Incubation Of Neurointermediate Lobes Research Articles

Insulin and release of neurohypophysial hormones: in vivo and in vitro studies.

No significant effect of intracerebroventricular administration of insulin on the bioassayed neurohypophysial storage of vasopressin and oxytocin could be found. This finding, however, only indirectly reflects possible changes of vasopressin and oxytocin release. Incubation of neurointermediate lobes in Locke's solution containing 3.3 mumol/l insulin resulted in an inhibition of oxytocin release under resting conditions as well as in a decrease of both vasopressin and oxytocin release during depolarization due to excess potassium. These results seem to suggest a possible regulatory role of brain insulin in the mechanisms of release of neurohypophysial hormones.

Inhibition of Prostaglandin Synthesis and the Release of Vasopressin and Oxytocin from the Rat Neurohypophysis: In Vitro Studies1)

The basal and K(+)-evoked release of vasopressin (VP) and oxytocin (OT) from the neurointermediate lobes in vitro was determined under conditions of indomethacin-induced inhibition of prostaglandin (PG) synthesis. The in vivo treatment with indomethacin (IM), administered to donor animals, did not modify the release of VP during resting conditions as well as during the potassium stimulation; on the contrary, both base-line and K(+)-evoked release of OT was significantly inhibited under such conditions. Incubation of neurointermediate lobes in a solution containing indomethacin resulted in an inhibition of VP and OT release both during resting conditions and during depolarization due to excess potassium.

(6R)-5,6,7,8-tetrahydr-α-biopterin Affects Vasopressin and Oxytocin Release from Rat Neurointermediate Lobe In Vitro*

Incubation of neurointermediate lobes in Locke's solution containing 0.13 mumol.l-1 (6R)-5,6,7,8-tetrahydro-alpha-biopterin dihydrochloride (THB4) resulted in an inhibition of bioassayed vasopressin secretion and in an increase of that of oxytocin both under resting conditions as well as during depolarization due to excess potassium. It is suggested that some events related to THB4 and localized in the neural lobe are involved in the mechanism of vasopressin and oxytocin release.

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

Biosynthesis of two structurally different pro-opiomelanocortins in the pars intermedia of the amphibian pituitary gland.

This study reports the biosynthesis of two forms of pro-opiomelanocortin in the pars intermedia of the pituitary gland of the African clawed toad Xenopus laevis. The two forms could be resolved by dodecyl sulphate gel electrophoresis on a 9-16% acrylamide gradient and their molecular weights were 38200 and 37300. Incubation of neurointermediate lobes with [3H]glucosamine followed by tryptic digestion of the newly synthesized glycoproteins, revealed that both prohormones have only one glycosylated site, namely within the region corresponding to gamma 3-melanotropin. Biosynthesis of proteins in lobes treated with tunicamycin to prevent glycosylation again resulted in the production of two pro-opiomelanocortins (Mr 35000 and 34200), indicating that the two forms differ in their primary structure. This notion was corroborated by the results of tryptic mapping of the newly synthesized prohormones. The maps showed that the primary structures of the two forms of pro-opiomelanocortin differ in at least two parts of the molecules, one part concerning the endorphin region.

Studies on the melanocyte-stimulating hormones of the neurointermediate lobe of the American bullfrog, Rana catesbeiana: I. Electrophoretic and chromatographic seprration and identification of the intraglandular, acid activatable, and secreted forms of melanotropic peptides

Peptides of the neurointermediate (NI) lobe of the American bullfrog ( Rana catesbeiana) were separated by polyacrylamide disc gel electrophoresis (PDGE) and by gel filtration. Those which had melanotropic activity (MSH) were identified using the Anolis skin bioassay. Five forms of MSH were detected on acrylamide gels. One region exhibited characteristics of an acid-activatable aggregate. Two regions had electrophoretic mobilities similar to mammalian α- and β-melanocyte-stimulating hormone. A high molecular weight form, which was also acid activatable, was separated by gel filtration on Sephadex G-50. A secreted form which had electrophoretic properties similar to mammalian β-MSH, was detected by PDGE of medium from a culture of NI lobes for 1 day. Tritiated amino acids were actively incorporated into the secreted form during incubation of NI lobes. These results indicate that the pars intermedia of the bullfrog contains multiple forms of MSH but it secretes predominantly only one form which resembles mammalian β-MSH. The gland contains a high molecular weight form which is converted to smaller forms by acid treatment.