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.

Abstract: Direct intrastriatal injection of N‐methyl‐D‐aspartate (NMDA; 100 μg/rat) increased striatal dopamine (DA) release in vivo. However, parenteral administration of (±)‐3‐(2‐carboxypiperizin‐4‐yl)propyl‐1‐phosphonic acid (CPP) and cis‐4‐phosphonomethyl‐2‐piperidine carboxylic acid (CGS‐19755) did not alter DA metabolism and release in several brain regions in the rat and mouse. Intracerebroventricular administration of the competitive NMDA antagonists CPP, CGS‐19755, 2‐amino‐5‐phosphonopentanoate, and 2‐amino‐7‐phosphonoheptanoate did not alter rat striatal DA metabolism and release but profoundly reduced cerebellar cyclic GMP (cGMP) levels in the same animals. CPP and CGS‐19755 decreased basal cerebellar cGMP levels in the mouse with ED50 values of 6 and 1 mg/kg, i.p., respectively. CPP antagonized the harmaline‐induced increases in cGMP levels with an ED50 value of 5.0 mg/kg, i.p. CPP (25 mg/kg, i.p.) also decreased basal cGMP levels in mouse cerebellum for up to 3 h, a result suggesting brain bioavailability and a long duration of NMDA receptor antagonism in vivo. These contrasting patterns suggest that NMDA receptors exert a tonic excitatory tone on the guanine nucleotide signal transduction pathway in the cerebellum while exerting a phasic control over nigrostriatal dopaminergic neurotransmission. These results also indicate that competitive NMDA antagonists, unlike phencyclidine receptor agonists, may not mediate biochemical and behavioral effects via dopaminergic mechanisms.

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.

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)

In addition to reconstituting immune competence, the thymus gland preparation, thymosin fraction 5 (TSN‐5), has recently been shown to stimulate secretion of hormones from the hypothalamic‐pituitary adrenal axis in vivo and from pituitary corticotropes in vitro. The purpose of the present study was to investigate the effects of TSN‐5 on secretion of immunoreactive β‐endorphin (iβ‐E) by mouse corticotropic tumor cells. The release of iβ‐E by AtP‐20 pituitary tumor cells was increased in a dose‐dependent manner by concentrations trations of 30–600 pg/ml of TSN‐5, whereas concenconcentrations greater than 1,000 μg/ml were increasingly less effective in stimulating secretion. TSN‐5 (600 μg/ml) significantly stimulated iβ‐E release within 7 min; maximal secretory responses (up to 275% of control release) occurred by 4 hr. The secretory response of AtP‐20 cells to 600 μg/ml TSN‐5 (37.9 ± 2.0 vs. 16.1 ± 1.0 ng iβ‐E/ml/4 hr, mean ± SE) was similar in magnitude to release evoked by 0.1 μM corticotropin‐releasing factor (CRF). Combining TSN‐5 and CRF treatment increased secretion of iβ‐E to nearly 600% of control levels, an effect greater than an additive influence of the two independent treatments. Whereas CRF treatment reduced the levels of iβ‐E in AtP‐20 cell extracts after 24‐hr treatment by 45% (231.8 ± 24.7 vs. 417.2 ± 17.8 ng iβ‐E/mg protein, CRF vs. vehicle treatments, respectively), TSN‐5 did not significantly alter cellular hormone content. Neither TSN‐α 1 nor TSN‐β 4 , two of the component peptides of TSN‐5, affected basal or CRF‐stimulated releast of iβ‐E, indicating that an unidentified constituent(s) is corticotropic. This study demonstrates that TSN‐5 directly stimulates hormone release from AtT‐20 cells and potentiates the secretory actions of CRF. These findings support the concept of interactions between neuroendocrine and immune regulators of the hypothalamic‐pituitary adrenal axis. Additionally, corticotropic tumor cells appear to be a useful model in which to differentiate a hormonally active constitutent(s) of TSN‐5 and evaluate the cellular mechanisms of action of TSN‐5 on corticotropic cells.

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 primary role in the pathophysiology of AD.

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.

Administration of apomorphine (0.3–3.0 mg/kg s.c.) evoked a rapid increase (10 min) in circulating levels of immunoreactive β-endorphin (iβ-END) in rats. As judged by gel filtration chromatography, virtually all of the increase corresponded to iβ-END resembling β-lipotropin (β-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 μg/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.

Glucocorticoid control of pituitary beta-endorphin (beta-END) release was investigated in vitro and in vivo. Cultured cells of both rat anterior (AL) and neurointermediate (NIL) lobe released beta-END-like immunoreactivity (beta-END-LI) in response to epinephrine (10(-7) M); however, only the response of AL cells was prevented by corticosterone (10(-8)-10(-6) M) or dexamethasone (10(-9)-10(-7) M). Gel chromatographic analysis (Sephadex G-50) revealed that the major forms of beta-END-LI released by AL cells corresponded to beta-END and beta-lipotropin (beta-LPH) in molecular size, whereas virtually all of the immunoreactivity released by NIL cells resembled beta-END. In vivo administration of dexamethasone attenuated the stress-induced release of beta-END-LI in a dose- and time-related fashion, having a more pronounced effect on plasma levels of beta-END-LI corresponding to beta-LPH in molecular size. Metyrapone (100 mg/kg), an inhibitor of glucocorticoid synthesis, evoked a rapid (20-40 min) four- to sixfold increase in total plasma beta-END-LI and 75% of this rise was due to immunoreactivity resembling beta-LPH in size. This response was diminished by coadministration of either dexamethasone (0.05-1.25 mg/kg) or corticosterone (0.05-1.25 mg/kg) and completely prevented by 4-hr pretreatment with dexamethasone (50 micrograms/kg). The briskness of the plasma beta-END-LI response to acute changes in glucocorticoid status suggests that a "rapid" feedback mechanism operates in the physiologic control of pituitary beta-END-LI secretion. Moreover, the ability of glucocorticoids to selectively inhibit AL release of beta-END-LI in vitro and their pronounced effect on plasma levels of beta-END-LI resembling beta-LPH, a marker of AL secretion, together indicate that glucocorticoids exert a selective influence over the secretion of AL corticotrophs in vivo. This demonstration of differential regulation of the AL versus IL secretion of beta-END-LI in vivo most likely reflects a phenomena having biologic importance related to the different physiologic actions of the several molecular forms of beta-END-LI secreted by the two tissues.

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.

Using gel filtration chromatography (Sephadex G-50) and radioimmunoassay for β-endorphin (β-END) and β-lipotropin (β-LPH) we investigated the site [anterior lobe (AL) vs. intermediate lobe (IL)] for serotonergic control of pituitary β-END-like immunoreactivity (β-END-LI) in the rat. Since the secretion of β-LPH in vitro clearly distinguishes β-END-LI release by the AL as compared to the IL, we interpreted changes in plasma levels of immunoreactivity resembling β-LPH to reflect β-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 β-END-LI was due to the form of immunoreactivity resembling β-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 β-LPH-sized immunoreactivity with little effect on β-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 β-END-LI; however, the immunoreactive peak coeluting with β-LPH was primarily affected, being increased 9.5-fold while that resembling β-END was increased less than 1-fold.