Effect of acetylcholine on the electrical and secretory activities of frog pituitary melanotrophs

The activity of melanotroph cells of the amphibian pars intermedia is regulated by multiple factors including classical neurotransmitters and neuropeptides. In this study, we have examined the possible involvement of acetylcholine (ACh) in the regulation of electrical and secretory activities of frog pituitary melanotrophs. Electrophysiological recordings were conducted on cultured cells by using the patch-clamp technique in the whole-cell configuration. In parallel, alpha-MSH release from acutely dispersed pars intermedia cells was studied by means of the perifusion technique. In all cells tested in the current-clamp mode, superfusion with ACh (10(-6) M) gave rise to a depolarization associated with an enhanced frequency of action potentials. Administration of ACh (10(-6) M) to perifused cells also induced stimulation of alpha-MSH release. These results indicate that the neurotransmitter ACh exerts a direct stimulatory effect on pituitary melanotrophs. The action of ACh on electrical and secretory activities was mimicked by muscarine (10(-5) M), while ACh-induced alpha-MSH secretion was completely abolished by the muscarinic antagonist atropine (10(-6) M). The depolarizing effect of muscarine was suppressed by the specific M1 muscarinic antagonist pirenzepine (10(-5) M), indicating the existence of a M1 subtype muscarinic receptor in frog pars intermedia cells. In addition, using a monoclonal antibody against calf muscarinic receptors, we have visualized, by the immunofluorescence technique, the presence of muscarinic receptor-like immunoreactivity in cultured intermediate lobe cells. Electrophysiological recordings showed that nicotine (10(-5) M) induces membrane depolarization associated with an increase of the frequency of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of thyrotropin-releasing hormone (TRH) immunoreactivity in the brain of urodele amphibians

To improve knowledge of the peptidergic systems in the brain of amphibians we have conducted a comparative analysis of the distribution of TRH immunoreactive cell bodies and fibers in three species of urodeles. Fiber labeling was observed in all main brain subdivisions suggesting different control functions for TRH in extrahypothalamic systems. However, as in other vertebrates, TRH neurons were abundant in the hypothalamic nuclei that presumably project to the median eminence and the neural lobe of the hypophysis. Considerable interspecies differences were noted mainly related to innervation of the olfactory and visual centers (thalamus and mesencephalic tectum) and the precise localization of immunoreactive cell bodies, which was assessed by double labeling with tyrosine hydroxylase. The comparison of the distribution of TRH immunoreactive neurons and fibers found in urodeles with those reported for other vertebrates, in particular with anamniotes, reveals a strong resemblance but also notable variations not only across vertebrate classes but also within the same class. In this respect, the virtual lack in urodeles of TRH innervation of the intermediate lobe of the hypophysis clearly contrasts with the innervation found in anurans. Therefore, the important role of skin color adaptation proposed for TRH in anurans on the basis of the direct innervation of the intermediate lobe is not applicable for urodeles.     

GABA-ergic control of alpha-melanocyte-stimulating hormone (alpha-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 parenchymal cells of the pars intermedia. The localization of GAD-like material in the intermediate lobe of the frog pituitary suggested a possible role of gamma-aminobutyric acid (GABA) in the regulation of melanotropic cell secretion. Administration of GABA (10(-6) to 10(-4) M), to perifused neurointermediate lobes caused a brief stimulation of alpha-melanocyte stimulating hormone (alpha-MSH) release followed by an inhibition. Picrotoxin (10(-4) M), a Cl- channel blocker, abolished only the stimulatory effect of GABA (10(-4) M), whereas bicuculline (10(-4) M), a specific antagonist of GABAA receptors, totally inhibited the effects of GABA (both stimulatory and inhibitory phases). Bicuculline induced by itself a slight stimulation of alpha-MSH release, suggesting that GABA-ergic nerve fibers present in the intermediate lobe are functionally active in vitro. The GABAA agonist muscimol (10(-7) to 10(-4) M) mimicked the biphasic effect of GABA on alpha-MSH release. Administration of baclofen, a specific GABAB agonist (10(-7) to 10(-4) M) induced a dose-dependent inhibition of alpha-MSH secretion. In contrast to GABA or muscimol, baclofen did not cause any stimulatory effect whatever the dose. Taken together these result suggested that GABAA and GABAB receptors were present on frog melanotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.     

