Hisayuki Funahashi

ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells1

The ABCA3 gene, of the ABCA subclass of ATP‐binding cassette (ABC) transporters, is expressed exclusively in lung. We report here the cloning, molecular characterization, and distribution of human ABCA3 in the lung. Immunoblot analysis using the specific antibody reveals a 150‐kDa protein in the crude membrane fraction of human lung. Immunohistochemical analyses of alveoli show that ABCA3 is expressed only in the type II cells expressing surfactant protein A. At the ultrastructural level, ABCA3 immunoreactivity was detected mostly at the limiting membrane of the lamellar bodies. Since members of the ABCA transporter family are known to be involved in transmembrane transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant in type II cells.

Orexins (hypocretins) directly interact with neuropeptide Y, POMC and glucose-responsive neurons to regulate Ca2+ signaling in a reciprocal manner to leptin: orexigenic neuronal pathways in the mediobasal hypothalamus

Orexin‐A and ‐B (hypocretin‐1 and ‐2) have been implicated in the stimulation of feeding. