Topographical distribution of pro-opiomelanocortin-derived peptides (ACTH/β-END/α-MSH) in the rat median eminence

The detailed distribution of adrenocorticotropin (ACTH), beta-endorphin (beta-END) and alpha-melanotropin (alpha-MSH) immunoreactivity was examined in the rat median eminence (ME) and pituitary stalk using light microscopic immunocytochemistry and radioimmunoassay (RIA). Nerve fibers and varicosities immunoreactive for ACTH/beta-END/alpha-MSH had identical distributions in the ME suggesting that they are part of the same arcuate proopiomelanocortin neuronal (POMC) system. The quantitative image analysis of POMC immunoreactive varicosities in the ME indicates no significant differences between the various rostro-caudal segments. In the main (preinfundibular) portion of the ME, a moderate density of immunoreactive elements was located in the lateral part of the internal zone and throughout the postinfundibular ME. Very few scattered varicosities were observed in the neurohemal (external) zone and in the pituitary stalk. By RIA, alpha-MSH is present in a substantially higher concentration than ACTH and beta-END throughout the ME. Knife cuts between the arcuate nucleus and ME indicate that proopiomelanocortin (POMC) fibers enter the ME in its whole rostro-caudal extent. Thus POMC neurons seem to provide innervation of structures in the internal zone but not in the neurohemal/external/zone where the portal capillary system is located. Moreover, the observation that the density of immunoreactive elements is substantially lower in the pituitary stalk than in the ME, suggests that the majority of immunoreactive fibers in the internal zone are not fibers of passage directed towards the neurohypophysis.     

GABA-ergic control of α-Melanocyte-Stimulating Hormone (α-MSH) release by frog neurointermediate lobe in vitro

Measurement of glutamate decarboxylase (GAD) activity in the intermediate lobe of the frog pituitary and brain showed that neurointermediate lobe extracts represented 12% of the GAD activity detected in the whole brain. No significant activity was measured in distal lobe extracts. Immunocytochemical studies revealed GAD-containing fibers among the parenchyma! cells of the pars intermedia. The localization of GAD-like material in the intermediate lobe of the frog pituitary suggested a possible role of γ-aminobutyric acid (GABA) in the regulation of melanotropic cell secretion. Administration of GAB A (10⁻⁶ to 10⁻⁴ M), to perifused neurointermediate lobes caused a brief stimulation of alpha-melanocyte stimulating hormone (α-MSH) release followed by an inhibition. Picrotoxin (10⁻⁴ M), a Cl⁻ channel blocker, abolished only the stimulatory effect of GAB A (10⁻⁴ M), whereas bicuculline (10⁻⁴ M), a specific antagonist of GABA_A receptors, totally inhibited the effects of GABA (both stimulatory and inhibitory phases). Bicuculline induced by itself a slight stimulation of α-MSH release, suggesting that GABA-ergic nerve fibers present in the intermediate lobe are functionally active in vitro. The GABA_A agonist muscimol (10⁻⁷ to 10⁻⁴ M) mimicked the biphasic effect of GABA on α-MSH release. Administration of baclofen, a specific GABA_BB agonist (10⁻⁷ to 10⁻⁴ M) induced a dose-dependent inhibition of α-MSH secretion. In contrast to GABA or muscimol, baclofen did not cause any stimulatory effect whatever the dose. Taken together these result suggested that GABA_A and GABa_b receptors were present on frog melanotrophs. Since bicuculline totally inhibited GABA effects (stimulation and inhibition) on α-MSH release, it appears however that the effect of GABA is mainly achieved through activation of G AB A_A receptors.     

Adenosine receptor agonists inhibit the release of γ-aminobutyric acid (GABA) from the ischemic rat cerebral cortex

The effects of CPA (a selective A1 receptor agonist), NECA (a mixed A1 and A2 receptor agonist), and CGS 21680 (a selective A2 receptor agonist) on the ischemia-evoked release of gamma-aminobutyric acid (GABA) from rat cerebral cortex was investigated with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. In control animals, superfusate GABA increased from a basal level of 206 +/- 26 nM (mean +/- S.E.M., n = 18) to 10,748 +/- 3,876 nM during the reperfusion period. Pretreatment with adenosine receptor agonists failed to affect basal levels of GABA release. However, CPA (10(-10) M), NECA (10(-9) M), and CGS 21680 (10(-8) M) significantly suppressed the ischemia-evoked release of GABA. The ability to block the ischemia-evoked release of GABA was not evident when the adenosine receptor agonists were administered at higher concentrations. Thus, the selective activation of either A1 or high-affinity A2a adenosine receptors results in an inhibition of ischemia-evoked GABA release.     

