Phoenixin: a novel brain-gut-skin peptide with multiple bioactivity.

In this brief review we summarize the current fndings relative to the discovery of a small peptide ligand, phoenixin (PNX). Using a bioinformatic approach, two novel peptides PNX-14 and PNX-20 containing 14 and 20 amino acids, respectively, were isolated from diverse tissues including the brain, heart, lung and stomach. Mass spectrometry analysis identified a major and minor peak corresponding to PNX-14 and PNX-20, in rat or mouse spinal cord extracts. With the use of a rabbit polyclonal antiserum, phoenixin immunoreactivity (irPNX) was detected in discrete areas of the rodent brain including several hypothalamic subnuclei and dorsal motor nucleus of the vagus. In addition, irPNX was detected in a population of sensory ganglion cells including dorsal root ganglion, nodose ganglion and trigeminal ganglion, and in cell processes densely distributed to the superficial layers of the dorsal horn, nucleus of the solitary tract and spinal trigeminal tract. irPNX cell processes were also detected in the skin and myenteric plexus, suggesting a brain-gut and/or brain-skin connection. Pharmacological studies show that PNX-14 injected subcutaneously to the nape of the neck of mice provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. Our result suggests that the peptide PNX-14 and/or PNX-20, may serve as one of the endogenous signal molecules transducing itch sensation. Additionally, results from other laboratories show that exogenous PNX may affect a number of diverse behaviors such as memory formation, depression, reproduction, food-intake and anxiolytic-like behaviors.

A total solid-phase synthesis of DILP8.

We have developed a cysteine anchoring method for the synthesis of DILP8 and its analogues. The first is to synthesis of DILP8A SS13-18, C14-MeOBzl, C24-Acm and activate it as DILP8A S13-18, C14-SSPyr C24-Acm. A next step is to synthesize the DILP8BC16-Acm. The desired peptide, DILP8 with Cys(Acm) at A-24 and B-16, was then dissolved in 75% HOAc by addition of Iodine in MeOH and 4M HCl in dioxane. The reaction mixture was monitored by HPLC and the excess iodine was reduced with ascorbic acid. Purification of the peptide was achieved by HPLC. Pure synthetic DILP8 showed a single peak on analytical HPLC with corrected molecular ion. By using the above methods, enough peptide and highly homogenous pure DLP8 were generated.

Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions.

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.

State-dependent calcium mobilization by urotensin-II in cultured human endothelial cells.

Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.

Obestatin inhibits vasopressin secretion: evidence for a physiological action in the control of fluid homeostasis.

Obestatin, a product of post-translational processing of the ghrelin prohormone, has been reported to act in the brain to inhibit thirst. We extended our initial studies on water drinking by examining the effects of obestatin on hypovolemia-induced water and saline drinking and vasopressin release in male rats. Intracerebroventricular administration of obestatin significantly inhibited water, but not saline (0.3 M NaCl) drinking in response to a hypovolemic challenge. Obestatin also inhibited, in a dose-related fashion, dehydration-induced vasopressin secretion without affecting plasma oxytocin levels. Vasopressin release induced by central angiotensin II administration was attenuated significantly by prior administration of obestatin. Finally, central administration of an antiserum specific to obestatin resulted in an exaggerated basal vasopressin release and an increased vasopressin response to overnight water deprivation. Antiserum treatment also resulted in significantly increased ad libitum water drinking and drinking in response to dehydration. We conclude that this product of post-translational processing of the ghrelin prohormone may be an important contributor to the physiologic regulation of fluid and electrolyte homeostasis.

Nesfatin-1: distribution and interaction with a G protein-coupled receptor in the rat brain.

Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1-82) (100 nm) was assessed by the Ca(2+) microfluorometric method. Nesfatin-1 elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 microm) and omega-conotoxin MVIIC (100 nm). The protein kinase A inhibitor KT 5720 (1 microm) suppressed nesfatin-1-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.

Smooth muscle-associated protein 8: distribution and biological activity in the rat brain.

