Alopecia areata - hyperactivity of the hypothalamic-pituitary-adrenal axis is a myth?

INTRODUCTION: Psychological stress is known to cause exacerbation of different skin pathologies including alopecia areata (AA). A hyperactivity of the hypothalamic-pituitary-adrenal axis (the HPA axis) in patients affected by AA (AA patients) was proposed to be a neuroendocrine response to stress. Still little is known about melanocyte-stimulating hormone (MSH) and cortisol production in AA settings. AIM: The aim of the study was to compare trends in a production of MSH and cortisol in patients with AA patients and healthy controls. MATERIAL AND METHODS: Plasma concentrations of free cortisol and MSH were measured in 43 AA patients (35.5 ± 10.6 years) and 37 healthy subjects (35.9 ± 10.5 years) selected from the Dermatology Outpatient Clinic at Medical University of Silesia in Katowice, Poland. Results were submitted to statistical analysis with Shapiro-Wilk W-test and subsequently nonparametric (Mann-Whitney U-test) or parametric (Student's t-test) statistics were performed. RESULTS: Mean plasma level of MSH was 5.39 ng/mL in AA patients and 5.71 ng/mL in healthy controls. The difference between groups was non-significant (P = 0.435), but the control group manifested higher values of MSH (Q75 = 13.6 ng/mL vs Q75 = 5.98 ng/mL) and this tendency was especially stronger in females. AA patients had greater mean plasma level of cortisol (157.63 ± 91.16 µg/L) than healthy controls (123.32 ± 71.28 µg/L); however, the difference between them was also non-significant (P = 0.063). No sex-dependent tendency to a greater production of cortisol was found. CONCLUSIONS: Expectations of disturbances in production of MSH and cortisol were not fulfilled. Neither MSH nor cortisol plasma levels appear to be clearly changed in AA patients.

Central administration of insulin suppresses food intake in chicks.

Although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the anorexigenic action of peptide hormones is similar in both species. For example, central administration of peptide hormones such as leptin, cholecystokinin or glucagon has been shown to suppress food intake behavior in chickens and mammals. Central administration of insulin suppresses food intake in mammals. However, the anorexigenic action of insulin in chickens has not yet been identified. In the present study, we investigated the effects of central administration of insulin on food intake in chicks. Intracerebroventricular administration of insulin in chicks significantly suppressed food intake. Central administration of insulin significantly upregulated mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART) and corticotropin-releasing factor (CRF), but did not influence mRNA levels of neuropeptide Y and agouti-related protein in the hypothalamus. These results suggest that alpha-melanocyte stimulating hormone (alpha-MSH, an anorexigenic peptide from the post-translational cleavage of POMC), CART and CRF are involved in the anorexigenic action of insulin in chicks. Furthermore, central administration of alpha-MSH or CART significantly suppressed food intake. In addition, alpha-MSH significantly upregulated CRF mRNA expression, suggesting that the anorexigenic action of alpha-MSH is mediated by CRF. Our findings demonstrate that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system and suggest that this anorexigenic action is mediated by CART, alpha-MSH and CRF.

[Effect of Tribulus terrestris extract on melanocyte-stimulating hormone expression in mouse hair follicles].

OBJECTIVE: To observe the effect of Tribulus terrestris extract on melanocyte stimulating hormone (MSH) expression in C57BL/6J mouse hair follicles, and investigate the role of Tribulus terrestris extract in activation, proliferation, epidermal migration of dormant hair follicle melanocytes. METHODS: The aqueous extract of Tribulus terrestris was administered orally in specific pathogen-free C57BL/6J mouse at the daily dose equivalent to 1 g/1 kg in adult human, and the expression and distribution of MSH in the mouse hair follicles was observed with immunohistochemistry. RESULTS: The positivity rate of MSH expression in the hair follicle melanocytes was 75% in mice treated with the extract, significantly higher than the rate of only 18.75% in the control group (P<0.01). CONCLUSION: The aqueous extract of Tribulus terrestris can significantly increase MSH expression in the hair follicle melanocytes by activating tyrosinase activity and promoting melanocyte proliferation, melanine synthesis, and epidermal migration of dormant melanocytes.

