Diminished neuropeptide Y and dopamine beta-hydroxylase immunoreactivity in a guinea pig model of left ventricular hypertrophy.

OBJECTIVE: The aims of the study were to determine the effect of chronic pressure overload of the left ventricle on the density and distribution of neuropeptide-Y-like immunoreactive (NPY-LI) nerve fibres in heart and to compare any changes to those observed in adrenergic nerve fibres, identified by dopamine beta-hydroxylase immunoreactivity. METHODS: Pressure overload was produced in female adult guinea pigs by constriction of the abdominal aorta, using a modified Weck haemoclip. The same operation was performed on a separate group of animals except that no clip was placed around the aorta. Five weeks after surgery, animals were anaesthetised, and the hearts were fixed by perfusion for immunohistochemistry. Cryostat sections were stained, using an indirect peroxidase/antiperoxidase method, for NPY or dopamine beta-hydroxylase. RESULTS: Aortic stenosis caused a 45% increase in left ventricular weight and a 58% increase in left atrial weight at 5 weeks postsurgery. Pulmonary oedema, a sign of cardiac failure, was evident in most of the animals with aortic stenosis. Immunohistochemical studies showed that in atria and right ventricles from animals with abdominal aortic stenosis the distribution and density of NPY-LI nerve fibres were similar to those in the sham operated guinea pigs. However, the left ventricles obtained from the animals with aortic stenosis were nearly devoid of NPY-LI nerve fibres. The density of dopamine beta-hydroxylase-LI nerve fibres was also substantially reduced in the hypertrophied left ventricles. CONCLUSIONS: Aortic stenosis resulting in left ventricular hypertrophy caused a nearly complete loss of NPY-LI and dopamine beta-hydroxylase-LI nerve fibres from the left ventricle. The parallel reduction in both neuropeptide Y and dopamine beta-hydroxylase is in accordance with the association of neuropeptide Y with sympathetic (adrenergic) nerve fibres in the left ventricle and suggests that chronic left ventricular hypertrophy causes a severe degeneration of sympathetic axons supplying this chamber and/or reduces the ability of these sympathetic neurones to maintain normal levels of neurotransmitter related enzymes and neuropeptides.

Effects of differentiation on neuropeptide-Y receptors and responses in rat pheochromocytoma cells.

Undifferentiated rat pheochromocytoma PC12 cells resemble immature adrenal chromaffin cells, express neuropeptide-Y (NPY) receptors of the Y1 subtype, and synthesize catecholamines as well as NPY. In the present study, we examined how phenotypic alteration of PC12 cells by nerve growth factor (NGF) or glucocorticoid affected cellular responsiveness to NPY and related agonists, especially with regard to modulation of catecholamine overflow. Unlike undifferentiated PC12 cells, cells differentiated to a sympathetic neuronal phenotype with NGF were responsive to the Y2 receptor-selective agonist, NPY 13-36. NPY 13-36 1) inhibited binding of [125I]NPY 1-36, 2) inhibited accumulation of evoked cAMP, and 3) inhibited evoked catecholamine overflow. NGF-differentiated cells were also responsive to the Y1 receptor-selective agonist [Leu31,Pro34]NPY (LP-NPY). Like NPY-(13-36), LP-NPY inhibited binding of [125I]NPY-(1-36); however, LP-NPY and NPY-(13-36) exerted their effects through heterogeneous receptors, as LP-NPY enhanced while NPY 13-36 inhibited evoked catecholamine overflow in NGF-differentiated cells, despite the fact that both agonists inhibited the evoked cAMP. In contrast to NGF-differentiated cells, cells differentiated to a mature chromaffin phenotype with dexamethasone were unresponsive to NPY-(13-36), nor did the Y2 agonist inhibit binding of [125I]NPY-(1-36). Dexamethasone-differentiated PC12 cells were, however, responsive to LP-NPY, as this agonist enhanced evoked catecholamine overflow and inhibited binding of [125I]NPY-(1-36). Peptide-YY also enhanced catecholamine overflow, but only significantly at 100 nM. The data suggest differential expression of NPY receptor subtypes on neuronal and endocrine cells where catecholamine overflow is a key feature. These studies further demonstrate inhibitory or excitatory modulation of catecholamine transmission by NPY via distinct receptor subtypes in homogeneous sympathoadrenomedullary models resembling sympathetic neurons and chromaffin cells.