With the use of an antiserum directed against the human smooth muscle-associated protein 8 (SMAP8) fragment SMAP8(98-138), Western blot and immunohistochemical studies revealed SMAP8 expression in the rat brain. A band with a molecular size of about 45 kDa was detected in tissues from the rat hypothalamus and a weaker band from the cortex. SMAP8 immunoreactivity (irSMAP8) was detected in neurons of the hypothalamic paraventricular, supraoptic, and supraoptic retrochiasmatic nuclei; a few irSMAP8 cells were scattered in the zona incerta as well as the cerebral cortex. Immunoreactive cell processes were detected mostly in the internal layer of the median eminence. Double labeling the hypothalamic sections with SMAP8 and vasopressin (VP) or oxytocin (OT) antiserum revealed that a population of VP- and OT-immunoreactive neurons expressed irSMAP8. The biological activity of SMAP8 in rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. SMAP8 (100 nM) elevated cytosolic calcium concentrations [Ca2+]i in a population of dissociated and cultured rat hypothalamic neurons; the response was eliminated in Ca2+-free saline. This is the first evidence of irSMAP8 in a population of VP/OT-containing hypothalamic neurons in the rat, and the peptide is biologically active in hypothalamic neurons, as evidenced by mobilization of extracellular Ca2+.

Expression and activity of cocaine- and amphetamine-regulated transcript peptide(1-39) in the rat.

Cocaine- and amphetamine-regulated transcript (CART) peptide consists of a family of peptides. Expression of the peptide fragment CART(1-39) was explored in the rat using an antiserum directed against CART(1-39) of the short form of the human CART prohormone. CART(1-39)-immunoreactivity, herein referred to as irCART, was detected in the rat central and peripheral nervous tissues with a pattern similar to that labeled with the antiserum CART(55-102) or CART(79-102). For example, irCART cells were detected in the hypothalamus, pons, medulla oblongata, spinal cord, and adrenal medulla. In urethane-anesthetized rats, CART(1-39) (0.05 to 2 nmol) by intrathecal injection did not cause a significant change of blood pressure or heart rate, but potentiated the pressor effects of glutamate injected intrathecally. Lastly, the effect of CART(1-39) on intracellular calcium concentrations [Ca2+]i was assessed and compared to that caused by CART(55-102) in cultured rat cortical neurons using the microfluorimetric method. CART(1-39) (100 nM) induced two types of responses in a population of cortical neurons: 1) a slowly rising increase in [Ca2+]i superimposed with oscillations, and 2) a fast increase followed by a sustained increase of [Ca2+]i. CART(55-102) caused only a slowly rising increase in [Ca2+]i in cortical neurons. Our result shows that the expression pattern of irCART in the rat nervous system and the potentiating action of CART(1-39) on glutamate-induced pressor response is similar to that reported for CART(55-102); but the calcium mobilizing action of CART(1-39) differs from that of CART(55-102), suggesting the possible existence of multiple CART receptors coupled to different calcium signaling pathways.

Distribution and biological activity of obestatin in the rat.

Obestatin, a 23 amino acid peptide recently isolated from the rat stomach, is encoded by the same gene that encodes ghrelin. With the use of an antiserum directed against the mouse/rat obestatin, obestatin immunoreactivity (irOBS) was detected in cells of the gastric mucosa, myenteric plexus, and in Leydig cells of the testis in Sprague-Dawley rats. Double labeling the myenteric plexus with obestatin antiserum and choline acetyltransferase (ChAT) antiserum revealed that nearly all irOBS neurons were ChAT positive and vice versa. For comparative purposes, myenteric ganglion cells, cells in the gastric mucosa, and Leydig cells of the testis were shown to be immunoreactive to preproghrelin. The biological activity of obestatin on rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. Obestatin (100 nM) administered to dissociated and cultured rat cerebral cortical neurons elevated cytosolic calcium concentrations [Ca2+]i in a population of cortical neurons. The result provides the first immunohistochemical evidence that obestatin is expressed in cells of the gastric mucosa and myenteric ganglion cells, and also in Leydig cells of the testis; the peptide is biologically active on central neurons.

Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats.

