Effects of pharmacological agents on the hypothalamus of Rana pipiens in relation to the control of skin melanophores (2023)

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, α-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 α-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 α-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. Nicotine (10⁻⁷ − 10⁻⁴ M) also caused a dose-related stimulation of α-MSH release from perifused pars intermedia cells. Both electrophysiological recordings and perifusion experiments showed that nicotine-induced stimulation of pituitary melanotrophs was not sensitive to various classical nicotinic antagonists including hexamethonium (10⁻⁴ M), α-bungarotoxin (10⁻⁵ M),d-tubocurarine (10⁻⁵ M), dihydro- β-erythroïdine (10⁻⁵ M) and toxin F (10⁻⁶ M). In addition, the nicotinic agonists cytisine (10⁻⁵ M) and 1,1-dimethyl-4-phenylpiperazinium (10⁻⁵ M) did not stimulate α-MSH release. In conclusion, the present results show that ACh acts as a neurohormone to stimulate the electrical and secretory activities of frog pars intermedia cells through M₁-muscarinic receptors. Our data also suggest the existence, in our model, of a subtype of nicotinic receptors unrelated to identified nicotinic binding sites.

Melanophore-stimulating hormone (MSH) release from the pars intermedia of the pituitary gland is probably regulated by multiple factors of hypothalamic origin. We have examined a number of potential regulatory factors for their effects on MSH release from the amphibian Xenopus laevis. Serotonin and acetylcholine have no effect on MSH release. Both adrenaline and noradrenaline inhibit release of MSH in a dose-dependent manner. Studies with specific receptor agonists and antagonists reveal that these neurotransmitters exert their in vitro effects primarily through a dopamine D-2 receptor, although and α-adrenergic receptor could not be excluded. We further conclude that the pars intermedia of X. laevis lacks a β-adrenergic receptor for the regulation of MSH secretion from the pars intermedia. In mammals, this receptor activates the adenylate cyclase system. Our studies reveal that despite the lack of β-adrenergic receptors, cyclic-AMP is likely an intracellular factor involved in the stimulation of MSH release.

Adaptation of the frog, Rana temporaria, to a white background for 12 hr has resulted in an intense formaldehyde-induced fluorescence (FIF) in the neurons of the preoptic recess organ (PRO), paraventricular organ (PVO), nucleus infundibularis dorsalis (NID) and their basal processes permitting visualization of the PRO- and PVO-hypophysial tracts that extend into the median eminence (ME) and pars intermedia (PI); the FIF is reduced in all the structures by 3 days. In frogs adapted to a black background, for 12 hr and 3 days, there was a general reduction in the FIF of the PRO neurons and PRO-hypophysial tract. By 12 hr black background adaptation, the PVO/NID neurons and only their adjacent basal processes show FIF which was sharply reduced by 3 days, making the PVO-hypophysial tract undetectable. In the PI fibers the fluorescence was more intense in black-adapted frogs than in white-adapted ones at both the intervals studied. The simultaneous changes in the FIF of the hypothalamic nuclei, tracts and PI suggest that the PRO and PVO/NID neurons participate in PI control through release of neurotransmitter(s) at the axonal ends.

Frogs (Rana pipiens) adapted to an intermediate state of melanin dispersion were treated with phentolamine and with propranolol in order to block, respectively, alpha and beta adrenergic receptors. Changes in fine structure of the pars intermedia gland cells indicate that adrenergic receptors of both types are involved in the process of MSH release from the pituitary. It is probable that the adrenergic inhibitory control of MSH release in this species is mediated by receptors of the beta type. It is also suggested that alpha adrenergic receptors are involved in the modulation and perhaps facilitation of MSH output.

Biochemical and Biophysical Research Communications, Volume 458, Issue 1, 2015, pp. 174-179

Vessel sprouting from pre-existing vasculature is a key step for the formation of a functional vasculature. The low level of vascular endothelial growth factor (VEGF) induces normal and stable angiogenesis, whereas high level of VEGF causes irregular and over sprouted vasculature. Intermedin (IMD) is a novel member of calcitonin family, and was found to be able to restrict the excessive vessel sprouting. However, the underlying mechanism had not been elucidated. In this study, using invitro and invivo angiogenic models, we found that the loosening of endothelial junction could significantly increase the ability of low-dose VEGF to induce vessel sprouting. IMD inhibited the junction dissociation-induced vessel sprouting by re-establishing the complex of vascular endothelial cadherin on the cell–cell contact. Our findings suggested a novel mechanism through which IMD could restrict the excessive vessel sprouting by preventing the endothelial junction from dissociation, and provide new insight into the understanding of the regulation of sprouting angiogenesis.

Journal of Investigative Dermatology, Volume 127, Issue 3, 2007, pp. 605-613

Intermedin (IMD), also called adrenomedullin-2, is a peptide that belongs to the calcitonin/calcitonin gene-related peptide/amylin peptide family. IMD exerts many effects on the cardiovascular system, gastrointestinal tract, and central nervous system. Here, we analyzed the expression of the IMD peptide in human skin of healthy controls, in biopsies from lesional and non-lesional areas of atopic dermatitis (AD) skin, in cultured human keratinocytes, and in the HaCaT keratinocyte cell line at the transcriptional (quantitative reverse transcription-PCR) and translational (immunohistochemistry) level. IMD messenger RNA (mRNA) and protein could be detected in keratinocytes and human skin. Keratinocytes, nerve fibers, periglandular cells, arterial/arteriolar smooth muscle cells, and pericytes of dermal microvessels were intensely IMD-immunoreactive. The IMD mRNA was, compared to healthy skin, significantly reduced in lesional and non-lesional areas of AD skin. This was accompanied by a reduction of IMD immunoreactivity in pericytes of the upper dermis indicating that skin from AD patients is generally affected, and downregulation of IMD in AD skin is not a secondary phenomenon caused by acute inflammation but is a general characteristic of AD skin. These data further point to a role of IMD expressed by pericytes in conferring higher susceptibility of the skin of AD patients to inflammatory stimuli.

American Journal of Obstetrics and Gynecology, Volume 47, Issue 4, 1944, pp. 561-571

Peptides, Volume 76, 2016, pp. 1-13

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM₁ receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1nmolL⁻¹, applied either throughout ischaemia (3h) and re-oxygenation (1h) or during re-oxygenation (1h) alone, attenuated HCM injury (P<0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P<0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P<0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM₁ receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Journal of Investigative Dermatology, Volume 34, Issue 6, 1960, pp. 377-391

Journal of Investigative Dermatology, Volume 32, Issue 6, 1959, pp. 651-659