Dopaminergic inhibition of pituitary beta-endorphin-like immunoreactivity secretion in the rat.

A pharmacologic approach was used to examine the possible role of dopamine neurons in the regulation of pituitary beta-endorphin-like immunoreactivity (beta-END-LI) secretion in the rat. Blockade of dopamine receptors by haloperidol or pimozide treatment evoked dose- and time-related increases in plasma levels of beta-END-LI. Physical immobilization increased circulating beta-END-LI six-fold and pretreatment with dopamine receptor agonists (bromocriptine or pergolide) significantly attenuated this rise without affecting plasma beta-END-LI levels in non-stressed animals. Dopaminergic drugs and immobilization produced similar effects on circulating PRL as on beta-END-LI. However, the magnitude of change in levels of PRL was generally greater than the change in beta-END-LI. The present findings suggest that dopaminergic neurons inhibit the release of pituitary beta-END-LI as well as PRL in the rat.

Evidence that a serotonergic mechanism stimulates the secretion of pituitary beta-endorphin-like immunoreactivity in the rat.

A pharmacological approach was used to investigate serotonergic control of the secretion of pituitary beta-endorphin-like immunoreactivity (beta-END-LI) in the rat. The administration of 75 or 200 mg/kg L-tryptophan (ip, over 30 min) increased brain serotonin by 17% and 19%, respectively, and increased circulating beta-END-LI from 0.30 +/- .06 to 0.56 +/- 0.7 and 0.64 +/- 0.8 ng/ml, respectively. D,L,5-Hydroxytryptophan (30 mg/kg, ip, over 30 min) produced a 4.9-fold increase in brain serotonin content and a 3.4-fold rise in plasma beta-END-LI. The administration of a serotonin reuptake blocker, fluoxetine (10 mg/kg, ip, over 15 min), elevated basal levels of plasma beta-END-LI from a control value of 0.38 +/- 0.02 to 1.21 +/- 0.32 ng/ml. Exposure to ether increased circulating beta-END-LI to 1.08 +/- 0.18 ng/ml, and fluoxetine treatment further increased this rise to 1.69 +/- 0.09 ng/ml (P less than 0.05). Quipazine, a serotonin receptor agonist, evoked a dose-related (2.5-5.0 mg/kg, ip) increase in circulating beta-END-LI levels by 15-45 min post injection. By contrast, intraventricular injection of the neurotoxin 5,7-dihydroxytryptamine (75 microgram free base, for 10 days) caused a 77% depletion of brain serotonin and attenuated the rise in beta-END-LI levels in response to immobilization (3.28 +/- 0.20 vs. 1.83 +/- 0.25 ng/ml). A higher dose of 5,7-dihydroxytryptamine (200 microgram free base, for 10 days) significantly decreased resting levels of beta-END-LI from 0.65 +/- 0.14 to 0.36 +/- 0.08 ng/ml. We conclude that brain serotonin neurons exert a stimulatory influence over the basal secretion of pituitary beta-END-LI and mediate, in part, the stress-induced release of this hormone.

N-methyl-D-aspartate treatment increases circulating adrenocorticotropin and luteinizing hormone in the rat.

Excitatory amino acids have been known to increase pituitary secretion of LH in vivo and are probably involved in the neuroendocrine regulation of the hypothalamic-pituitary-gonadal axis. We have found that systemic administration of the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) evokes a transient and profound increase in circulating levels of ACTH as well. Treatment of adult male Long-Evans rats with NMDA (30 mg/kg, sc) maximally increased plasma ACTH and immunoreactive beta-endorphin from 7-15 min after injection, and levels of both remained significantly elevated until 60 min into the time course. Corresponding increases in corticosterone were observed 15 and 30 min after treatment, while LH, similar to other pituitary hormones, was increased from 7-30 min after NMDA. Stimulation of the pituitary-adrenal and pituitary-gonadal neuroendocrine axes by NMDA was monitored in subsequent studies by plasma ACTH and LH, respectively; both were increased in a dose-related manner after the administration of 3-60 mg/kg NMDA, although stimulation of ACTH (800%) was more pronounced than that of LH (200%). The increases in ACTH and LH due to NMDA were inhibited by pretreatment with the competitive NMDA antagonist (+/-)3-(2-carboxypiperazin-4- yl)propyl-1-phosphonic acid, CPP (6 and 10 mg/kg, ip, for 21 min); by contrast, dexamethasone pretreatment (50 micrograms/kg, ip, for 4 h) blocked only the NMDA-evoked increase in circulating ACTH. These findings indicate that an NMDA receptor mechanism might be involved in the acute activation of the hypothalamic-pituitary-adrenal axis in the rat.