Intermedin is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family peptide, which has vasodilatory and hypotensive actions identical to those of adrenomedullin and CGRP. Cleavage sites located between 2 basic amino acids at Arg93-Arg94 result in the production of prepro-intermedin95-147, namely intermedin1-53. The bioactive action of intermedin1-53 and its physiological significance are unclear. In this work, we aimed to explore the effects of intermedin1-53 on acute myocardial injury induced by isoproterenol. Myocardial ischemia injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of intermedin1-53 was observed. Plasma lactate dehydrogenase activity, myocardial and plasma malondialdehyde content were higher in the isoproterenol group than that in controls. Isoproterenol-treated rats showed lower maximal rate of increase and decrease of left-ventricle pressure development (+/-left-ventricle dp/dtmax) and higher left-ventricle end-diastolic pressure (all P<0.01), which suggested severe heart failure and myocardial injury. Semi-quantitative RT-PCR analysis showed that the gene expression of calcitonin receptor-like receptor and receptor-activity-modifying protein (RAMP)1, RAMP2 and RAMP3 in ventricular myocardia were up-regulated by 79% (P<0.01), 48% (P<0.01), 31% (P<0.05) and 130% (P<0.01), respectively, compared with controls. In myocardial sarcolemmal membranes, the maximum binding capacity for [125I]-intermedin1-53 was increased by 118% (P<0.01) in the isoproterenol group compared with controls. Rats treated with low dosage intermedin1-53 (5 nmol/kg/day, 2 days) showed 21% (P<0.05) higher myocardial cAMP content, 18% and 31% higher+left-ventricle dp/dtmax and -left-ventricle dp/dtmax respectively, 288% lower left-ventricle end-diastolic pressure (all P<0.01), and attenuated myocardial lactate dehydrogenase leakage and malondialdehyde formation (all P<0.01). Treatment with high dosage intermedin1-53 (20 nmol/kg/day, 2 days) gave better results than that with low dosage intermedin1-53. These results suggest that the intermedin receptor system was up-regulated in isoproterenol-induced myocardial ischemic injury and intermedin1-53 might play a pivotal cardioprotective role in such injury.

Salusin beta is a surrogate ligand of the mas-like G protein-coupled receptor MrgA1.

The mas-like G protein-coupled receptors form a subfamily of G protein-coupled receptors that includes variable member numbers across different species and that have been shown to bind a wide variety of ligands from peptides to amino acid derivatives. While screening a library of peptides against different orphan G protein-coupled receptors, we found that human salusin beta activates the mouse mas-like G protein-coupled receptor, mMrgA1 with an EC(50) of about 300 nM. Salusin beta is a bioactive peptide recently discovered through bioinformatics analysis which stimulates arginine-vasopressin release from rat pituitary and causes rapid and profound hypotension and bradycardia. However, when we further analyzed the generality of the mMrgA1 activation, we found that human salusin beta does not activate corresponding human mas-like G protein-coupled receptors. Our results show that human salusin beta is a surrogate ligand of the mouse MrgA1 and raises a cautionary flag for experiments that analyze the pharmacological profiles of mas-like G protein-coupled receptors from different species.

Cocaine- and amphetamine-regulated transcript peptide and sympatho-adrenal axis.

Cocaine- and amphetamine-regulated transcript peptide (CART) is constitutively expressed in discrete regions of the mammalian central and peripheral nervous system. Immunohistochemical studies reveal a well-defined network of CART-immunoreactive (irCART) neurons organized along the sympatho-adrenal axis. Sympathetic preganglionic neurons, but not parasympathetic preganglionic neurons, in the lateral horn area are CART-positive; which in turn innervate postganglionic neurons in the paravertebral and prevertebral sympathetic ganglia as well as the adrenal medulla. A population of chromaffin cells in the adrenal medulla is CART-positive; whereas, postganglionic neurons are not. Sympathetic preganglionic neurons themselves are contacted by irCART cell processes arising from neurons in the arcuate nucleus, the retrochiasmatic nucleus and the rostral ventrolateral medulla. Results from several recent studies suggest CART directly excites neurons along the sympathetic neural axis or indirectly by potentiating the action of glutamate on NMDA receptors, as evidenced by an elevation of blood pressure and heart rate following intracerebroventricular, intracisternal or intrathecal administration of the peptide to anesthetized rats or conscious rabbits.

Insulin-like peptide 5: expression in the mouse brain and mobilization of calcium.

Insulin-like peptide 5 (INSL5) mRNA was detected in the mouse hypothalamus by RT-PCR. Immunohistochemical studies using an antiserum against the mouse INSL5 peptide revealed INSL5-immunoreactive (irINSL5) neurons in the paraventricular, supraoptic, accessory secretory, and supraoptic retrochiasmatic nuclei and immunoreactive cell processes in the internal layer of the median eminence. In the pituitary, irINSL5 was detected in terminal-like elements of the posterior lobe and in cells of the anterior lobe. Double-labeling experiments showed that irINSL5 is expressed in vasopressin-, but not oxytocin-containing neurons. INSL5 (100 nm) administered to dissociated and cultured mouse hypothalamic neurons elevated cytosolic calcium concentrations [Ca(2+)](i), as assessed by the microfluorimetric fura-2 method. In a Ca(2+)-free medium, INSL5 induced in dissociated neurons an increase of [Ca(2+)](i), which was sensitive to the endoplasmic reticulum calcium pump inhibitor thapsigargin (1 microm) and the IP(3) receptor blocker 2-aminoethoxydiphenyl borate (100 microm) or xestospongin C (5 microm). Our result provides the first evidence that INSL5 is expressed in a population of cells in the mouse hypothalamus and pituitary and that it elevates [Ca(2+)](i) by a mechanism involving both Ca(2+) influx and Ca(2+) release from intracellular stores. The concentration of irINSL5 in the hypothalamic-pituitary axis suggests a neuroendocrine function of this insulin superfamily member.

