The role of prolactin in the suppression of Crh mRNA expression during pregnancy and lactation in the mouse.

Maternal stress is associated with negative health consequences for both the mother and her offspring. To prevent these adverse outcomes, activity of the hypothalamic-pituitary-adrenal (HPA) axis is attenuated during pregnancy and lactation. Although the mechanisms generating this adaptive change have not been defined fully, the anterior pituitary hormone prolactin may play a significant role. The present study investigated the role of prolactin in regulating the basal activity of the HPA axis during pregnancy and lactation in the mouse, focussing upon the corticotrophin-releasing hormone (CRH) neurones. Using in situ hybridisation, a decrease in Crh mRNA-expressing cell number in pregnant (55.6±9.0 cells per section) and lactating (97.4±4.9) mice compared to virgin controls was characterised (186.8±18.7, P<.01 Tukey-Kramer test; n=6-7 per group). Removal of the pups (24 hours) and thus the associated suckling-induced prolactin secretion, restored CRH neurone number (180.1±19.7). To specifically test the role of prolactin in suppressing Crh mRNA expression in lactation, prolactin levels were selectively manipulated in lactating mice. Lactating mice were treated with ovine prolactin (1500 µg day-1 , osmotic minipump, s.c.; n=7) or vehicle (n=6) for 24 hours following pup removal. This was sufficient to suppress Crh mRNA expression from 108.0±13.5 to 53.7±16.7 cells per section (P<.05 Student's t-test). Additional cohorts of lactating mice were treated with bromocriptine (300 µg over 24 hours, s.c.; n=7) or vehicle (n=5) to suppress endogenous prolactin secretion; however, no change in Crh mRNA expression was detected. Thus, although prolactin was sufficient to suppress Crh mRNA expression in the paraventricular nucleus, it does not appear to be required for the ongoing regulation of the CRH neurones in lactation.

Restraint stress increases prolactin-mediated phosphorylation of signal transducer and activator of transcription 5 in the hypothalamus and adrenal cortex in the male mouse.

Prolactin is a pleiotropic peptide hormone produced by the lactotrophs in the anterior pituitary. Its rate of secretion is primarily regulated by a negative-feedback mechanism where prolactin stimulates the activity of the tuberoinfundibular dopaminergic (TIDA) neurones, increasing their release of dopamine, which accesses the pituitary via the median eminence to suppress further prolactin secretion. In addition to its well established role in lactation, circulating prolactin is secreted in response to stress, although the mechanism by which this is achieved or its cellular targets remains unknown. In the present study, we show that 15 minutes of restraint stress causes an approximately seven-fold increase in circulating prolactin concentration in male mice. Monitoring prolactin receptor activation, using immunohistochemistry to determine the level and distribution of tyrosine phosphorylated signal transducer and activator of transcription 5 (pSTAT5), we show that this stress-induced increase in prolactin interacts with both central and peripheral targets. Restraint stress for 15 minutes significantly increased pSTAT5 staining in the arcuate nucleus, median eminence and the zona fasciculata of the adrenal cortex. In each case, this response was prevented by pretreating the animals with bromocriptine to block prolactin secretion from the pituitary. Interestingly, in contrast to many cells in the arcuate nucleus, stress reduced pSTAT5 staining of the TIDA neurones (identified by dual-labelling for tyrosine hydroxylase). This suggests that there is reduced prolactin signalling in these cells and thus potentially a decline in their inhibitory influence on prolactin secretion. These results provide evidence that prolactin secreted in response to acute stress is sufficient to activate prolactin receptors in selected target tissues known to be involved in the physiological adaptation to stress.

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells.

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P << 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.

Feedback regulation of PRL secretion is mediated by the transcription factor, signal transducer, and activator of transcription 5b.

