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Lactotrophs, gonadotrophs, thyrotrophs and somatotrophs of the rat anterior pituitary (AP) express 68-kDa neurofilaments (NF68) and other neuronal markers. NF68 expression in the AP appears to be estrogen-dependent, but its significance is unknown. The aims of this work were: (1) to establish the expression pattern of NF68 immunoreactivity in the mouse AP, and (2) discover if corticotrophs and melanotrophs from both rodent species also express NF68. Primary cultures and frozen sections of AP from sexually mature mice were immunolabeled with anti-NF68 antibodies. In separate experiments, samples were immunostained for NF68 and AP hormones. Here we report that mouse lactotrophs, gonadotrophs, thyrotrophs and somatotrophs also express NF68 in a sexually dimorphic manner. The percentages of non-expressing, weakly expressing and strongly expressing cells were similar between both rodent species, although NF68+ cells were about 50% less abundant in the mouse compared to the rat pituitary. Remarkably, our study shows for the first time that rodent pituitary cells from the proopiomelanocortin lineage nearly completely lack NF68 immunoreactivity. In this regard, they differ from the rest of the AP population. Our findings establish a foundation for experiments aimed at investigating the functional significance of estrogen-dependent regulation of NF68 expression in rodent AP cells.

A patch-clamp study was conducted on cultured frog pituitary melanotrophs, in order to investigate the effects of adrenaline on the electrical activity of these cells. In the whole-cell configuration, adrenaline (1 microM) caused hyperpolarization that was accompanied by a fall in membrane input resistance and a blockage of spontaneous action potentials. Under voltage clamp, adrenaline elicited a net-outward current. The hyperpolarization became undetectable at a command voltage of -100 mV which corresponded to the equilibrium potential of potassium ions. The effect of adrenaline on membrane potential and spontaneous activity was blocked by the alpha 2-adrenergic receptor antagonist yohimbine (1-10 microM) but could not be mimicked by the alpha 2-adrenergic agonist clonidine (1-10 microM). In the cell-attached configuration, exposure of the extra-patch membrane to adrenaline increased the occurrence of single-channel currents with a slope conductance of 100 pS. The deduced reversal potential of these currents corresponded to the equilibrium potential of potassium ions. These results suggest that frog melanotrophs display an alpha 2-adrenergic receptor subtype coupled to potassium channels involved in hyperpolarization.

The proopiomelanocortin (POMC) gene is expressed predominantly in corticotrophs of the pituitary anterior lobe, melanotrophs of the intermediate lobe and neurons of the arcuate nucleus of the hypothalamus. The different ontogeny of POMC mRNA as well as the complicated hormonal regulation of POMC gene expression in the three different cell types suggests a concerted interaction between several cis-acting elements in the POMC gene and transcription factors located in each of the three cell types. To investigate cell-specific elements in the POMC gene we tested two different constructs in transgenic mice. The construct –4000rPOMCLacZ, carrying 4 kb of the rat POMC promoter fused to the Escherichia coli β-galactosidase gene, showed appropriate expression in melanotrophs in 50% of the mice analyzed. β-Galactosidase activity was less evident in corticotrophs under basal environmental conditions. In brain, 7 out of 15 independently derived transgenic founders had ectopic expression of the transgene in different areas; however, none of the animals analyzed expressed β-galactosidase in neurons of the arcuate nucleus. The construct HAL*, a ‘tagged’ 10.2-kb mouse genomic fragment, was more efficiently targeted to the pituitary. Using in situ hybridization, we detected uniform expression of HAL* in melanotrophs in 100% of the 6 pedigrees analyzed and transgenic mRNA levels paralleled those of the endogenous POMC mRNA. In corticotrophs, basal expression was low but after adrenalectomy HAL* mRNA levels were comparable to those of POMC. None of the 6 pedigrees had appropriate expression of HAL* in the brain; however, 2 lines had ectopic expression in the dentate gyms of the hippocampus. These data, together with previously reported studies, suggest that accurate neuronal expression of the POMC gene requires DNA elements in addition to the sequences that are sufficient for expression in pituitary melanotrophs and corticotrophs. The consistently lower level of transgene expression in corticotrophs compared to melanotrophs under basal conditions indicates that these two pituitary cell types may also differ in their requirements for POMC gene regulatory elements.