Regulation of α-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus by serotonin and the interaction of serotonin with the dopaminergic system inhibiting peptide release

Release of α-melanocyte-stimulating hormone (α-MSH) from frontal slices of rat hypothalamus superfused with oxygenated artificial cerebrospinal fluid (ACSF) was quantified by radioimmunoassay. Control depolarisations with 50 mM KCl-containing ACSFm produced significant increases in α-MSH release which were partially blocked by 10⁻⁶ M cinnaserin, a serotonin (5-HT) receptor antagonist. Superfusion of the tissues with varying concentrations of 5-HT *10⁻⁷ M to 10⁻⁴ M) resulted in an inverted U-shaped dose-response curve, maximum α-MSH release being obtained with 10⁻⁶ M 5-HT. Addition of 10⁻⁶ M cinanserin shifted the 5-HT dose-response curve to the right whilst the presence of 10⁻⁸ M flupenthixol, a dopamine receptor antagonist, resulted in a sigmoidal 5-HT dose-response curve. Superfusion with ACSF containing either 10⁻⁷ M fluoxetine, a 5-HT re-uptake inhibitor, or 10⁻⁷ M p-chloroamphetamine, an agent releasing 5-HT, induced significant increases in α-MSH release which were abolished in the presence of 10⁻⁶ M cinanserin. These data demonstrate the presence of an endogenous 5-HT system that exerts a biphasic effect on α-MSH release. A stimulatory effect caused by lower 5-HT concentrations appears to be a direct action whilst an inhibitory effect at higher concentrations is mediated through an inhibitory endogenous dopaminergic system. A significant proportion of K⁺-stimulated peptide release is 5-HT-mediated.     

Somatostatin Plays a Dual, Stimulatory/Inhibitory Role in the Control of Growth Hormone Secretion by Two Somatotrope Subpopulations from Porcine Pituitary

Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low- (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH-releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time- and dose-dependent effects of SRIF-14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF-14 and SRIF-28 on cultures of LD and HD cells. SRIF-14 (10^?7 M) induced a rapid (30 min) and sustained (4 h) 2-fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10^?15 M) stimulated GH release from both LD (154.1±8.2% of basal, P<0.05) and HD (337.2±55.5% of basal, P<0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF-28 elicited similar responses to those observed for SRIF-14 in both somatotrope subpopulations, yet 10^?15 M SRIF-28 was less potent than the same dose of SRIF-14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.     

Morphine and norepinephrine but not 5-hydroxytryptamine and γ-aminobutyric acid inhibit the potassium-stimulated release of substance P from rat spinal cord slices

We studied whether morphine, norepinephrine (NE), 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) inhibit the potassium-stimulated release of substance P (SP) from rat spinal cord slices. Male Sprague-Dawley rats were decapitated and a 2-cm segment of lumbosacral spinal cord was removed, chopped into 0.5 × 0.5 mm pieces, weighed, placed in a perfusion chamber and perfused at 37°C with a modified Krebs bicarbonate buffer. Perf USAte was collected, lyophilized, then assayed for SP using radioimmunoassay. Exposure of spinal cord tissue to 50 mM KCl for 8 min produced a calcium-dependent increase in the release of SP from a basal level of approximately 0.1 pg/mg tissue/min to 0.3 pg/mg tissue/min. Morphine and NE at concentrations of 10⁻⁴ and 10⁻⁵ M did not alter basal release but caused a significant reduction in the potassium-stimulated release of SP. Naloxone (10⁻⁵M) and phentolamine (10⁻⁵M) did not affect SP release but attenuated the effects of morphine and NE, respectively. Naloxone did not antagonize the inhibition of release produced by NE nor did phentolamine block the effect of morphine, suggesting that the actions of the agonists are independent. In contrast, 5-HT and GABA at concentrations of 10⁻⁴ M and 10⁻⁴ M did not significantly alter the basal or potassium-stimulated release of SP. These results demonstrate a differential regulation of SP release in the spinal cord and support the hypothesis that morphine and NE may modify nociception, in part, by inhibiting the release of SP in the spinal cord.     