Electroacupuncture suppresses expression of gastric ghrelin and hypothalamic NPY in chronic food restricted rats.

Electroacupuncture (EA) has been reported to reduce body weight in overweight subjects in clinical practice, as well as in rats and mice with diet-induced obesity. In the present study, this effect of EA was tested in lean rats subjected to long-term food restriction (FR, food was offered only 1 h/day). Two hertz EA administered once every other day produced a further reduction in body weight in FR rats. Exploration of the mechanism involved revealed significant downregulation of the orexigenic peptides: ghrelin in the stomach, and neuropeptide Y (NPY) but not Agouti-related peptide (AgRP) in the hypothalamus, which was in line with the reduction in food intake in rats receiving EA stimulation as compared with those receiving restraint only. Uncoupling protein 3 (UCP3), involved in accelerating energy expenditure, was not significantly altered. These results suggest that the EA-induced body weight reduction was due mainly to a decrease in food intake rather than an increase in energy expenditure. A reduction in the orexigenic peptides ghrelin and NPY may be involved in the underlying mechanism.

Distribution of beacon immunoreactivity in the rat brain.

Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.

Apelin protects myocardial injury induced by isoproterenol in rats.

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.

Effect of relaxin on myocardial ischemia injury induced by isoproterenol.

The omnipresent 6-kDa polypeptide relaxin (RLX) is emerging as a multifunctional endocrine and paracrine factor in a broad range of target tissues including cardiovascular tissues. To explore the pathophysiological roles of RLX in ischemic cardiovascular diseases, we studied the changes in RLX mRNA level in the myocardium and the effect of RLX supplements in rats with isoproterenol (ISO)-induced myocardial injury. In ISO-treated rats, RLX levels in myocardia and plasma increased 3.7- and 6.9-fold, respectively (P<0.01), the mRNA level increased significantly in myocardia compared with controls. Co-administration of RLX (0.2 and 2.0 microg/kg/d) and ISO increased left-ventricular pressure development and decreased left ventricular end-diastolic pressure (LVDEP) (all P<0.01). Malondialdehyde content in myocardia and lactate dehydrogenase and creatine phosphokinase activities in plasma in RLX-treated rats decreased markedly compared with that in ISO-treated alone rats (P<0.01 or P<0.05). In the high-dose RLX group, fibroblastic hyperplasia was relieved in myocardia, hydroxyproline level was lower, by 33% (P<0.05), and endothelin content in plasma was lower, by 31% (P<0.01) than in the ISO-alone group. Compared with control group, any indexes in sham rats treated with high-dose RLX were unaltered (all P>0.05). These results showed an up-regulation of myocardial RLX during ISO-induced myocardial ischemia injury and the protective effect of RLX on ISO-induced cardiac inhibition and fibrosis, which suggests that RLX could be an endogenous cardioprotective factor in ischemic heart diseases.

Cardiovascular effects of newly discovered peptide intermedin/adrenomedullin 2.