(+) 3-[3-hydroxyphenyl-N-(1-propyl) piperidine] selectively differentiates effects of sigma ligands on neurochemical pathways modulated by sigma receptors: evidence for subtypes, in vivo.

The effects of sigma ligands, (+)3PPP 3-[3-hydroxyphenyl-N(1-propyl) piperidine] and (-)butaclamol, were evaluated in vivo on the metabolism of dopamine (DA) and in the striatum release of adrenocorticotrophic hormone (ACTH) and prolactin in the rat and changes in levels of cyclic guanosine monophosphate (cGMP) in the cerebellum of the mouse and compared with the effects of (+)NANM (N-allyl-normetazocine, SKF 10,047) and (+)pentazocine. Both (+)3PPP and (-) butaclamol decreased the release of prolactin and did not affect the metabolism of DA. N-Allyl-normetazocine and (+)pentazocine increased release of prolactin and have been shown previously to increase the metabolism of DA. All four ligands increased release of ACTH; however, only the increases caused by (+)NANM and (+)pentazocine were reversed by pretreatment with CPP, a N-methyl-D-aspartate (NMDA) receptor antagonist. (+)Pentazocine and (+)NANM inhibited the NMDA receptor-mediated changes in levels of cGMP in the cerebellum of the mouse, while (+)3PPP and (-)butaclamol did not attenuate the response to NMDA. In addition to further confirming a functional interaction between sigma receptors and NMDA receptors, these studies divide the observed effects of putative sigma ligands into two groups, characterized by benzomorphan compounds and non-benzomorphan compounds, suggesting the possibility of subtypes at sigma receptor in vivo.

Sigma receptors modulate the hypothalamic-pituitary-adrenal (HPA) axis centrally: evidence for a functional interaction with NMDA receptors, in vivo.

The present report investigates the modulation of the hypothalamic-pituitary-adrenal (HPA) axis in the rat by sigma receptors, using a selective ligand, (+) pentazocine, and comparing the effects with (+) SKF 10, 047. Both compounds stimulate ACTH release potently after central and peripheral administration. These effects are centrally mediated, since they did not release ACTH from anterior pituitary primary cultures. The effects are not blocked by naloxone, but are blocked by the NMDA antagonist, CPP, indicating a centrally mediated functional interaction between NMDA and sigma receptors, in vivo.

Activation of distinct second messenger systems in anterior pituitary corticotrophic tumor cells alters the phosphorylation states of both shared and distinct cytosolic proteins.

The purpose of the present study was to investigate the effects of activation of different second messenger systems on protein phosphorylation in pituitary corticotrophic tumor cells (AtT-20/D16-16). Using two-dimensional gel analysis of cytosolic extracts from AtT-20 cells, several phosphoproteins exhibited alterations in 32P incorporation in response to stimulation of the cells with either forskolin--an activator of adenylate cyclase--or 12-O-tetradecanoyl phorbol-13-acetate (TPA)--a tumor promoting phorbol ester linked to protein kinase C activation. Alterations in phosphorylation levels were seen for phosphoproteins of the following apparent molecular weights and pIs: 87 kDa (pI 4.4-4.6), 67 kDa (pI 4.7-4.9), 43 kDa (pI 4.8-5.0), 39 kDa (pI 4.9-5.1), 33 kDa (pI 4.8-5.0), 19.5 kDa (pI 5.7-5.9), 19 kDa (pI 5.8-6.0), 16 kDa (pI 5.2-5.4) and 14 kDa (pI 5.1-5.3). For individual phosphoproteins, 32P incorporation varied over time and was also modulated by concentrations of Ca2+ and Mg2+ in the incubation medium. Treatment of the cells with forskolin led to statistically significant changes in the phosphorylation states of the 19.5 and 14 kDa proteins. Treatment of the cells with TPA also produced statistically significant changes in the 19.5 and 14 kDa proteins but, in addition, the 87 kDa, the 39 kDa and the 16 kDa phosphoproteins also exhibited significant changes. Alterations in the phosphorylation states of the 19.5 and the 14 kDa proteins were significantly correlated with alterations in beta-endorphin release from the cells. The primary finding of the present study was that activation of distinct second messenger systems can lead to alterations in the phosphorylation states of both shared and distinct phosphoproteins.

