositol phosphates (IPX) elicited by thyrotropin-releasing hormone (TRH), angiotensin II, neurotensin, bombesin and gonadotropin-releasing hormone (GnRH) in normal anterior pituitary and prolactin-secreting 7315a tumor cells. 4 alpha-Phorbol 12,13-didecanoate (PDD), an inactive analog of PMA, was found to have no effect on IPX levels; the PKC inhibitor H-7 attenuated the PMA-related inhibition of TRH-induced IPX. To examine whether PMA attenuates IPX generation or increases IPX metabolism, the effects of PMA on the levels of inositol phosphates and phosphoinositides were determined. TRH increased inositol trisphosphate, inositol bisphosphate and inositol monophosphate, and decreased PIP2 and phosphatidylinositol 4-phosphate levels. PMA had no effect on basal phosphoinositide or inositol phosphate levels, but attenuated the effects of TRH on these parameters. Thus PMA and DAG, by a mechanism involving PKC-mediated attenuation of secretagogue-induced hydrolysis of PIP2, decreases IPX production, and therefore PKC activation may exert negative feedback regulation on anterior pituitary secretory activity.

Animals↗