Seizures and Thyrotropin-Releasing Hormone (TRH) Neural System in the Rat Brain

Abstract. In order to study the relationship between seizures and the thyrotropin-releasing hormone (TRH) neural system, immunoreactive TRH (IR-TRH) and TRH receptor binding activity were determined by pentylenetetrazol (PTZ)-induced seizures and amygdaloid (AM) kindling. IR-TRH markedly increased in the septum 40 and 150 seconds after the PTZ injection. A significant increase in the IR-TRH concentrations was also noted in the hippocampus and thalamus/midbrain 40 and 150 seconds after the PTZ injection, respectively. However, no significant changes were observed in the TRH receptor binding before, during or after the PTZ-induced seizures. In addition, a lasting change in the striatal TRH receptors after AM kindling as well as a transient IR-TRH increase in the limbic structures were seen 48 hours after AM-kindled convulsions. TRH and its analog (DN-1417) inhibited PTZ-induced generalized seizures dose-dependently. These findings indicate the involvement of the TRH neural system in seizure mechanisms, and suggest that endogenous TRH may be an antiepileptic substance in the brain.     

Post-translational processing of thyrotropin-releasing hormone precursor in rat brain: identification of 3 novel peptides derived from pro TRH

The sequence of rat hypothalamic pro-thyrotropin releasing hormone, deduced by sequencing of cDNA, in addition to 5 TRH progenitor genitor sequences contains leader, trailer and 4 intervening sequences separated by paired basic amino acid sequences. We have developed radioimmunoassays to synthetic peptides corresponding to portions of these cryptic proTRH sequences and have used these assays to identify and partially characterize proTRH peptides, distinct from TRH, in extracts of rat brain. Two of these peptides correspond closely in size to one intervening sequence and the car☐y-terminal sequence of proTRH. Three other peptides correspond to the intact amino-terminal leader sequence and two peptides formed by a further cleavage of the leader sequence at an internal paired basic amino acid sequence.     

Effects of melanocortin receptor ligands on thyrotropin-releasing hormone release: evidence for the differential roles of melanocortin 3 and 4 receptors

The hypothalamic melanocortin system is important in the central regulation of food intake and body weight. We have previously demonstrated that intracerebroventricular administration of alpha-melanocyte stimulating hormone (alpha-MSH), a nonselective MC3 and MC4 receptor agonist, stimulated plasma thyroid-stimulating hormone, and agouti-related protein (AgRP), an MC3 and MC4 receptor antagonist, suppressed it. In this study, we investigated the effects of MC3 and MC4 receptor (MC3-R and MC4-R) selective agonists and antagonists on the release of thyrotropin-releasing hormone (TRH) from hypothalamic explants in vitro. alpha-MSH stimulated TRH release from the rat hypothalamic explants (alpha-MSH 100 nm 230 +/- 22.9% basal, P < 0.005). In contrast, gamma 2-MSH, a selective MC3-R agonist, suppressed TRH release (gamma 2-MSH 10 microns 76.2 +/- 7.4% basal, P < 0.05). AgRP (83-132), a nonselective MC3/4-R antagonist, induced no change in TRH release whilst JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH 11-22), a selective MC4-R antagonist, suppressed it (JKC-363 10 nm 57.2 +/- 11.5% basal, P < 0.05). Both AgRP (83-132) and JKC-363 blocked alpha-MSH stimulated TRH release but only AgRP (83-132) blocked the inhibitory effect of gamma 2-MSH on TRH release. These data suggest differential roles for the MC3 and MC4 receptors in TRH release; MC3-R agonism inhibiting and MC4-R agonism stimulating TRH release.     

Distribution of Somatostatin Receptors 1, 2 and 3 mRNA in Rat Brain and Pituitary

In this study sequence-specific antisense oligonucleotide probes have been used to investigate the distribution of the mRNAs coding for the somatostatin receptor subtypes termed somatostatin receptor 1, somatostatin receptor 2 and somatostatin receptor 3 in the rat brain and pituitary using in situ hybridization techniques. The three receptor subtype mRNAs were found to be widely distributed in the brain with different patterns of expression, but with some overlap. Somatostatin receptor 1 mRNA was particularly concentrated in the cerebral and piriform cortex, magnocellular preoptic nucleus, hypothalamus, amygdala, hippocampus, and several nuclei of the brainstem. Somatostatin receptor 3 mRNA was very abundant in the cerebellum and pituitary (in contrast to somatostatin receptor 1), but it was also found in hippocampus, amygdala, hypothalamus and in motor nuclei of the brainstem. Somatostatin receptor 2 mRNA levels were very low relative to the other two mRNAs evaluated. Receptor 2 mRNA was observed in the anterior pituitary, and in the brain it was found in the medial habenular nucleus, claustrum, endopiriform nucleus, hippocampus, some amygdala nuclei, cerebral cortex and hypothalamus. None of the three somatostatin receptor mRNAs studied here was found in the caudate nucleus. Northern analysis revealed distinct sizes of mRNAs for each subtype, and displacement experiments showed that each probe sequence was subtype-specific.     