The effects of pro-opiomelanocortin peptides on cyclic AMP and tyrosinase in melanoma cells.

Des-, mono-, and diacetylated melanotropin (des-, mono-, and di-Ac MSH, respectively) were compared for their dose-related effects on content of adenosine 3':5'-monophosphate (cAMP) and tyrosinase activity in the Cloudman S91 mouse melanoma tumor. Des-Ac MSH was more potent than the acetylated forms of MSH at increasing cellular levels of cAMP; mono- and di-Ac MSHs, however, were more potent than des-Ac MSH at elevating the activity of the enzyme, tyrosinase. Lysine-gamma1 MSH, a melanotropin from the amino terminus of pro-opiomelanocortin, exhibited slight stimulatory effects on tyrosinase and these actions were less than additive to those of mono-Ac MSH. Unlike their actions on amphibian skin-darkening or in mammalian behavior, neither beta-endorphin1-31 nor its derivatives, N-Ac-beta-endorphin1-27 or beta-endorphin30-31 (glycylglutamine), exhibited any influence on tyrosinase activity evoked by mono-Ac MSH in the tumor cells.

CRF and cAMP regulation of POMC gene expression in corticotrophic tumor cells.

The effects of corticotropin releasing factor (CRF) on pro-opiomelanocortin (POMC) gene expression were investigated in an anterior pituitary corticotrophic tumor cell line, AtT-20/D16-16. The results of mRNA dot blot hybridization assays suggested that CRF, at a concentration of 10(-7) M, positively regulates the expression of the POMC gene in AtT-20 cells in a concentration-dependent fashion. Evaluation of the time course of this effect indicated that CRF had a biphasic mode of action. CRF and alpha-amanitin (inhibitor of RNA polymerase II activity) were also found to affect POMC mRNA levels in a concentration-dependent fashion. Eight-bromo-cyclic adenosine monophosphate (8-Br-cAMP) produced biphasic effects on POMC mRNA levels, supporting evidence of a role for cAMP as a second messenger in the regulation of POMC gene expression. It was also found that alpha-amanitin negatively regulated basal and CRF-stimulated POMC mRNA levels at both the 2 hr and 24 hr time periods, supporting evidence for positive regulation of POMC by CRF at the transcriptional level.

Spinal fluid CRF reduction in Alzheimer's disease.

Abnormalities in several neurotransmitters, including neuropeptides, have been found in postmortem studies of Alzheimer's disease (AD). Recently, corticotropin-releasing factor (CRF) was found to be diminished in cerebral cortex. In this study spinal fluid CRF-immunoreactivity (CRF-I) was determined in 16 patients with mild to moderate AD and 9 age-matched controls. Mean CRF-I levels were significantly lower in Alzheimer patients compared with controls. Furthermore, a tendency for a CRF-I increment with successive spinal fluid aliquots in control subjects was absent in Alzheimer patients. CRF-I levels failed to correlate with measures of disease severity or various tests of cognitive function. These results suggest that involvement of CRF containing neurons may play a secondary rather than a primary role in the pathophysiology of AD.

Apomorphine selectively stimulates opiocortin hormone release from the pars distalis in rats.

Administration of apomorphine (0.3-3.0 mg/kg s.c.) evoked a rapid increase (10 min) in circulating levels of immunoreactive beta-endorphin (i beta-END) in rats. As judged by gel filtration chromatography, virtually all of the increase corresponded to i beta-END resembling beta-lipotropin (beta-LPH) in molecular size. The apomorphine response was inhibited by pretreatment with either the dopamine antagonist, haloperidol (0.1 mg/kg i.p., 2 h) or the glucocorticoid, dexamethasone (50 micrograms/kg i.p., 4 h). Together, these results indicate that dopamine-receptor activation stimulates the release of opiocortin hormones from corticotrophs of the pars distalis.

