Proopiomelanocortin (POMC) and testing the phylogenetic position of turtles (Testudines)

Morphological and molecular studies have inferred multiple hypotheses for the phylogenetic relationships of Testudines. The hypothesis that Testudines are the only extant anapsid amniotes and the sister taxon of diapsid amniotes is corroborated by morphological studies, while the hypothesis that Testudines are diapsid amniotes is corroborated by more recent molecular and morphological studies. In this study, the placement of Testudines is tested using the full length cDNA sequence of the polypeptide hormone precursor proopiomelanocortin (POMC). Because only extant taxa have been used, the hypotheses being tested are limited to the following (1) Testudines as the sister taxon of Archosauria, (2) Testudines included in Archosauria and the sister taxon of Crocodilia, (3) Testudines as the sister taxon of Lepidosauria, (4) Testudines as the sister taxon of Sauria, and (5) Testudines as the sister taxon of a monophyletic Mammalia–Sauria clade. Neither Maximum likelihood, Bayesian, or maximum parsimony analyses are able to falsify the hypothesis of (Archosauria (Lepidosauria, Testudines)) and as such is the preferred inference from the POMC data.     

Proopiomelanocortin (POMC)-related peptides in the brain of the rainbow trout, Salmo gairdneri

We have investigated the presence of ACTH, -MSH and β-endorphin, three peptides which derive from the multifunctional precursor protein proopiomelanocortin (POMC) in the brain of the rainbow trout Salmo gairdneri. Using both the indirect immunofluorescence and peroxidase-antiperoxidase techniques, a discrete group of positive cells was identified in the hypothalamus, within the anterior part of the nucleus lateralis tuberis. -MSH-containing neurons represented the most abundant immunoreactive subpopulation. Coexistence of -MSH, ACTH and β-endorphin was observed in the lateral part of the nucleus. ACTH- and β-endorphin-containing cells were mainly distributed in the rostral and caudal regions of the nucleus. In the medial portion of the nucleus lateralis tuberis, numerous cells were only stained for -MSH. Moderate to dense plexuses of immunoreactive fibers were observed in the ventral thalamus and the floor of the hypothalamus. Some of these fibers projected towards the pituitary. The concentrations of ACTH, -MSH and β-endorphin-like immunoreactivities were measured in microdissected brain regions by means of specific radioimmunoassays. Diencephalon, mesencephalon and medulla oblongata extracts gave dilution curves which were parallel to standard curves. The highest concentrations of POMC-derived peptides were found in the diencephalon (-MSH: 4.28±0.43 ng/mg prot.; ACTH: 1.08±0.09 ng/mg prot.; β-endorphin: 1.02±0.1 ng/mg prot.), while lower concentrations were detected in the mesencephalon, medulla oblongata and telencephalon. The present results demonstrate that various peptides derived from POMC coexist within the same cell bodies of the fish hypothalamus. Taken together, these data suggest that expression and processing of POMC in the fish brain is similar to that occurring in pituitary melanotrophs.     

Proopiomelanocortin (POMC) mRNA and POMC-derived peptides immunolocalization in the skin of Protopterus annectens, an African lungfish

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.     

Interactions between gut peptides and the central melanocortin system in the regulation of energy homeostasis

Genetic and pharmacological studies have shown that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Animals and humans with defects in the central melanocortin system display a characteristic melanocortin obesity phenotype typified by increased adiposity, hyperphagia, metabolic defects and increased linear growth. In addition to interacting with long-term regulators of energy homeostasis such as leptin, more recent data suggest that the central melanocortin system also responds to gut-released peptides involved in mediating satiety. In this review, we discuss the interactions between these systems, with particular emphasis on cholecystokinin (CCK), ghrelin and PYY(3-36).     

Role of melanocortin signaling in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis

The melanocortin signaling system is orchestrated by two, independent groups of neurons in the hypothalamic arcuate nucleus with opposing functions that synthesize either alpha-melanocyte stimulating hormone (alpha-MSH) or agouti-related protein (AGRP). These neurons exert regulatory control over hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (PVN) at least in part through direct, overlapping, monosynaptic projections to the PVN. Alpha-MSH has an activating effect on hypophysiotropic TRH neurons via the phosphorylation of CREB, and when administered exogenously, can completely reverse fasting-induced suppression of TRH mRNA in the PVN. AGRP has a potent inhibitory effect on the hypothalamic-pituitary-thyroid axis in normally fed animals, mediated through actions at melanocortin 4 receptors. Inhibition of the HPT axis by fasting may be explained by inhibition of melanocortin signaling as a result of a reduction in alpha-MSH and increase in AGRP. Neuropeptide Y may also modulate the effects of the melanocortin signaling system during fasting by potentiating the inhibitory actions of AGRP on hypophysiotropic TRH neurons to prevent the phosphorylation of CREB and through direct inhibitory effects on alpha-MSH-producing neurons in the arcuate nucleus.     