Mutational analysis of evolutionarily conserved ACTH residues.

alpha-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1-24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1-24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15-18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15-18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20-24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.

Potassium-induced secretion of melanocyte-stimulating hormone from the melanotrophs of the neurointermediate lobe of the lizard Anolis carolinensis.

Secretion of melanocyte-stimulating hormone (MSH) from the melanotrophs of the neurointermediate lobe (NIL) of the lizard Anolis carolinensis was studied to investigate the role of membrane potential and extracellular calcium ions in the control of secretion in this species. MSH secretion was monitored from perifused NILs which had been in organ culture for 7-14 days prior to experiment to allow the nerve terminals present in the tissue to degenerate. Elevation of the K(+) concentration in the perifusate induced a marked increase in MSH secretion. Perifusion of the cultured NILs with a nominally Ca-free solution did not reduce basal MSH secretion but blocked K-induced secretion. Moreover, nimodipine, an antagonist of voltage-gated Ca(2+) channels, inhibited K-induced secretion, whereas BAY K 8644, a Ca(2+) channel agonist, potentiated it; but neither drug affected basal secretion. High ¿K(+) perifusate also stimulated MSH secretion from freshly excised (acute) NILs. Furthermore, in these preparations nominally Ca-free solution reduced basal secretion by 40% in addition to blocking K-induced secretion. As in the cultured NILs, nimodipine blocked and BAY K 8644 potentiated K-induced secretion in the acute NILs while not affecting basal secretion. The results from the cultured NILs are consistent with the hypothesis that stimulated MSH secretion from the melanotrophs of the anole is dependent upon Ca(2+) influx through voltage-gated calcium channels. The qualitatively similar results obtained from the acute NILs in response to high ¿K(+), nimodipine, and BAY K 8644 suggest that, for the most part, what is being observed are the direct effects of these substances on the melanotrophs. Basal secretion of MSH in cultured NILs is significantly less than that in the acute preparations. The calcium-sensitive fraction of basal secretion present in acute NILs may require the presence of nerve terminals.

Expression of proopiomelanocortin (POMC)-derived melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) peptides in skin of basal cell carcinoma patients.

We proposed that local expression and production of proopiomelanocortin (POMC) peptides may play a role in human skin physiology and pathology, including the development and progression of skin cancers. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting hybridization techniques were used to study gene expression. Reversed-phase (RP) high-pressure liquid chromatography (HPLC) separation with subsequent radioimmunoassays were used to identify alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) peptides. Immunocytochemistry (IHC) was used to localize ACTH, alpha-MSH, and beta-MSH antigens in skin. RT-PCR, RP-HPLC, and IHC analyses documented the expression of POMC mRNA and production of ACTH and alpha-MSH peptides in lesional and perilesional skin of basal cell carcinoma (BCC) patients and in cultured keratinocytes, which was accompanied by the expression of the MC1-R gene encoding the receptor activated by MSH and ACTH. Thirty specimens were analyzed by IHC. Immunoreactive alpha-MSH, beta-MSH, and ACTH were detected, in 21 of 21, in 11 of 20, and in 6 of 8 of lesional skin, and in 6 of 6, in 5 of 7, and in 6 of 8 perilesional skin specimens analyzed, respectively. Antigen distribution was heterogenous and present in BCC, epidermis, hair follicles, dermal mononuclear cells, and extracellular matrix. We conclude that messenger RNA for POMC, MC1-R, and the peptides MSH and ACTH are produced in skin of BCC patients. Because keratinocytes are a target for MSH and ACTH bioregulation, the production of these peptides is stimulated by UVB, and the peptides can act as immunosupressors, we suggest that MSH and ACTH may facilitate development of BCC.