Systemic NMDA treatment increases plasma ACTH in the neonatal female and male rat.

The present study was designed to examine the adrenocorticotropic hormone (ACTH) response to N-methyl-D-aspartate (NMDA) in neonatal rats. Subcutaneous injection of NMDA (30 mg/kg) was found to increase plasma ACTH concentrations two-fold after 15 min in 9-10 and 20-21 day-old female and male rats. Pretreatment with the competitive NMDA receptor antagonist CPP (10 mg/kg) failed to attenuate the ACTH response to NMDA in the younger rats, yet reduced the response in older ones. These findings indicate that NMDA can elevate plasma ACTH in both female and male neonatal rats, however this response is not sensitive to CPP antagonism until the end of the neonatal period.

Evidence for serotonergic stimulation of pituitary beta-endorphin release: preferential release from the anterior lobe in vivo.

Using gel filtration chromatography (Sephadex G-50) and radio-immunoassay for beta-endorphin (beta-END) and beta-lipotropin (beta-LPH) we investigated the site [anterior lobe (AL) vs. intermediate lobe (IL)] for serotonergic control of pituitary beta-END-like immunoreactivity (beta-END-LI) in the rat. Since the secretion of beta-LPH in vitro clearly distinguishes beta-END-LI release by the AL as compared to the IL, we interpreted changes in plasma levels of immunoreactivity resembling beta-LPH to reflect beta-END-LI release from the AL. Following the administration of L-tryptophan (200 mg/kg, 30 min, ip), a serotonin precursor, nearly all of the rise in total plasma beta-END-LI was due to the form of immunoreactivity resembling beta-LPH in molecular size. Similarly, 5-hydroxytryptophan (30 mg/kg, 30 min, ip), a serotonin precursor, and fluoxetine (10 mg/kg, 15 min, ip), a serotonin reuptake blocker, predominantly increased circulating levels of beta-LPH-sized immunoreactivity with little effect on beta-END-sized immunoreactivity. Quipazine (2.5 and 5.0 mg/kg, 30 min, ip), a serotonin receptor agonist, elevated plasma levels of both forms of beta-END-LI; however, the immunoreactive peak coeluting with beta-LPH was primarily affected, being increased 9.5-fold while that resembling beta-END was increased less than 1-fold. Immobilization stress (30 min) dramatically elevated plasma levels of both forms of immunoreactivity, however, a greater relative rise in beta-LPH than beta-END was observed. Intraventricular administration of 5,7-dihydroxytryptamine (75 micrograms, free base, 10 d), a serotonin neurotoxin, lowered plasma levels of both forms of immunoreactivity about equally in stressed animals. Further, dexamethasone, a synthetic glucocorticoid which selectively inhibits AL corticotroph secretion in vitro, attenuated the beta-LPH response to serotonergic activation in vivo. Together, these findings indicate that serotonergic drugs predominantly influence the release of beta-END-LI resembling beta-LPH and further suggest that serotonin neurons preferentially regulate the release of beta-END-LI from AL corticotrophs in vivo.

A comparison of domperidone and haloperidol effects on different dopaminergic neurons in the rat brain.

Domperidone, a dopamine (DA) receptor antagonist with reportedly preferential actions outside of the blood-brain barrier, and haloperidol, a centrally active DA antagonist, were compared with respect to their abilities to increase the activity of dopaminergic neurons in the rat brain. The activity of nigrostriatal, mesolimbic, tuberohypophyseal and tuberoinfundibular dopamine nerves was estimated by measuring the in vivo rate of DA synthesis (dihydroxyphenylalanine accumulation following administration of an inhibitor of aromatic L-amino acid decarboxylase) in the striatum, olfactory tubercle, posterior pituitary and median eminence, respectively. In an initial study, the rates of DA synthesis in striatum, olfactory tubercle, and posterior pituitary were determined at 2, 8, and 16 h after subcutaneous administration of 0.25, 2.5, or 25 mg/kg domperidone. At the lowest dose of domperidone, DA synthesis was increased only in the posterior pituitary at 8 and 16 h; at the intermediate dose, DA synthesis increased in the posterior pituitary at 8 and 16 h and in the olfactory tubercle at 8 h. Only at 8 h after the highest dose of domperidone was DA synthesis increased in the striatum. When 2.5 mg/kg of domperidone or haloperidol were administered, DA synthesis in posterior pituitary and median eminence was increased in a similar fashion (in the latter region only at 16 h). In contrast, domperidone promoted only modest and delayed increases in DA synthesis in the olfactory tubercle and had no effect in the striatum. These results indicate that systemically administered domperidone preferentially increases DA synthesis in neurons terminating outside the blood-brain barrier, but after a pronounced delay, high doses of the drug can also activate DA neurons which project to the forebrain.