Adenosine inhibits alpha-melanocyte-stimulating hormone release from frog pituitary melanotrophs. Evidence for the involvement of a(1) adenosine receptors negatively coupled to adenylate cyclase

Adenosine is recognized as an important modulator of cell activity. In particular, adenosine regulates the secretion of adrenocorticotropin from anterior pituitary cells. However, the possible role of adenosine on the pars intermedia has never been investigated. In the present study, we have examined the effect of adenosine on α-melanotropin (α-MSH) secretion from the intermediate lobe of the pituitary of the frog (Rana ridibunda), using the perifusion technique. When whole neurointermediate lobes were exposed to graded doses of adenosine (10(-9) to 10(-4) M), a dose-dependent inhibition of a-MSH release was observed. Repeated pulses of adenosine (5 ± 10(-5) M) induced a reproducible inhibition of α-MSH secretion without any desensitization phenomenon. The effect of adenosine was mimicked by the non-selective agonist 5'-N-ethylcarboxamide-adenosine and the highly specific adenosine A, receptor agonist N(6) -[R-phenylisopropyl]-adenosine (R-PIA). In contrast the selective adenosine A(2) receptor agonist, CGS 21680, induced a slight stimulation of α-MSH release. Adenosine-induced inhibition of α-MSH secretion was blocked by the non-selective adenosine antagonist, 8-(p-sulfophenyl)-theophyline. Adenosine and R-PIA also inhibited α-MSH secretion from acutely dispersed pars intermedia cells. Adenosine did not block thyrotropin-releasing hormone-induced α-MSH release from perifused neurointermediate lobes. In contrast, adenosine inhibited both acetylcholine-evoked and muscarine-evoked α-MSH secretion. Finally, R-PIA induced a significant inhibition of basal and forskolin-stimulated cyclic AMP levels in whole neurointermediate lobes. The present results demonstrate that adenosine exerts a direct inhibitory effect on α-MSH release from melanotrope cells through activation of the A(1) receptor subtype, negatively coupled to adenylate cyclase. These data suggest that adenosine may play a physiological role in the regulation of hormone release from the intermediate lobe of the pituitary.     

Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides

A large family of TRH-like peptides in the limbic region of rat brain including pGlu-Glu-Pro-NH(2) (EEP), pGlu-Val-Pro-NH(2) (Val(2)-TRH), Leu(2)-TRH, Phe(2)-TRH and Tyr(2)-TRH has recently been discovered. TRH (pGlu-His-Pro-NH(2)) has antidepressant, neuroprotective, analeptic, anticonvulsant, antiamnesic and euphoric properties, and other TRH-like peptides such as EEP exert several of these effects. A new TRH receptor (TRHR2) has been reported which is highly expressed in regions of rat brain that regulate attention and learning, arousal, sleep and processing of sensory information. The TRHR1 predominates in limbic structures involved in regulation of mood and in pituitary. This study examined the possibility that some of the newly discovered TRH-like peptides bind with high affinity to TRHR2, and that this receptor acts as the transducer for some of the CNS effects of this new class of neuropeptides. EEP, Val(2)-TRH and Leu(2)-TRH were analeptics, like TRH, but Phe(2)-TRH and Tyr(2)-TRH were not. The affinity and efficacy of TRH-like peptides for TRHR1 and TRHR2 were measured in HEK293 cells stably expressing these receptors. The IC(50) values of TRH-like peptides for displacement of [3H]TRH from TRHR2 were TRH<(Leu(2)-, Phe(2)-TRH)<(Gln(2)-, Ser(2)-TRH)<(Val(2)-, Tyr(2)-, Arg(2)-, Thr(2)-, and Glu(2)-TRH). The IC(50) for Leu(2)-TRH was about 100 times that for TRH. When tested at the calculated IC(50) values, TRH-like peptides stimulated calcium responses in cells expressing TRHR1 and TRHR2, indicating that the peptides act as weak agonists at both receptors. These results indicate that TRHR1 and TRHR2 do not mediate the behavioral effects of TRH-like peptides.     

Evidence for a role of brain thyrotropin-releasing hormone (TRH) on stress gastric lesion formation in rats

Specific polyclonal antibodies raised against synthetic thyrotropin-releasing hormone (TRH) infused intracerebroventricularly (ICV) significantly decreased gastric lesions induced by cold restraint stress. The antiulcer effect of immunologic blockade of brain TRH was specific. Normal rabbit serum or antibodies raised against somatostatin, alpha-MSH, Leu-enkephalin, gonadotropin-releasing hormone and atrial natriuretic factor were ineffective. These findings suggest that brain TRH may play an important role in experimental stress ulcer formation.     

Differential prolactin responses to Haloperidol and TRH in normal adult men

(1) Prolactin (PRL) responses to low dose intravenous (i.v.) haloperidol differed among individuals, but PRL responses to 25 μg and 500 μg i.v. thyrotropin-releasing hormone (TRH) in the same subjects were similar. (2) Serum concentrations of haloperidol accounted for only 36% of the variability in PRL response. (3) It is concluded that the inter-individual variability in PRL response to haloperidol does not result from differences in pituitary PRL-secreting capacity, nor can it be attributed in a major way to differences in drug metabolism. It is proposed that the variable PRL responses to haloperidol reflect individual differences in dopamine receptor sensitivity to blockade by this drug.     