Inhibition of voltage-dependent calcium channels by prostaglandin E2 in rat melanotrophs.

The effects of PGE2 on voltage-dependent Ca2+ channel currents were studied in dissociated rat melanotrophs by the whole-cell configuration of the patch-clamp technique. In about 90% of melanotrophs examined, PGE2 reversibly inhibited voltage-dependent Ba2+ currents elicited by voltage steps from a holding potential of -80 to 0 mV, with an ED50 of 68 nM. The maximum inhibition of Ba2+ currents by 1 microM PGE2 (35.3%) was comparable with that by the maximally effective concentration (100 nM) of dopamine. The EP1/EP3 PGE (EP) agonists, 17PT-PGE2 and sulprostone, and the EP2/EP3 agonist, misoprostol, mimicked the inhibition by PGE2, whereas the selective EP2 agonist, butaprostol, had little effect. The inhibition by PGE2 was partially, but significantly, reduced by the selective EP1 antagonist, SC-51322. The magnitude of the PGE2-induced inhibition of Ba2+ currents was greatly reduced by pretreatment with pertussis toxin, or by a depolarizing prepulse, to +80 mV, lasting for 50 msec. Although four distinct types (N-, P/Q-, L-, and R-types) of high-threshold Ba2+ currents were observed, PGE2 (1 microM) caused significant inhibition of only P/Q- and L-type currents, which were 17.3 and 10.1%, respectively, of the total Ba2+ currents. These results suggest that PGE2 inhibits P/Q- and L-type Ca2+ channels of rat melanotrophs via EP1 and EP3 receptors, which are coupled to pertussis toxin-sensitive G proteins, and produces both voltage-sensitive and -insensitive inhibition of Ca2+ channels.

Glucocorticoid regulation of hypothalamic proopiomelanocortin.

Although glucocorticoids clearly inhibit proopiomelanocortin (POMC) gene transcription and peptide synthesis in the anterior pituitary, the effects of glucocorticoids on POMC in the hypothalamus are still unclear, even though most POMC neurons in the arcuate nucleus are known to have glucocorticoid receptors. In this study, we have therefore examined the effect of adrenalectomy (ADX) and glucocorticoid replacement on POMC mRNA and peptide (beta-EP and alpha-MSH) levels in the medial basal hypothalamus (MBH) of the rat. POMC mRNA was measured by a sensitive solution hybridization S1 nuclease protection assay, and beta-EP and alpha-MSH were measured by radioimmunoassay. In a first experiment, animals were studied 7 days after ADX or sham surgery. The mean POMC mRNA concentration was 1.01+/-0.14 pg/microg RNA (means+/-SE) in the intact animals and decreased to 0.55+/-0.07 pg/microg RNA in the MBH of the ADX animals (p < 0.005). Beta-EP levels decreased in parallel from 4.30+/-0.18 to 3.36+/-0.11 ng/mg protein (p < 0.001); alpha-MSH levels decreased from 3.25+/-0.21 to 2.41+/-0.16 ng/mg protein (p < 0.005). In a second experiment, animals were studied 2 weeks after ADX. POMC mRNA levels again fell significantly from 1.15+/-0.19 pg/microg RNA in the intact animals to 0.51+/-0.06 pg/microg in the ADX animals (p < 0.01). Beta-EP levels fell also, but this was not significant. In a third experiment, all animals underwent ADX, and half of them received daily subcutaneous injections of dexamethasone (20 microg). Nine days after ADX, the mean POMC mRNA level was 0.66+/-0.04 pg/microg RNA in the saline-treated animals and increased to 0.98+/-0.08 pg/microg RNA in the dexamethasone-treated animals (p < 0.005). A parallel increase in beta-EP levels from 5.03+/-0.41 to 6.01+/-0.53 ng/mg protein was also noted, but this was not statistically significant. We conclude that POMC gene expression is significantly inhibited in the MBH at 1 and 2 weeks after ADX. This effect was reversed by glucocorticoid replacement with doses close to the physiological range. The parallel changes in POMC mRNA and peptide levels strongly suggest that, in contrast to the anterior pituitary, low doses of glucocorticoids stimulate the biosynthesis of POMC in the MBH of ADX rats.

Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [seecomments].

The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Deciphering posttranslational processing events in the pituitary of a neopterygian fish: cloning of a gar proopiomelanocortin cDNA.

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The beta-endorphin sequence had 91% sequence identity with sockeye A beta-endorphin and 71% sequence identity with Xenopus laevis beta-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar alpha-MSH sequence was identical to the alpha-MSH sequence in rat POMC. The gar beta-MSH sequence had 77% sequence identity with salmonid forms of beta-MSH and 53% sequence identity with tetrapod forms of beta-MSH. The gamma-MSH region of gar POMC only had 26% primary sequence identity with tetrapod gamma-MSH sequences. Gar gamma-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar gamma-MSH is discussed in light of the absence of this sequence in salmonid fish.

Obesity, diabetes and functions for proopiomelanocortin-derived peptides.

Melanocortin peptides (adrenocorticotropin (ACTH), alpha-,beta-, and gamma-melanocyte stimulating hormone (MSH), and fragments thereof) derived from proopiomelanocortin (POMC) have a diverse array of biological activities, many of which have yet to be fully elucidated. The recent cloning of a family of five distinct melanocortin receptors through which these peptides act has provided the tools to further our understanding of melanocortin peptide functions. Early work on melanocortin peptides focused on their roles in pigmentation, adrenocortical function, the immune, central and peripheral nervous systems. Although melanocortin peptides have long been known to affect lipolysis, characterisation of the melanocortin receptors has opened up several lines of evidence for important roles in the development of obesity, insulin resistance and type II diabetes. We present here a review of the current evidence for melanocortin peptides playing such a role, and based on this evidence, a model for melanocortin peptides and their receptors in maintaining energy balance.

Postnatal expression of melanocortin-3 receptor in rat diencephalon and mesencephalon.

In situ hybridization was applied to examine the postnatal expression of melanocortin-3 (MC-3) receptor mRNA in the rat brain. Very weak and limited signals were seen in the hypothalamus on postnatal day 0 (P0) and in the dorsal lateral thalamus on P4. A marked increase was noted in several regions of the diencephalon and mesencephalon on P7. The highest levels were reached on P21, which was the time when an adult-like pattern was established. On P21, intense signals were seen in the ventromedial nucleus and the arcuate nucleus of the tuberal hypothalamus, the habenular nucleus of the epithalamus and the ventral tegmental area. [125I] Nle4, D-Phe7-alpha-MSH showed overlapping, but wider labelling of melanocortin receptors, that followed a similar developmental course. alpha-MSH-like immunoreactivity was seen widely in the forebrain and midbrain from P14. In contrast to the staining of alpha-MSH in neurons and their process, gamma 2-MSH-like immunoreactivity was detected strongly in the blood vessels. The neuronal localization of MC-3 receptor mRNA suggests that this receptor may mediate the neurotropic actions of melanocortin peptides in the developing brain.

Sturgeon proopiomelanocortin has a remnant of gamma-melanotropin.

For the investigation of the evolution of the proopiomelanocortin (POMC) gene in early ray-finned fishes, nucleotide sequence of POMC cDNA from a chondrostean fish, the sturgeon has been determined. POMC cDNA was amplified by PCR from double-strand cDNA prepared from sturgeon pituitary and ligated with lambdaZAP II. The POMC cDNA consists of 1079 bp without a poly-A. An open reading frame of the POMC cDNA encodes 263 amino acid residues. Sturgeon POMC contains ACTH, alpha-melanotropin (MSH), beta-MSH and beta-END at positions (115-153), (115-127), (186-202) and (205-238), respectively. Location of POMC(51-72) is homologous to gamma-MSH, whereas the third residue of MSH-core sequence, His-Phe-Arg-Trp, is changed to His. Moreover, there are no basic amino acids to serve as a processing signal on the N-terminal side of POMC(51-72). These structural characteristics suggest that an ancestor of the ray-finned fishes had gamma-MSH, whereas significant mutations occurred during the evolution of chondrostean fish.