PRL secretion from the anterior pituitary gland is inhibited by dopamine produced in the tuberoinfundibular dopamine neurons of the hypothalamus. The activity of tuberoinfundibular dopamine neurons is stimulated by PRL; thus, PRL regulates its own secretion by a negative feedback mechanism. PRL receptors are expressed on tuberoinfundibular dopamine neurons, but the intracellular signaling pathway is not known. We have observed that mice with a disrupted signal transducer and activator of transcription 5b gene have grossly elevated serum PRL concentrations. Despite this hyperprolactinemia, mRNA levels and immunoreactivity of tyrosine hydroxylase, the key enzyme in dopamine synthesis, were significantly lower in the tuberoinfundibular dopamine neurons of these signal transducer and activator of transcription 5b-deficient mice. Concentrations of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence were also significantly lower in signal transducer and activator of transcription 5b-deficient mice than in wild-type mice. No changes were observed in nonhypothalamic dopaminergic neuronal populations, indicating that the effects were selective to tuberoinfundibular dopamine neurons. These data indicate that in the absence of signal transducer and activator of transcription 5b, PRL signal transduction in tuberoinfundibular dopamine neurons is impaired, and they demonstrate that this transcription factor plays an obligatory and nonredundant role in mediating the negative feedback action of PRL on tuberoinfundibular dopamine neurons.

The distribution of opioid binding subtypes in the bovine adrenal medulla.

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1-13). Specific [3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1-13). Delta opioid sites, labelled with [3H][D-Ala2,D-Leu5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [3H][D-Ala2, NMePhe4,Gly-ol5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

Localization of angiotensin II binding sites in the bovine adrenal medulla using a labelled specific antagonist.

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [125I]-[Sar1,Ile8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.

The effects of neosurugatoxin on evoked catecholamine secretion from bovine adrenal chromaffin cells.

1. The effect of neosurugatoxin (NSTX), a toxin from the Japanese ivory mollusc (Babylonia japonica), on the nicotinic response of bovine adrenal chromaffin cells was examined. 2. NSTX inhibited acetylcholine- and nicotine-induced catecholamine secretion from the cultured cells with an IC50 against 5 microM nicotine of 30 nM. 3. This inhibitory effect was reversible and independent of the presence of agonist. 4. NSTX had no effect on the catecholamine release from cultured cells evoked by 50 mM K+, or 1 microM histamine. 5. NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 microM muscarine. 6. These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where alpha-bungarotoxin is ineffective.

Opioid inhibition of nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells.

The ability of a number of opioid agonists and antagonists to affect nicotine-induced 45Ca2(+)-uptake into cultured bovine adrenal medullary cells has been investigated. High (10 microM) concentrations of the opioid agonist bremazocine produced a significant inhibition of nicotine-induced 45Ca2(+)-uptake throughout the 15 min time course examined. The opioid subtype-selectivity of this inhibition was investigated; mu and delta selective agonists produced only minor effects whereas the kappa selective agonist U50-488H and the endogenous opioid peptides dynorphin(1-13) and metorphamide almost abolished nicotine-induced 45Ca2(+)-uptake. The U50-488H inhibition was significant at 10 nM concentrations with an IC50 of approximately 1 microM. U50-488H inhibition could not be reversed or reduced by the opioid antagonists naxolone, diprenophine or Mr2266. Furthermore, Mr2266 and its optical isomer Mr2267 also produced marked inhibition of 45Ca2(+)-uptake. The inhibition was specific to nicotine-induced 45Ca2(+)-uptake in that a similar level of uptake evoked by potassium depolarization was unaffected by high concentrations of U50-488H. These data indicate that opioid inhibition of nicotine-induced 45Ca2(+)-uptake does not involve classical, stereospecific opioid receptors and suggests the involvement of a pharmacologically distinct opioid recognition site. It is speculated that this may be associated with the nicotine receptor-ionophore complex.

Effects of opioid compounds on basal and muscarinic induced accumulation of inositol phosphates in cultured bovine chromaffin cells.