Lack of effect of interleukins 1α and 1β, during in vitro perifusion,on anterior pituitary release of adrenocorticotropic hormone and β endorphin in the male rat

It has been demonstrated that interleukin 1 (IL1) injection provokes a great variety of biological effects, notably an activation of the corticotropic axis, increasing plasma adrenocorticotropic hormone (ACTH) and corticosterone. However, the primary site of action of IL1 is still controversial. In the present study, we first verified the in vivo capability of human interleukins 1α (hIL1α) and 1β (hIL1β) to release ACTH and β endorphin (β EP) in the normal male rat, before investigating, through an anterior pituitary (AP) perifusion system, the hIL1α and hIL1β effects on basal and corticotropin-releasing factor (CRF)-induced ACTH and β EP secretions. This system enabled the examination of a dynamic profile of hormones secretion, avoiding the possibility of feedback mechanisms, as is the case with the use of regular but very often longtime incubations. The results showed that in a perifusion system, with a short duration treatment (below 2 hr) compatible with the kinetics of action observed in vivo, basal and CRF-induced ACTH and β EP release were not modified in the presence of a broad range of concentrations (from 10^?12 to 10^?9 M) of hIL1α or hIL1β. Taken together, these results clearly show that in an in vitro situation close to physiological conditions, the primary site of action of hIL1α and hIL1β on ACTH and β EP release is not located at the AP level in the male rat. © 1993 Wiley-Liss, Inc.     

Regulation of alpha-melanocyte-stimulating hormone release from superfused slices of rat hypothalamus by serotonin and the interaction of serotonin with the dopaminergic system inhibiting peptide release

Release of alpha-melanocyte-stimulating hormone (alpha-MSH) from frontal slices of rat hypothalamus superfused with oxygenated artificial cerebrospinal fluid (ACSF) was quantified by radioimmunoassay. Control depolarisations with 50 mM KCl-containing ACSF produced significant increases in alpha-MSH release which were partially blocked by 10(-6) M cinanserin, a serotonin (5-HT) receptor antagonist. Superfusion of the tissues with varying concentrations of 5-HT (10(-7) M to 10(-4) M) resulted in an inverted U-shaped dose-response curve, maximum alpha-MSH release being obtained with 10(-6) M 5-HT. Addition of 10(-6) M cinanserin shifted the 5-HT dose-response curve to the right whilst the presence of 10(-8) M flupenthixol, a dopamine receptor antagonist, resulted in a sigmoidal 5-HT dose-response curve. Superfusion with ACSF containing either 10(-7) M fluoxetine, a 5-HT re-uptake inhibitor, or 10(-7) M p-chloroamphetamine, an agent releasing 5-HT, induced significant increases in alpha-MSH release which were abolished in the presence of 10(-6) M cinanserin. These data demonstrate the presence of an endogenous 5-HT system that exerts a biphasic effect on alpha-MSH release. A stimulatory effect caused by lower 5-HT concentrations appears to be a direct action whilst an inhibitory effect at higher concentrations is mediated through an inhibitory endogenous dopaminergic system. A significant proportion of K+-stimulated peptide release is 5-HT-mediated.     

α-Melanotropin, β-endorphin and adrenocorticotropin-like immunoreactivities are colocalized within duodenal myenteric plexus perikarya

Adrenocorticotropin (ACTH) immunoreactivity was localized at the ultrastructural level as positive 'cores' within large dense-cored vesicles (LDVs) of axons and dendrites of the rat duodenum. The immunostained vesicle 'cores' were 35-50 nm in mean diameter, corresponding to 'cores' of LDVs with a mean diameter of 80-90 nm. alpha-melanotropin (alpha-MSH) was detected also within LDVs, expressing the same mean diameter as ACTH-stained vesicles. alpha-MSH and ACTH were localized only within structures belonging to the enteric nervous system of the rat duodenum. alpha-MSH and ACTH, as detected by immunostaining, were absent in endocrine cells of the rat duodenum. These findings suggest the possibility that these peptides may have important physiological roles in the rat duodenum.     