A D-2 dopaminergic agonist stimulates secretion of anterior pituitary immunoreactive beta-endorphin in rats.

Hypothalamic dopamine neurons are known to control circulating levels of immunoreactive beta-endorphin (i beta-END) by inhibiting hormone secretion by the intermediate lobe (IL) of the pituitary gland. We examined the ability of the D-2 selective dopaminergic agonist, LY141865, to influence circulating levels of i beta-END in rats and found that in contrast to inhibiting IL secretion, LY141865 increased release of i beta-END from the anterior lobe (AL). Intraperitoneal injection of 1 mg/kg LY141865 transiently increased plasma levels of i beta-END by 7-30 min after drug treatment; plasma prolactin levels were maximally reduced within 15 min and throughout the remaining 2-hour time course of treatment. Doses of 0.3 and 1.0 mg/kg of LY141865 increased circulating i beta-END to 440 and 690%, respectively, of control levels (0.38 +/- 0.12 ng/ml, mean +/- SEM, n = 6). Lower doses of the D-2 agonist (0.01-0.1 mg/kg) failed to significantly affect plasma i beta-END. Sephadex G-50 chromatography of plasma pools revealed that virtually all of the increase due to LY141865 treatment was immunoreactivity resembling beta-lipotropin in molecular size, the principal component of AL secretion of i beta-END. Furthermore, LY141865-evoked release was blocked by pretreatment of rats with dexamethasone (50 micrograms/kg i.p., 4 h) which inhibits AL but not IL secretion of pro-opiomelanocortin-derived peptides. Stimulation of i beta-END release by LY141865 was also inhibited by the general dopamine antagonist, haloperidol, (0.1-3.0 mg/kg i.p., 2 h) and by the D-2 selective antagonist, sulpiride (100 micrograms/rat i.c.v., 4 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin and dopamine independently regulate pituitary beta-endorphin release in vivo.

Serotonin and dopamine neurons have been shown to exert a stimulatory and inhibitory control, respectively, over pituitary release of beta-endorphin-like immunoreactivity (beta-END-LI). In the present study we sought to determine whether an interaction exists between these two reciprocal mechanisms regulating beta-END-LI in the rat. The intraperitoneal (i.p.) administration of 5 mg/kg quipazine, a serotonin receptor agonist, or 2.5 mg/kg haloperidol, a dopamine receptor antagonist, each elevated circulating levels by beta-END-LI 5-fold over control levels by 30 min post-injection. Pretreatment (1 h) with 5 mg/kg, i.p., cinanserin, a serotonin receptor antagonist, completely blocked the quipazine-induced rise in beta-END-LI without affecting the elevated levels of beta-END-LI in haloperidol-treated animals. Conversely, pretreatment (2 h) with 1 mg/kg, i.p., bromocriptine, a dopamine receptor agonist, had no effect on quipazine-induced release of beta-END-LI but did completely prevent the rise in plasma beta-END-LI due to haloperidol treatment. Gel filtration chromatography revealed that quipazine and haloperidol treatments elevated plasma levels of both beta-END-size immunoreactivity and beta-lipotropin (beta-LPH)-sized immunoreactivity though to different relative degrees. However, since circulating levels of beta-LPH serve as a marker for anterior lobe (AL) beta-END-LI secretion, serotonin and dopamine appear to exert stimulatory and inhibitory control, respectively, over AL beta-END-LI release. Further, the quipazine-induced rise in total plasma beta-END-LI primarily resembled beta-LPH in size and was blocked by cinanserin but not bromocriptine pretreatment. And conversely, bromocriptine but not cinanserin prevented the haloperidol-induced rise in circulating beta-END-LI.(ABSTRACT TRUNCATED AT 250 WORDS)

Phencyclidine increased release of beta-endorphin from anterior lobe of the pituitary.