The mammalian adrenal medulla expresses a variety of both opioid peptides and opioid receptors. The function of this adrenal opioid system is, however, largely unknown. We have examined the ability of a number of opioid compounds to influence basal and muscarinic stimulated accumulation of inositol phosphates in cultured bovine chromaffin cells. Muscarine produced a dose-dependent 1.5-fold increase in total inositol phosphates. This response was sensitive to atropine inhibition. The ten opioid compounds examined were chosen because between them they possess selectivity for all of the identified opioid receptor subtypes. However, none of these opioids in the concentration range 10nM-10 microM had any significant effect on either basal or muscarinic induced total inositol phosphate accumulation. We conclude that it is unlikely that opioid peptides released from either the chromaffin cells themselves or the splanchnic nerve can modulate the inositol phosphate second messenger system within the adrenal chromaffin cells.

Guanylate cyclase activities in enriched preparations of neurones, astroglia and a synaptic complex isolated from rat cerebellum.

Guanylate cyclase activity was measured in homogenates of a number of freshly isolated and cultured cell preparations derived from the 8-day old rat cerebellum. The freshly isolated fractions enriched in astrocyte perikarya, exhibited twice the basal activity of the Purkinje neurone enriched fraction. In contrast, cultured granule neurones and astrocytes displayed approximately equivalent activities. In freshly isolated cells guanylate cyclase activity could be stimulated approx 15-fold by 1.0 mM sodium nitroprusside. Cultured neurones and astroglia were relatively insensitive to this activator. The excitatory amino acid analogue N- methyl- d -aspartate produced a 150-fold increase in the cGMP content of intact freshly isolated cells, suggesting an enzyme stimulation of a similar order of magnitude to that produced by sodium nitroprusside. Significantly, the cGMP content of intact cultured cells did not increase in the presence of excitatory amino acids or potassium stimulation. These studies demonstrate the presence of high levels of guanylate cyclase activity within astrocytes, and suggest that these cells may make a major contribution to the cGMP synthesizing capacity of the cerebellum. Activation of guanylate cyclase in freshly isolated cells by excitatory amino acids or sodium nitroprusside may involve a mechanism which is absent or ineffective in cultured cells.

Effects of angiotensin II on cultured, bovine adrenal medullary cells.

Primary cultures of bovine adrenal medullary cells have been used to study the effects of angiotensin II on catecholamine secretion and inositol phosphate accumulation. Angiotensin II induced a weak secretion of both adrenaline and noradrenaline, with a threshold of 10-100 pM and a shallow concentration-dependence up to 10 microM. The response was fully dependent on extracellular Ca++, was partially inhibited by 100 nM nifedipine, was completely blocked by [Sar1, Ala8]-angiotensin II (IC50 5-10 nM) and was unaffected by 0.1 mM hexamethonium. Angiotensin II also increased inositol phosphate accumulation over the range 1 pM-10 microM. Inositol trisphosphate levels increased in a biphasic manner after 15 sec and 1 min exposure to 10 nM angiotensin II, but were not significantly increased at 30 sec or 5, 15 or 30 min stimulation. Inositol bisphosphate was significantly increased after 1 min. Inositol monophosphate levels only increased after 1 min stimulation, but continued to rise during 30 min stimulation. Removal of extracellular Ca++ or addition of EGTA reduced basal inositol phosphate accumulation but not the ability of angiotensin II to stimulate inositol phosphate accumulation relative to basal. Nifedipine (100 nM) had no effect on basal or angiotensin II-induced inositol phosphate accumulation. The inositol phosphate response to angiotensin II was abolished by 1 microM [Sar1, Ala8]-angiotensin II. The results suggest that secretion of adrenal medullary catecholamines can be evoked by angiotensin II, at concentrations that are compatible with a role for circulating angiotensin II or for angiotensin II generated locally within the adrenal medulla. They do not support the suggestion that the secretory actions of angiotensin II on chromaffin cells are mediated by mobilization of intracellular Ca++ stores.