Previous studies have indicated that 30 min of swimming in room temperature water is a potent stimulus for the secretion of beta-endorphin (beta E) from the intermediate lobe (IL) of the pituitary in rodents. Repeated daily challenge with this paradigm over days to weeks leads to a progressive increase in proopiomelanocortin (POMC)-derived IL peptides and POMC mRNA levels as well as an increase in the stimulated secretion of beta E in response to rechallenge with swim. The current studies were undertaken to examine the possible role of dopamine systems in mediating swim stress-induced changes in IL beta E biosynthesis and release. Confirming previous results, a 30 min swim stress exposure caused plasma concentrations of beta E to increase several fold. Apomorphine (APO), a dopaminergic agonist, completely blocked this effect, suggesting that dopamine receptors may mediate the acute IL response to swim stress. Animals that swam once daily for 14 days displayed elevated beta E levels in both the IL and plasma 24 h after the last swim session. In these animals, repeated administration of APO did not reverse swim-stress-induced changes in beta E. Immediately following an acute-swim rechallenge, animals which had been previously swim-stressed for 14 days demonstrated significantly greater beta E release than naive animals. Again, an acute injection of APO inhibited the acute increase in IL secretion, suggesting that repeatedly swum animals are still responsive to the acute effects of APO even though repeated coadministration of APO with swim exposure had no effect on IL beta E peptide stores or plasma beta E concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to provide neurochemical and endocrinological evidence that dopamine (DA) neurons terminating in the intermediate lobe of the rat pituitary originate in the periventricular nucleus of the hypothalamus. One week following surgical separation of the periventricular nucleus from the mediobasal hypothalamus, DA and 3,4-dihydroxyphenyl-acetic acid (DOPAC) concentrations in the intermediate lobe were reduced by 50%, and this was accompanied by an increase in plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentrations. In contrast, this procedure had no effect on concentrations of prolactin in the plasma, or DA or DOPAC in the median eminence, the region of the mediobasal hypothalamus containing terminals of tuberoinfundibular DA neurons. Electrical stimulation of the periventricular nucleus increased the ratio of DOPAC/DA in the intermediate lobe and reduced the concentrations of alpha-MSH in the plasma, whereas in these same animals the DOPAC/DA ratio in the median eminence and concentrations of prolactin in the plasma were unaltered. These results indicate that approximately 50% of all the DA neurons terminating in the intermediate lobe of the rat pituitary originate in or project through the periventricular nucleus of the hypothalamus, and that these DA neurons regulate the secretion of alpha-MSH from intermediate lobe melanotrophs.

Voltage-gated currents through Ca channels in rat melanotrophs in short-term primary culture were investigated in whole cell voltage clamp recordings, using Ba2+ ions as charge carrier. At potentials higher than -50 mV a transient current (It) was activated, which inactivated entirely during a 150-ms command pulse and reached a maximal amplitude at -10 mV. At potentials higher than -20 mV a second component (Is) appeared, which inactivated slowly and had its maximal amplitude between 0 and +10 mV. The relative amplitudes of It and Is differed between cells. Application of 40 microM NiCl2 reversibly blocked It while leaving Is intact, whereas 20 microM CdCl2 reversibly blocked Is, but not It. Nifedipine blocked Is concentration dependently, but did not affect It. The steady state inactivation of It occurred between -80 and -40 mV. The steady-state inactivation curve of Is was located between -60 and 0 mV. The latter inactivation curve showed two phases, which were fitted best by the sum of two Boltzmann equations, suggesting the existence of two populations of Is channels.