Phencyclidine (PCP) has been found to affect neuroendocrine function by altering the release of the anterior pituitary hormones, adrenocorticotrophin, luteinizing hormone and prolactin. The purpose of this study was to examine the effect of PCP on release of the two pituitary hormones also derived from the adrenocorticotropin precursor, namely, alpha-melanocyte-stimulating hormone and beta-endorphin (beta-E), synthesized in the neurointermediate and anterior lobes of the pituitary. At behaviorally active doses, PCP administered i.c.v. increased plasma levels of immunoreactive beta-E (i beta-E) without affecting the concentration of immunoreactive alpha-melanocyte-stimulating hormone, suggesting that PCP increased the release of beta-E from only the anterior lobe of the pituitary. Dexamethasone pretreatment blocked the PCP-induced increase in i beta-E which indicated further the anterior lobe effects of PCP. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate), a selective PCP ligand, at behaviorally active doses also increased the plasma concentration of i beta-E. The dose-response curves for induction of behavior was very different from that for increasing the concentration of i beta-E in plasma. The increase in release of i beta-E was stereoselective as (+)-(1-(1-phenylcyclohexyl)-3 methylpiperidine but not (-)-(1-(1-phenylcyclohexyl)-3 methylpiperidine increased release of i beta-E. The increase in plasma levels of beta-E was not due to an interaction with opioid receptors because naloxone did not block PCP-induced release of beta-E. In vitro, PCP also significantly increased release of i beta-E from anterior lobe of the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear protein kinase substrates in AtT-20 cells: translocation of a 14 kDa phosphoprotein.

In this work we characterize some acidic nuclear substrates of protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA), using intact anterior pituitary corticotrophic tumor cells (AtT-20/D16-16). It was found that, as in the cytosolic fraction, substrates for both PKC and PKA exist in the nucleus and that changes in the phosphorylation states of a few of these phosphoproteins are mediated by both kinases. One of the phosphoproteins examined, a 14 kDa phosphoprotein (pp14) described previously, exhibited a phorbol-ester induced translocation from nucleus to cytosol in pulse-chase experiments utilizing 35S-methionine labeling. These results suggest that pp14 may be involved in signal transduction in AtT-20 cells. Although its identity remains to be determined, a 14 kDa DNA-binding protein was also seen in nuclear extracts of AtT-20 cells.

Characterization of substance P-like immunoreactivity and tachykinin-encoding mRNAs in rat medullary thyroid carcinoma cell lines.

Rat thyroid tissue and three rat medullary thyroid carcinoma cell lines, 6-23, WE4/2, and CA77, have been examined for substance P (SP) and SP-like peptide expression. Analysis by combined HPLC and radioimmunoassay revealed the presence of SP in thyroid and 6-23 cell extracts. The presence of SP-encoding mRNAs was also detected in 6-23 cells by solution hybridization-nuclease protection analysis. SP-encoding mRNA expression was increased (fourfold) by maintaining the 6-23 cells in low serum (2%) for 4 or 10 days. The 6-23 cells also expressed other SP-like immunoreactive species, which were chromatographically and immunologically distinct from established tachykinin peptides. WE4/2 cells did not contain SP but did display SP-like immunoreactivity (SPLI), which migrated like the unidentified SPLI in 6-23 cells. CA77 cells did not contain SP or SP-encoding mRNA but did contain SPLI that migrated identically to the unidentified SPLI in the other cell lines. This novel SPLI was detected with an antiserum directed against the SP carboxyl terminus and to a lesser extent with an antiserum directed against the neurokinin A carboxyl terminus, but it showed minimal cross-reactivity using an antiserum directed against the midportion of SP. Treatment with 50 mM KCl resulted in secretion of this SPLI from CA77 cells. Gel filtration analysis demonstrated that this novel SPLI had an apparent molecular weight of approximately 1,000. These results are discussed in terms of cell lines that express tachykinin peptides and in terms of the molecular nature of the new SPLI detected in CA77 cells.