Receptor stimulated formation of inositol phosphates in cultures of bovine adrenal medullary cells: the effects of bradykinin, bombesin and neurotensin.

The ability of a number of drugs and neuropeptides to stimulate phosphoinositide metabolism in cultured bovine adrenal medullary cells has been assessed. Low concentrations (10 nM) of angiotensin II, bradykinin, histamine, arginine-vasopressin, and bombesin, and high (10 microM) concentrations of oxytocin, prostaglandins E1, and E2, beta-endorphin, and neurotensin stimulated significant accumulation of [3H]inositol phosphates in adrenal medullary cells preloaded with [3H)]inositol. Bradykinin stimulated a significant response at concentration as low as 10pM, with an EC50 of approximately 0.5 nM. The response was markedly inhibited by the bradykinin B2 antagonist [Thi5,8,D-Phe7] bradykinin but not the B1 antagonist [Des-Arg9,Leu8] bradykinin. Higher concentrations of bombesin and neurotensin were required to elicit a response (10 nM and 10 microM respectively). The bombesin response was sensitive to inhibition by the bombesin antagonist [D-Arg1,D-Pro2,D-Trp7,9Leu11]-substance P. In contrast, the neurotensin response was not reduced by the NT1 antagonist [D-Trp11]-neurotensin. These results indicate there are a number of agents that can stimulate phosphatidylinositide hydrolysis in the adrenal medullary cells by acting on different classes of receptors. Such a range of diverse agonists that stimulate inositol phosphate formation will facilitate further analysis of the phosphatidylinositide breakdown in chromaffin cell function.

Simultaneous measurement of tyrosine hydroxylase activity and phosphorylation in bovine adrenal chromaffin cells.

A method for simultaneous measurement of tyrosine hydroxylase (TH) activation and phosphorylation in permeabilised and intact bovine adrenal chromaffin cells (BACCs) was established. Permeabilised cells were stimulated with cyclic AMP (1--10 microM) in the presence of [32P]ATP and L-[carboxyl-(14)C]tyrosine. Intact BACCs were preincubated with 32P(i) for 3 h and stimulated with forskolin (1--5 microM) in the presence of L-[carboxyl-(14)C]tyrosine. On stimulation each well was covered with a sealed 'chimney' fitted with a small plastic cup containing 300 microl of 1.0 M NaOH that trapped the 14CO(2) released. TH activity was determined by measuring 14C radioactivity. TH phosphorylation was measured in the same cells by separating the solubilized proteins on SDS PAGE followed by autoradiography and/or HPLC analysis. It was found that H89, a protein kinase A inhibitor, significantly blocked both TH phosphorylation and activation in response to cyclic AMP in permeabilised cells. However, in intact cells, H89 was effective only in respect to forskolin-stimulated TH activity and did not block the forskolin-stimulated TH phosphorylation of Ser-40. The reason(s) for this lack of correlation between TH activation and phosphorylation is presently not understood.

In vivo treatment with mu and delta, but not kappa-selective opioid agonists reduces [3H]spiperone binding to the guinea-pig striatum: autoradiographic evidence.