Effect of morphine on proopiomelanocortin gene expression and peptide levels in the hypothalamus.

Opiates have been reported to suppress POMC in the medial basal hypothalamus (MBH) but studies have been complicated by the fact that acutely, in the rat, opiates stimulate corticosterone and inhibit gonadal steroid release, which could both affect POMC in brain. We have therefore examined POMC gene expression and peptide levels in the MBH of castrated rats after 10 days of treatment with subcutaneous morphine or placebo pellets and after pellet removal. POMC mRNA was measured by solution hybridization assay and beta-endorphin (beta-EP) and alpha-MSH were measured by RIA. In castrated male rats, the mean POMC mRNA concentration in the MBH was 1.67 +/- 0.11 pg/microgram RNA in the control animals and decreased to 1.17 +/- 0.11 pg/microgram RNA in the morphine-treated animals (P < 0.01). Similarly in castrated, estradiol replaced female rats, the mean POMC mRNA level in the MBH was 1.36 +/- 0.19 pg/microgram RNA and decreased to 0.82 +/- 0.08 pg/microgram RNA after morphine treatment (P < 0.05). beta-EP levels were not significantly different in either study. When castrated male rats were similarly morphine pelleted and killed either on day 10 or 2 days later after pellet removal, the mean POMC mRNA level again fell from 1.83 +/- 0.21 in the controls to 1.28 +/- 0.20 pg/microgram RNA after 10 days of morphine; 2 days after pellet removal levels remained suppressed at 0.80 +/- 0.08 pg/microgram RNA (P < 0.01). In this study the concentrations of beta-EP and alpha-MSH were both noted to decline in the MBH after morphine treatment (P < 0.05). When the forms of beta-EP in the MBH were characterized by HPLC, a decrease in the concentration of beta-EP was again seen after morphine but no significant differences in the pattern of beta-EP processing or in the relative amounts of beta-EP1-31 compared to beta-EP1-27 and beta-EP1-26 were noted in morphine-treated animals. There was also no significant effect of 10(-6)-10(-4) M morphine on basal or KCl-stimulated release of beta-EP or gamma 3-MSH release from the perifused rat hypothalamus in vitro. We conclude that morphine suppresses POMC gene expression in the hypothalamus of chronically treated male and female rats. Persistent changes were also noted during morphine withdrawal. In some cases this was accompanied by a fall in beta-EP peptide content. These effects were seen in castrated animals with and without sex steroid replacement and are thus independent of the effects of morphine on the pituitary-gonadal axis. These results show that opiate drugs modify endogenous opioid systems in the brain and provide further support for the hypothesis that such changes may contribute to mechanisms of opiate dependence and withdrawal.

Block of BK (maxi K) channels of rat pituitary melanotrophs by Na+ and other alkali metal ions.

The block of large-conductance calcium-activated potassium (BK) channels by internal and external alkali metal ions was studied in adult rat melanotrophs. Internal but not external 20 mM Na+ produced a strongly voltage-dependent, flickery block that was well-fitted to the Woodhull model by using a value of 140 mM for the dissociation rate constant at 0 mV [Kd(0)] and an equivalent valence (zdelta) of 0.9. At a concentration of 20 mM external K+, Cs+ and Rb+, but not Li+, caused a rightward shift of the voltage dependence of the intracellular Na+ (Na+i ) block. This effect of K+, Cs+ and Rb+ was modelled by an equilibrium knock-out mechanism in which the block-relieving ion binds to a site located within the voltage field and consequently increases the off-rate of Na+. Internal Li+ caused little or no block whereas internal Cs+ caused a voltage-dependent block [Kd(0) approximately 150 mM]. Flickery channel block observed in cell-attached patches was consistent with a cytoplasmic Na+ activity between 1 and 10 mM.