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP). The present study aimed to investigate the cardiovascular effects of IMDs (IMD1-47 and IMD8-47) in rats. Intravenous administration of 150 nmol IMDs continuously decreased mean arterial pressure and inhibited cardiac function. Administration with IMDs decreased left ventricular end-systolic pressure (LVESP) and maximal rate of left-ventricle pressure development (+/-LVdp/dt(max)), and elevated left ventricular end-diastolic pressure (LVEDP). Changes with IMD1-47 treatment were close to that with IMD8-47 (P>0.05). Perfusion of isolated rat hearts in vitro with IMD8-47 (10(-8) and 10(-7)mol/L) resulted in lower LVSP, by 40 and 56% (P<0.01); lower +LVdp/dt (max), by 33 and 47% (P<0.01); lower -LVdp/dt(max), by 25 and 39% (P<0.01); but higher coronary perfusion flow (CPF), by 25% (P<0.05) and 33% (P<0.01), respectively, than controls. However, both IMD8-47 and IMD1-47 (from 10(-13) to 10(-7)mol/L) relaxed preconstricted aortic rings in a dose-dependent manner. Intravenous administration of IMD1-47 and IMD8-47 (10(-7)mol/L) in vivo increased the cyclic adenosine monophosphate (cAMP) content by 68 and 150% (both P<0.01), respectively, in myocardia and 320 and 281% (both P<0.01), respectively, in aortas, compared with controls. Perfusion of isolated hearts with IMD1-47 and IMD8-47 (10(-7)mol/L) enhanced cAMP content by 24% (P<0.05) and 73% (P<0.01), respectively, compared with controls. IMDs inhibited 3H-Leucine incorporation in cardiomyocytes in a concentration-dependent manner. IMD1-47 and IMD8-47 (10(-7) and 10(-8)mol/L) decreased 3H-Leucine incorporation by 12-25% (P<0.01) and 14-18% (P<0.01), respectively. IMD mRNA was detected in cultured neonatal cardiomyocytes and isoproterenol-induced hypertrophic myocardia but not normal myocardia of adult rats. These results suggest that IMD might be a regulatory factor for cardiovascular function and myocardial hypertrophy as a cardiovascular active peptide.

Up-regulation of the expression of cocaine and amphetamine-regulated transcript peptide by electroacupuncture in the arcuate nucleus of diet-induced obese rats.

It was reported that acupuncture or electro-acupuncture (EA) is effective in reducing the body weight for obese patients, although the mechanisms remain obscure. In a previous study, we have found that rats fed with high-fat (HIF) diet developed diet-induced obesity (DIO) with a concomitant decrease in the hypothalamic content of the cocaine and amphetamine-regulated transcript (CART) peptide, a peptide with anorexiogenic effect. To assess the central effect of EA on DIO rat, we revealed that EA up-regulated the expression of CART peptide in the arcuate nucleus (ARC) of the DIO rats. After feeding with HIF diet for 14 weeks, the DIO rats received EA stimulation three times per week for 4 weeks. The expression of CART peptide in ARC was measured using immunohistochemistry. The plasma ACTH was measured with ELISA. EA caused a reduction of both body weight and energy intake in DIO rats and increased the expression of CART peptide in ARC. The plasma ACTH was increased in response to restraint stress, but EA produced no further increase in ACTH levels. The results suggest that EA can up-regulate the expression of CART peptide to approach normal level, resulting in an inhibition of food intake and a reduction of body weight in DIO rats.

Neuropeptide B immunoreactivity in the central nervous system of the rat.

Neuropeptide B (NPB) is a recently identified endogenous ligand for the orphan G protein-coupled receptors GPR7 and GPR8. NPB mRNA is expressed in the human, rat, and mouse brain. With the use of an antiserum directed against the rat NPB, immunoreactivity to NPB (irNPB) was detected in several discrete areas of the hypothalamus and midbrain. In the hypothalamus, irNPB cells were present in the medial preoptic area and nucleus, ventromedial preoptic nucleus, retrochiasmatic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, accessory neurosecretory nuclei, periventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, supraoptic retrochiasmatic nucleus, lateral hypothalamic area, posterior hypothalamic area, dorsal hypothalamic area, and zona incerta. A few irNPB perikarya were noted in the arcuate nucleus, whereas a dense network of nerve fibers was present in the median eminence. In the midbrain, irNPB somata were noted in the substantia nigra (compact, reticular, medial, and lateral parts), paranigral nucleus, ventral tegmental area, interfascicular nucleus, and dorsal raphe nucleus. Neurons in the Edinger-Westphal were strongly labeled. Labeled cells were not detected in the cortex, medulla oblongata, and spinal cord; few lightly labeled cells were occasionally seen in the hippocampus. Double labeling the hypothalamic sections with NPB antiserum and vasopressin or oxytocin antibody revealed that a population of vasopressin- but not oxytocin-immunoreactive cells was irNPB. Tyrosine hydroxylase-positive neurons in the midbrain, presumably dopaminergic, were irNPB. The distribution of irNPB neurons in several areas of the hypothalamus and midbrain together with the colocalization with vasopressin or tyrosine hydroxylase suggests that the peptide may subserve neuroendocrine, autonomic, and motor functions.