Properties of γ-glutamyltransferase in developing rat brain

The activity and properties of brain γ-glutamyltransferase (EC 2.3,2.2) were studied in 7-, 14-and 90-day-old rats. The enzyme activity was highest in the pons-medulla and lowest in the cerebellum in each age group. The activity of glycylglycine, 10 protein amino acids, GABA and taurine as acceptor of the γ-glutamyl group was studied with 7-day-old and adult rats. The best acceptors were glycylglycine, lysine and methionine and the poorest taurine, valine and isoleucine. The relative acceptor activity of lysine changed most during development. K_m for the γ-glutamyl donor, γ-glutamyl-p-nitroanilide, with glycylglycine was about 3 mM in all experimental groups. It did not change during development but V increased about fivefold in all brain areas studied. A mixture of serine and borate strongly inhibited γ-glutamyltransferase in each age group. Potassium and magnesium ions had no measurable effect on the enzyme activity but sodium ions were stimulatory.     

Stimulation of hypothalamic β-endorphin and dynorphin release by corticotropin-releasing factor (in vitro)

Corticotropin-releasing factor (CRF) at doses of 10⁻¹²–10⁻⁸ M significantly stimulated the release of β-endorphin and dynorphin from superfused rat hypothalamic slices. These effects were shown to be mediated by the CRF receptor since they were antagonized by the CRF receptor antagonist α-helical CRF_9–41 (10⁻⁶ M). The two opioid peptides showed different time courses of response and in the case of β-endorphin, an attenuation of the response upon continued exposure to CRF was observed.     

Characteristics of NMDA receptors that stimulate release of hypothalamic alpha-MSH.

N-methyl-D-aspartic acid (NMDA) 10(-4) M stimulated release of immunoreactive alpha-melanocyte-stimulating hormone (alpha-MSH) from superfused slices of rat hypothalamus through receptors which shared common features with other central NMDA-type glutamate receptors. The receptors possessed inhibitory sites for both Mg2+ and ketamine; basal and NMDA-stimulated alpha-MSH release was reduced by high (5 mM) Mg2+ ion concentrations and by 10(-4) M ketamine, whilst use of Mg(2+)-free media led to a prolongation of the NMDA-stimulated response. The receptors were also shown to possess an allosteric glycine site. The glycine site agonist D-serine 10(-4) M potentiated basal and NMDA-stimulated alpha-MSH release whilst the antagonist, 7-chlorokynurenic acid 10(-4) M, reduced NMDA-stimulated release, an effect which was partially reversed by 10(-4) M D-serine.     

Leukotrienes C4 and D4 stimulate the release of luteinizing hormone-releasing hormone from rat median eminence in vitro

The release of luteinizing hormone-releasing hormone (LH-RH), somatostatin (SRIF) and growth releasing factor (GRF) by male rat median eminences (MEs) incubated in vitro for 30 min, in the presence of leukotrienes (LT) C4, D4, E4 and B4 was estimated by radioimmunoassay (RIA). Leukotrienes, with the exception of LTE4 stimulated the release of LH-RH. The dose-response curve was bimodal for LTC4 with two maxima at 10(-8) and 10(-16) M (X2.2 and 1.9, respectively), biphasic for LTD4 with a maximum (X2) at 10(-8) M; LTB4 was active only at 10(-6) M (X1.9). These different curves suggest a specific effect on the release of LH-RH. Moreover, these effects were selective since no alteration of SRIF and GRF secretions was observed. No additive effect on LH-RH release was observed when LTC4 and LTD4 were added simultaneously at 10(-8) M. FPL-55712, a drug supposed to be an antagonist of LTC4, showed an unexpected stimulatory effect (X4.2 and 1.7-fold) on LH-RH release at 3.10(-5) and 10(-6) M, respectively. However, FPL-55712 did not alter the release of LH-RH induced with 10(-8) or 10(-16) M LTC4. These results extend our previous observations on the stimulatory action of LTC4 and are the first evidence of the stimulatory effect of LTD4 and LTB4 on the LH-RH release.     