Oxytocinergic regulation of cardiovascular function: studies in oxytocin-deficient mice

Oxytocin (OT) has been implicated in the cardiovascular responses to exercise, stress, and baroreflex adjustments. Studies were conducted to determine the effect of genetic manipulation of the OT gene on blood pressure (BP), heart rate (HR), and autonomic/baroreflex function. OT knockout (OTKO -/-) and control +/+ mice were prepared with chronic arterial catheters. OTKO -/- mice exhibited a mild hypotension (102 +/- 3 vs. 110 +/- 3 mmHg). Sympathetic and vagal tone were tested using beta(1)-adrenergic and cholinergic blockade (atenolol and atropine). Magnitude of sympathetic and vagal tone to the heart and periphery was not significantly different between groups. However, there was an upward shift of sympathetic tone to higher HR values in OTKO -/- mice. This displacement combined with unchanged basal HR led to larger responses to cholinergic blockade (+77 +/- 25 vs. +5 +/- 15 beats/min, OTKO -/- vs. control +/+ group). There was also an increase in baroreflex gain (-13.1 +/- 2.5 vs. -4.1 +/- 1.2 beats x min(-1) x mmHg(-1), OTKO -/- vs. control +/+ group) over a smaller BP range. Results show that OTKO -/- mice are characterized by 1) hypotension, suggesting that OT is involved in tonic BP maintenance; 2) enhanced baroreflex gain over a small BP range, suggesting that OT extends the functional range of arterial baroreceptor reflex; and 3) shift in autonomic balance, indicating that OT reduces the sympathetic reserve.     

Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides alpha-, beta-, and gamma-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue 'back' to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.     

The role of the central melanocortin system in the regulation of food intake and energy homeostasis: lessons from mouse models

A little more than a decade ago, the molecular basis of the lipostat was largely unknown. At that time, many laboratories were at work attempting to clone the genes encoding the obesity, diabetes, fatty, tubby and agouti loci, with the hope that identification of these obesity genes would help shed light on the process of energy homeostasis, appetite and energy expenditure. Characterization of obesity and diabetes elucidated the nature of the adipostatic hormone leptin and its receptor, respectively, while cloning of the agouti gene eventually led to the identification and characterization of one of the key neural systems upon which leptin acts to regulate intake and expenditure. In this review, we describe the neural circuitry known as the central melanocortin system and discuss the current understanding of its role in feeding and other processes involved in energy homeostasis.     

Involvement of amygdala central nucleus in the regulation of cardiovascular functions

Effects of high frequency stimulation of the amygdala central nucleus involved a selective decrease in the activity: an increase in the activity of the inferior cardiac nerve and a simultaneous decrease in the activity of the vertebral nerve, as well as an obvious BP increase. Bilateral electrolytic lesions of the same amygdala structure accompanied with an overload of the higher nervous activity induced no hypertensions. The role of the amygdala central nucleus in control of cardiovascular functions is discussed.     

Function of nociceptin and opioid OP4 receptors in the regulation of the cardiovascular system.

Nociceptin is the endogenous ligand of the opioid OP4 or ORL1 (opioid receptor-like1) receptor. It decreases blood pressure and heart rate in anesthetized and conscious rats and mice after its intravenous and intracerebroventricular injection in a manner sensitive to OP4 but not to OP1-3 (or delta, kappa and mu opioid) receptor antagonists. OP4 receptors involved in the cardiovascular effects of nociceptin were identified on sensory afferent fibres, in brain areas including the nucleus tractus solitarii and the rostral ventrolateral medulla, on preganglionic and/or postganglionic sympathetic and parasympathetic nerve fibres innervating blood vessels and heart or directly on these target organs. These receptors do not seem to be tonically activated but may play a role in the pathophysiology of inflammation, arterial hypertension and cardiac or brain circulatory ischemia.     

Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.     