Formation and degradation of deamido-TRH (pyroglutamyl-histidyl-proline) in rat brain after intraventricular injection of TRH

Thyrotropin-releasing hormone (TRH) injected into the lateral ventricle of the rat's brain was rapidly metabolized to deamido-TRH (DA-TRH). Brain levels of TRH decreased with a half-life of 7 min, while the DA-TRH formed from its disappeared with a half-life of 2.5 min. To prevent the post-mortem degradation of DA-TRH it was necessary to sacrifice rats by directing focused microwave irradiation towards the brain. The short half-lives determined for TRH and DA-TRH in vivo were much shorter than those obtained using in vitro techniques. The in vivo formation and accumulation of DA-TRH was inhibited by bacitracin and unaffected by probenicid.     

Studies of substrate requirements, kinetic properties, and competive inhibitors of the enzymes catabolizing TRH in rat brain

The enzymes catalyzing the breakdown of TRH are soluble and dependent upon active sulfhydryl groups. One of these enzymes is a pyroglutamyl aminopeptidase (EC 3.4.11.8) (apparent molecular weight 60,000 daltons) with a specificity restricted to pyroglutamyl peptide bonds. The second enzyme is a prolyl endopeptidase (apparent molecular weight 70,000 daltons) with a specificity restricted to proline peptide bonds wherein the proline is preceded by an alpha adjacent amino acid possessing a blocked N-terminus. Substrate requirements for this latter enzyme are identical to those reported previously for the TRH deamidase of rat brain, the prolyl endopeptidase of rabbit brain and the post proline cleaving enzyme of rat brain. We conclude that this enzymatic activity variously described as TRH deamidase, post proline cleaving enzyme, and prolyl endopeptidase is that of a single enzyme best denoted as a prolyl endopeptidase (EC 3.4.22.16, proposed). The pyroglutamyl aminopeptidase has a Km for TRH of 45 micro M as compared to a Km for TRH of 2400 micro M for the prolyl endopeptidase. Total enzymatic activity of the prolyl endopeptidase, however, is approximately 3500 nmol/h/rat brain with the total enzymatic activity of the pyroglutamyl aminopeptidase being approximately 600 nmol/h/rat brain. The 5- to 6-fold higher affinity of the pyroglutamyl aminopeptidase for TRH suggests that of these two catabolic pathways, removal of the N-terminal pyroglutamyl moiety is likely to be the more important pathway for the initial catabolism of TRH in rat brain. Analysis of substrate specificity permitted the synthesis of several effective competitive inhibitors of the two enzymes. Of these, the most effective inhibitors of the pyroglutamyl aminopeptidase were p-glu-NH2 (Ki 185 micro M) and p-glu-his-OCH3 (Ki 16.5 micro M). The most effective inhibitors of the prolyl endopeptidase were Ac-gly-pro-NH2 (Ki 1143 micro M) and Z-gly-pro-NH2 (Ki 442 micro M).     

Adenosine A2A receptor gene disruption provokes marked changes in melanocortin content and pro-opiomelanocortin gene expression

A2A receptor knockout (A2AR-/-) mice are more anxious and aggressive, and exhibit reduced exploratory activity than their wild-type littermates (A2AR+/+). Because alpha-melanocyte-stimulating hormone (alpha-MSH) influences anxiety, aggressiveness and motor activity, we investigated the effect of A2AR gene disruption on alpha-MSH content in discrete brain regions and pro-opiomelanocortin (POMC) expression in the hypothalamus and pituitary. No modification in alpha-MSH content was observed in the hypothalamus and medulla oblongata where POMC-expressing perikarya are located. In the arcuate nucleus of the hypothalamus, POMC mRNA levels were not affected by A2AR disruption. Conversely, in A2AR-/- mice, a significant increase in alpha-MSH content was observed in the amygdala and cerebral cortex, two regions that are innervated by POMC terminals. In the pars intermedia of the pituitary, A2AR disruption provoked a significant reduction of POMC mRNA expression associated with a decrease in alpha-MSH content. By contrast, in the anterior lobe of the pituitary, a substantial increase in POMC mRNA and adrenocorticotropin hormone concentrations was observed, and plasma corticosterone concentration was significantly higher in A2AR-/- mice, revealing hyperactivity of their pituitary-adrenocortical axis. Together, these results suggest that adenosine, acting through A2A receptors, may modulate the release of alpha-MSH in the cerebral cortex and amygdala. The data also indicate that A2A receptors are involved in the control of POMC gene expression and biosynthesis of POMC-derived peptides in pituitary melanotrophs and corticotrophs.