β-Amyloid peptide fragment 25–35 potentiates the calcium-dependent release of excitatory amino acids from depolarized hippocampal slices

β-Amyloid protein (βAP) has been frequently associated with the neuropathology of Alzheimer's disease (AD), although the mechanisms by which it can induce neurodegeneration are still unknown. Some studies in hippocampal cultured neurons suggest that βAP, particularly its fragment 25ndash;35, may induce neural growth or render neurons more vulnerable to excitotoxic insults by a mechanism involving intracellular Ca²⁺ dyshomeostasis. We have studied the effect of fragment 25–35 on the release of endogenous amino acids from hippocampal slices of young adult (3–3.5-month-old) and aged (23–25-month-old) rats, under basal, K⁺ -depolarization, and post-depolarization conditions, in the presence and absence of Cat²⁺. In both young and aged tissue, the basal release of amino acids was not affected by the peptide. By contrast, 1-hr preincubation of slices from young animals with 10 μM 25–35 fragment resulted in a 140% increase of glutamate and aspartate release stimulated by K⁺ depolarization, compared with the control-stimulated release. These effects were strictly dependent on external Ca²⁺ Neither the K⁺ -stimulated release of γ-amino butyric acid (GABA) nor the release of glycine, glutamine, taurine, or alanine, which was not stimulated by high K⁺, were affected. Substance P and a scrambled sequence of the 25–35 fragment were without any effect per se, but substance P blocked the stimulatory effect of fragment 25–35 on glutamate and aspartate release. In slices from aged rats the basal release of glutamate was significantly higher (260%) than that in young tissue, and the K⁺ -induced release of both aspartate and glutamate was also higher. Fragment 25–35 also potentiated the K⁺ -induced release of these two amino acids, although to a lesser extent than in young tissue. These results indicate that glutamate is retained less by the aged hippocampus and that fragment 25–35 is able to augment the release of glutamate and aspartate under excitatory conditions, an effect that could be involved in the mechanisms of neurotoxicity of β-amyloid peptides. © 1995 Wiley-Liss, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Allicin and disulfiram enhance platelet integrin αIIbβ3-fibrinogen binding

Introduction

Activation of the platelet receptor αIIbβ3 (glycoprotein IIbIIIa) involves a change in the disulfide bonds pattern in the extra-cellular domain of the receptor. The disulfide-bond reducing agent, dithiothreitol (DTT), can increase integrin activity, and point mutations of specific cysteine residues of the integrin can cause its lockage at the high affinity state. The present study is aimed to support the hypothesis that prevention of specific αIIbβ3 intra-molecular disulfide bond formation increases receptor-ligand binding activity.

Methods

Platelet aggregation was induced by collagen or ADP and epinephrine. Integrin αIIbβ3-fibrinogen binding was evaluated on prostaglandins E₁ (PGE₁)-treated washed platelets or baby hamster kidney (BHK) cells expressing human αIIbβ3. Integrin was directly activated by an anti-ligand induced binding site (LIBS) PT25-2 antibody. The effect of sulfhydryl-reactive agents, such as allicin, glutathione, dithiobis nitrobenzoic acid (DTNB) and disulfiram, was tested on αIIbβ3 activity.

Results

Allicin (40 µM) completely inhibited washed platelets agonist-induced aggregation. Both allicin and disulfiram (40 µM) inhibited αIIbβ3-fibrinogen binding and P-selectin expression in washed platelets. However, there was an increase in αIIbβ3-fibrinogen binding but not P-selectin expression in PGE₁-treated washed platelets activated by PT25-2 antibody. At a high concentration (400 µM) both inhibited αIIbβ3-fibrinogen binding. Similarly, in BHK cells expressing αIIbβ3 activated by PT25-2 antibody, allicin at a low concentration increased αIIbβ3 activity.

Conclusions

Allicin and disulfiram inhibit agonist-induced washed platelet activation probably via inhibition of platelet signaling, but enhance PT25-2 antibody-induced αIIbβ3 integrin activity most likely by preventing reformation of disulfide bridges thereby stabilizing the active conformation of the integrin.     