Glutamate and GABA in the medial amygdala induce selective central sympathetic/parasympathetic cardiovascular responses

Glutamate and γ-aminobutyric acid (GABA) participate in central cardiovascular control, and are found in the rat posterodorsal medial amygdala (MePD), an area of the forebrain that modulates emotional/social behaviors. Here we tested whether these neurotransmitters in the MePD could change the basal activity, chemoreflex, and baroreflex cardiovascular responses in awake rats. Power spectral analysis and symbolic analysis were used to evaluate these responses. Microinjections of saline, glutamate (2 μg), or GABA (61 ng or 100 μg; n = 5-7 rats per group) did not affect basal parameters or chemoreflex responses. However, baroreflex responses showed marked changes. Glutamate increased power spectral and symbolic sympathetic indexes related to both cardiac and vascular modulations (P < 0.05). In turn, the displacement of the baroreflex half-maximal heart rate (HR) response was associated with a GABA (61 ng) mediated decrease in the upper plateau (P < 0.05). Administration of GABA (61 ng, but not 100 μg) also increased HR variability (P < 0.05), in association with parasympathetic activation. These data add novel evidence that the MePD can promote selective responses in the central regulation of the cardiovascular system, i.e., glutamate in the MePD evoked activation of a central sympathetic reflex adjustment, whereas GABA activated a central parasympathetic one.     

Population dynamics and regulation in the coypu Myocastor coypus in central Italy

A capture-recapture design was applied between January 1989 and June 1991 in a 37 5 ha area located in the central valley of the Tiber River (Latium), aimed at estimating the demographic parameters of a coypu population in the Mediterranean climate and at preliminarily investigating the factors that regulate its numbers in this region The population fluctuated between 27 and 137 individuals and followed a seasonal pattern It decreased after the winter and increased between summer and winter Density remained almost stable during a particularly mild winter Survival rates remained relatively high over the entire study period, reproductive activity and additions to the population from in situ reproduction, however, showed minimum peaks following colder winters Evidence of immigration was detected during periods of increasing density Key factor and preliminary density dependence analyses showed that pregnancy failure and newborn losses are cumulatively the most important factor contributing to varation in total mortality, and are positively related to coypu numbers in previous periods The failure to realize maximum potential fecundity is inversely related to previous coypu abundance We show that post-recruitment losses play only a minor role in determining population decline     

Activation of angiotensin II receptors in brain potentiates the stimulating effect of endogenous opioid neurons on central sympathetic outflow

Endogenous opioid peptides appear to have neurotransmitter or neuromodulator functions in brain mediating a wide variety of effects. We have reported that intracisternal administration of synthetic human beta-endorphin increases plasma concentration of catecholamines, apparently by acting at unknown brain sites to increase sympathetic outflow to the adrenal medulla and sympathetic nerves. In the present study we examined the possibility that angiotensin II, acting in brain, modulates endorphin-induced catecholamine secretion. Simultaneous intracisternal administration of angiotensin II 1.0 nmol together with synthetic human beta-endorphin 1.45 nmol potentiated the plasma epinephrine, norepinephrine and dopamine responses to intracisternal beta-endorphin. In contrast, simultaneous intracisternal administration of the angiotensin II antagonist, [Sar1, Val5, Ala8]-angiotensin II (saralasin), 1.1 nmol together with beta-endorphin, blunted the plasma epinephrine, norepinephrine and dopamine responses to beta-endorphin. These data are consistent with the hypothesis that activation of angiotensin II receptors in brain potentiates the endorphin-induced stimulation of central sympathetic outflow. It remains to be demonstrated whether angiotensin II acting in brain to modulate activity of opioid neurons is synthesized in brain or is derived peripherally.     

Secretory pathways and processing of human proopiomelanocortin (POMC) using transformed mouse cultured fibroblasts (L cells) and AtT20 cells by human POMC gene--preembedding immunoelectron microscopic studies.

In order to elucidate the relationship between secretory pathway and processing for precursor molecule of peptide hormones, we performed immunoelectron microscopic studies to localize POMC-derived peptides in mouse cultured L cells (fibroblasts without secretory granules) and in mouse AtT20 cells (ACTH secreting pituitary tumor cells with secretory granules) which had been transformed with human POMC gene. From the electron microscopic localization patterns, L42 cells were considered to serve as a model of constitutive pathway without processing of POMC, and A53 cells were considered to serve as a model of transgranular (regulated) pathway with processing of POMC. Immunoblotting supported these interpretations.