The aim of the present experiments was to determine whether some rationale for the presence of the several inhibitory neurotransmitters in the innervation to the toad melanotroph might be found in dif

As early as 1932, basophil cells have been considered to be involved in the corticotropic and melanotropic functions due to their proliferation during Cushing’s disease. Later on, opposite opinions were held and other cells suggested to be implied in the tumor process: chromophobe, acidophil or even follicular cells. Moreover, new categories of basophil cells have been evidenced through sophisticated staining methods, in animal and human pituitaries. At the same time, the prevalent dogma ‘one hormone-one cell’ raised the problem of the search for both a corticotroph and a melanotroph in the human pituitary, which is devoid of an intermediate lobe. Since 1960, immunocytochemistry became a reliable method and simultaneously new peptides have been found. However, these peptides share common sequences of amino acids, thus raising questions as to the specificity of the antisera. Most authors mention the reaction to the basophil cells previously considered as melanotrophs and called β or R [here β(R)]. These large rounded cells, showing red granules after Alcian blue-PAS staining, are mostly located in the median area of the anterior lobe, in the cystiform zone and in cell cords invading the neural lobe. In the fetal pituitary, reactivity is observed either in some differentiated β(R) cells or, earlier, in chromophobe cells.The question is now raised whether one cell may contain several peptides and, in this case, whether the different peptides are carried by the same granule. Technical procedures using numerous antisera on serial sections have shown associations at the level of a given cell (and of a given granule under the electron microscope). However, dissociations are also to be noticed, as far as ACTH and β-MSH (adult) and more particularly α-MSH (adult and fetus) are concerned.

This study was aimed at determining the alpha-MSH and ACTH contents of the neurointermediate lobe (NIL) of the mouse hypophysis during ontogenesis, as well as the ability of the gland, incubated in a perfusion system, to respond to dopamine (DA) and high potassium (K+). We showed that: (1) the NIL content of alpha-MSH exhibited a biphasic pattern of evolution, characterized by a dramatic increase appearing between postnatal days 3 and 5. By contrast, NIL ACTH content followed a completely different pattern of evolution; (2) both DA and high K+ reversibly inhibited MSH release from superfused NIL, the latter effect being more pronounced with the use of pituitaries from 7-day-old than from 1-day-old mice; (3) the inhibitory influence of high K+ was impaired by haloperidol pretreatment. From these data, as well as from morphological observations, it appears that functional DA receptors seem to be present on melanotrophs of the developing hypophysis in mice and that the increase in alpha-MSH in IL cells observed shortly after birth may result from the onset of inhibitory influence exerted by dopaminergic innervation on hormonal secretion. This view, however, does not preclude possible effects of other hypothalamic releasing and/or inhibiting factors.

Dihydropyridines that have been shown in studies on other tissues either to facilitate Ca influx (BAY K 8644) or depress it (nimodipine and nifedipine) were examined for their effects on the secretory activity of the melanotrophs. Isolated mouse neurointermediate lobes, cultured for a week or longer to allow the nerves to degenerate, were perifused and output of MSH activity was measured by bioassay. The Ca-channel agonist BAY K 8644 augmented secretion under basal conditions and potentiated secretion evoked by 30 mM K+, a submaximally depolarizing concentration. The stimulant effect on secretion under basal conditions persisted in the presence of tetrodotoxin (which blocks the action potentials, Na spikes, in the melanotrophs) but was lost when Ca2+ was omitted or nimodipine added. The results are considered to support the view that the prominent basal secretory activity in these cells, as well as that evoked by excess K+, involve the inward flux of Ca2+ through dihydropyridine-sensitive Ca channels. If the stimulant and inhibitory effects of the dihydropyridines on basal secretion are to be attributed to actions on voltage-regulated Ca channels, as the results from other tissues would suggest, then such channels in the melanotrophs are different from those previously described in other tissues.