Human antibody-dependent cellular cytotoxicity mediated by interferon γ-activated neutrophils is impaired by vasoactive intestinal peptide

The aim of the present study was to evaluate the effect of vasoactive intestinal peptide (VIP) on the expression and activity of receptors for the Fc portion of IgG (FcyR) in human neutrophils. Cells were assayed under basal conditions and following in vitro stimulation with interferon gamma (IFNγ). Antibody dependent-cellular cytotoxicity (ADCC) was chosen as a means of evaluating FcyR activity. The results indicated that incubation with VIP (10⁻⁶ M) during 18 h slightly diminished cytotoxicity of non stimulated neutrophils. In contrast, VIP exerted a marked inhibitory effect on neutrophils activated with IFNy. Similar results were obtained with forskolin, another agent that increases intracellular cAMP. Finally, using monoclonal antibodies and flow cytometry analysis, we found decreased membrane expression of FcyR after VIP incubation. Taken together, these results show that VIP is able to act on human neutrophils, partially blocking IFNγ-activation of FcyR mediated functions. Modulation of neutrophil cytotoxic response by VIP may have an important role in limiting tissue injury during inflammation.     

Clonidine releases immunoreactive β-endorphin from rat pars distalis

Clonidine (10⁻⁶, 10⁻⁷ M) evokes the release of β endorphin-like immunoreactivity (β-END-LI) from cell cultures of anterior (pars distalis) but not neurointermediate (pars nervosa plus pars intermedia) lobe of the rat pituitary. This drug-induced secretion is blocked by α-adrenergic (phenoxybenzamine, yohimbine; 10⁻⁵ M) but not β-adrenergic (propranolol, 10⁻⁵ M) antagonism. Gel filtration (Sephadex G-50) reveals that β-END-LI released from anterior lobe cells consists of 2 major forms of immunoreactivity which coelute with β-lipotropin or β-endorphin standards. Conversely, β-END-LI released spontaneously from neurointermediate lobe cells almost entirely corresponds to β-endorphin. The data show that α-adrenergic stimulation by clonidine releases β-END-LI selectively from cells of anterior but not neurointermediate lobe in vitro and suggests that the clonidine-induced release of pituitary β-END-LI we have observed in vivo occurs in part by direct action on the corticotrophs of the pars distalis.     

Immunocytochemically stained binding sites for oxytocin and α-melanocyte-stimulating hormone in rat brain following ventricular administration

Binding sites for oxytocin (OXT) and alpha-melanocyte-stimulating hormone (alpha-MSH) in brain of homozygous Brattleboro rats were immunocytochemically visualized after ventricular administration of the peptides by Accurel implants. Two patterns were found: 'ring type' staining in perineuronal structures was observed in CA1 and CA3 areas of ventral hippocampus and in subiculum for OXT implanted brains and a very weak staining in striatum for alpha-MSH-implanted brains; cytoplasmic staining of intracellular binding sites was observed in the bed nucleus of the stria terminalis (BST) in brains with OXT implants and in the anterodorsal thalamic nucleus (AD) and postcingulate cortex in brains with alpha-MSH implants. These localizations are different from those described for vasopressin binding sites in the same rat strain.     

Somatostatin release from dispersed hypothalamic cells—Effects of membrane depolarization, calcium and glucose deprivation

We have developed a new short term in vitro system to examine hypothalamic somatostatin (SRIF) release. Hypothalamic cells were obtained from normal rats after trypsin or collagenase aided dispersion and released immuno-reactive (IR) SRIF which eluted in 3 molecular weight (MW) forms on gel chromatography. The smallest MW form, which constituted the major peak, co-eluted with synthetic cyclic 1-14 SRIF on gel and reverse phase high pressure liquid chromatography (HPLC). After 24 h in culture in medium containing heat inactivated fetal calf serum, cell viability was demonstrated by two techniques, (1) vital staining with trypan blue, and (2) incorporation of 32Pi into phospholipids. SRIF release was also studied at this time which was optimal in terms of responsivity of the cells to depolarizing stimuli. SRIF release increased in a time dependent manner, over 3 h. Membrane depolarization, induced either by potassium chloride 56 mM or ouabain (the Na+, K+-ATPase inhibitor) 10(-6) M or greater, markedly stimulated SRIF release. Incubation at 4 degrees C, or in the presence of EDTA 0.05 M or verapamil, the calcium channel blocker, 50 microM abolished these stimulatory effects. Glucose deprivation was induced by the addition of 2-deoxy-D-glucose (2-DG) to the experimental medium. 2-DG, at concentrations of up to 200 mg%, had no significant effect on SRIF release during incubation periods of up to 1 h.