In guinea-pigs, acute treatment with mu and delta receptor opioid agonists induces sedation and immobility [1,5], and attenuates the behavioural activation produced by the dopamine D2 agonist quinpirole [5]. In contrast, kappa-selective opioid agonists induce dystonic-like movements [4,5,8]. This has led us to investigate the possibility of an interaction between acute opioid treatment and the dopamine D2 system. The effect of acute treatment with mu, delta and kappa opioid agonists on [3H]spiperone binding sites (dopamine D2) in guinea-pig brain was studied using receptor autoradiography. The mu preferring agonist morphine (15 mg/kg subcutaneously, SC) given for 2 h, and the delta receptor selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) (20 nM, intracerebroventricularly, ICV) given for 0.5 h, both decreased the density of specific (butaclamol displaceable) [3H]spiperone binding in the caudate putamen by 23.8 +/- 1.7% and 24.2 +/- 2.7% respectively, and in nucleus accumbens by 26.1 +/- 2.7% and 21.9 +/- 4.6% respectively compared to saline treated animals. There were no significant changes in the level of [3H]spiperone binding to other brain regions examined including frontal cortex, hippocampus, substantia nigra, ventral tegmental area, amygdala, hypothalamic nuclei and cerebellum. In other experiments, incubation of coronal slices from various brain regions with [3H]spiperone, in the presence of a high concentration of morphine (20 microM) or DPDPE (10 microM) did not affect the level of binding, thus precluding effects due to residual tissue levels of drugs after in vivo treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of somatostatin on perfused bovine adrenal glands and cultured bovine adrenal medullary cells.

The effects of somatostatin on catecholamine secretion and inositol phosphate accumulation have been studied using isolated perfused bovine adrenal glands and cultured bovine adrenal medullary cells. Somatostatin had no effect on basal adrenaline or noradrenaline secretion from either preparation. At concentrations above 1 microM, somatostatin inhibited the secretion of both catecholamines induced by 5 microM nicotine from cultured chromaffin cells. In contrast, over the concentration range 0.1 nM-10 microM, somatostatin had no effect on the secretory responses produced by 10 nM angiotensin II or 1 microM histamine. Inositol phosphate accumulation in cultured bovine adrenal medullary cells was unaffected by 0.1 nM-0.1 microM somatostatin, however at 1 and 10 microM somatostatin it was significantly increased, by 23% and 103% respectively. The effects of somatostatin (0.1 nM-10 microM) and of 50 microM muscarine on inositol phosphate accumulation were simply additive. Similarly, somatostatin at 0.1 nM and 10 nM together with 10 nM angiotensin II or 1 microM histamine produced additive inositol phosphate responses. In contrast, 1 microM somatostatin gave significantly more-than-additive (synergistic) inositol phosphate responses with angiotensin II and histamine. The results suggest that some adrenal medullary cells possess several types of receptors, and that these receptors may interact to produce non-additive responses.

Staurosporine inhibits inositol phosphate formation in bovine adrenal medullary cells.

The effect of protein kinase C activators and inhibitors on histamine-stimulated phospholipase C in bovine adrenal medullary cells has been investigated. The protein kinase C activators, phorbol 12,13-dibutyrate (PDB) or sn-1,2-dioctanoylglycerol (DOG), inhibited histamine-stimulation of phospholipase C. This inhibition was prevented by the protein kinase C-selective inhibitor Ro 31-8220 (3-[1-[3-(2-isothioureido) propyl]indol-3-yl]-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dio ne) but not the broad spectrum protein kinase inhibitor staurosporine. Indeed staurosporine on its own inhibited both the histamine-stimulated response and, in permeabilized cells, phospholipase C activated by Ca2+. Staurosporine inhibition of phospholipase C is unlikely to be mediated via protein kinase A or Ca2+/calmodulin-dependent protein kinase because it was not reproduced by selective inhibition of these kinases. Staurosporine treatment, however, reduced inositol phospholipid levels in stimulated cells. Thus staurosporine and Ro 31-8220, two widely used protein kinase C inhibitors, have quite different effects on phospholipase C activation. Furthermore, staurosporine may cause this inhibition through a reduction in the level of phospholipase C substrate.

Prostanoid responses of bovine adrenal medullary cells: lack of effect of opioids.