Adenosine inhibits L- and N-type calcium channels in pituitary melanotrophs. Evidence for the involvement of a G protein in calcium channel gating.

It has been previously demonstrated that activation of A1 adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A1 adenosine receptor agonist R-PIA (50 microM each) produced a decrease of the amplitude of the barium current, while the selective A2 adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTP gamma S (100 microM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 microgram/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 microM) together with the phosphodiesterase inhibitor IBMX (100 microM) to the intracellular solution did not modify the effect of R-PIA on the current. It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.

Identification of POMC processing products in single melanotrope cells by matrix-assisted laser desorption/ionization mass spectrometry.

The use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in identifying proopiomelanocortin (POMC) processing products in melanotrope cells of the pituitary intermediate lobe of Xenopus laevis was explored. Mass spectra were obtained with such a high sensitivity of detection that the peptides could be identified in a single melanotrope cell. In addition to known POMC processing products of the Xenopus melanotrope cell, the presence of previously unidentified POMC-derived peptides was demonstrated. Together these POMC processing products accounted for the entire length of the POMC precursor. Furthermore, Xenopus possesses two genes for POMC and the sensitivity and accuracy of the MALDI-MS technique allowed identification of processing products of both the POMCA and POMCB gene. In addition, differences were obtained between the mass spectra of melanotrope cells from Xenopus laevis adapted to different conditions of background illumination. These results show that MALDI-MS is a valuable tool in the study of the expression of peptides in single (neuroendocrine) cells.

Changes of activation and inactivation gating of the transient potassium current of rat pituitary melanotrophs caused by micromolar Cd2+ and Zn2+.

We studied the effects of Zn2+ and Cd2+ on the behaviour of IK(f), a transient outward potassium current in acutely dissociated melanotrophs of the pars intermedia of the rat pituitary gland. Micromolar concentrations of external Cd2+ or Zn2+ caused parallel and nearly equal rightward shifts along the voltage axis of the activation and steady-state inactivation curves for IK(f). The KD for the half-maximal shift of the activation curve was 278 microM for Cd2+ and 93 microM for Zn2+; the maximal shifts of the activation curve were 32.5 and 34 mV, for Cd2+ and Zn2+, respectively. The times to half-activation and half-inactivation were shifted rightward by 30-60 mV in both 500 microM Cd2+ and 500 microM Zn2+. We suggest that Cd2+ and Zn2+ interact specifically with a binding site on or electrically close to the IK(f) channel and in so doing modify the electric field "seen" by the voltage sensors.

Characterization of the GABA-induced current in frog pituitary melanotrophs.

The molecular mechanisms regulating GABAA receptor activity in cultured frog melanotrophs were studied using the patch-clamp technique. In the whole-cell configuration, application of GABA evoked a dose-related increase of inward chloride currents. The ED50 value, estimated from the sigmoidal dose-response curve was 2 x 10(-6) M and the Hill coefficient was 1.55. The amplitude of the GABA-induced current decayed with time. Kinetics analysis of the desensitization revealed that the time-course of the current decrement was fitted by one exponential. Graded doses of GABA or association of GABA with the benzodiazepine receptor agonist flunitrazepam accelerated the desensitization process. In contrast, the time-course of the current did not significantly vary at different holding potentials. In the outside-out configuration, GABA was found to activate channels which displayed three unitary conductance levels (8, 15 and 30 pS). The channel openings of the more frequent conductance level (30 pS) exhibited short and long lasting open states (1.2 and 28.3 ms at -60 mV). Altogether these data reveal that frog melanotrophs possess a single population of GABAA receptors which interconvert into a higher affinity state in the presence of benzodiazepine receptor agonists. Two GABA molecules must bind to the receptor to trigger long lasting channel openings. In addition, the activity of the GABAA receptor appears to be independent of the accumulation of intracellular chloride ions.