The effects of opioid compounds on catecholamine (CA) secretion and phosphatidylinositol turnover induced by prostaglandins E1 (PGE1) and E2 (PGE2) in cultured bovine adrenal medullary cells have been studied. PGE1 induced CA secretion at 100 nM and above. PGE2 was more potent, inducing CA secretion at 1-10 nM. Both prostaglandins required extracellular calcium to induce CA release. Neither etorphine nor diprenorphine (1 nM-10 microM) affected CA secretion induced by 1 microM PGE1 or 0.1 microM PGE2. PGE1 a small increase in phosphatidylinositol turnover at 10 microM, but had no effect at lower concentrations. PGE2 was effective at 1 and 10 microM. Etorphine and diprenorphine had no effect on phosphatidylinositol turnover induced by PGE1 or PGE2. The results indicate prostaglandins can facilitate CA secretion independently of their effects on phosphatidylinositol metabolism. They also indicate that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary cells to modify their responses to prostaglandins.

The role of protein kinase C in nicotinic responses of bovine chromaffin cells.

The effects of the protein kinase C inhibitor CGP 41251 (31-benzoyl-staurosporine) on nicotinic responses of cultured bovine adrenal chromaffin cells have been investigated. CGP 41251 inhibited tyrosine hydroxylase activation by phorbol 12,13-dibutyrate, with an IC50 of < 0.3 microM and complete inhibition at 1 microM. In contrast, it had little effect on nicotine-stimulated tyrosine hydroxylase activity up to 1 microM, and did not fully inhibit it even at 10 microM. From 1 to 10 microM, CGP 41251 caused a similar concentration-dependent inhibition of tyrosine hydroxylase activity stimulated by nicotine, K+, forskolin and 8-Br-cyclic AMP. CGP 42700 (19,31-dibenzoyl-staurosporine), a structural analogue of CGP 41251 that lacks activity as a protein kinase C inhibitor, had no effect on tyrosine hydroxylase activity stimulated by any of the agonists. CGP 41251 had no effect on catecholamine secretion induced by nicotine. The results suggest phorbol ester-sensitive protein kinase C isozymes do not play a major role in nicotinic stimulation of tyrosine hydroxylase activity or catecholamine secretion in chromaffin cells.

Ca(2+) influx stimulated phospholipase C activity in bovine adrenal chromaffin cells: responses to K(+) depolarization and histamine.

The role of Ca(2+) influx in activating phospholipase C in bovine adrenal chromaffin cells has been investigated. Phospholipase C activity in response to K(+) depolarization (56 mM) was blocked by the L-type Ca(2+) channel antagonist nifedipine and partially inhibited by the omega-conotoxins GVIA and MVIIC. In contrast, phospholipase C activity in response to histamine receptor activation was unaffected by omega-conotoxin GVIA and partially inhibited by omega-conotoxin MVIIC or nifedipine. This response was however markedly inhibited by the non-selective Ca(2+) channel antagonists La(3+) or 1-[beta-[3-(4-Methoxyphenyl)propoxy]-4-methoyphenethyl]-H-imidazol e (SKF-96365). Despite this Ca(2+) dependence phospholipase C activity was not increased during periods of "capacitative" Ca(2+) inflow generated by histamine-, caffeine- or thapsigargin-mediated depletion of internal Ca(2+) stores. Thus, while Ca(2+) influx in response to K(+) depolarization or G-protein receptor activation can increase phospholipase C activity in these cells, in the latter case it appears to be ineffective unless there is concurrent agonist occupation of the receptor.

Localisation and pharmacological characterisation of [3H]bremazocine binding in the bovine adrenal medulla.

[3H]Bremazocine (5 nM), in the presence of excess unlabelled mu and delta opioid ligands labelled two anatomically distinct populations of binding sites in the bovine adrenal medulla; a high density over the peripheral adrenaline-containing region of the medulla and a lower density over the central noradrenaline-containing region. This non-mu, non-delta opioid binding was specific (diprenorphine sensitive) but did not appear to involve classical kappa (kappa 1), sigma or PCP binding sites being insensitive to high concentrations of dynorphin (1-13), 3-PPP or MK-801. A significant proportion of the binding at both locations was however sensitive to competition by U50,488H or metorphamide. These data provide further evidence to support the existence of multiple opioid binding sites in the bovine adrenal medulla.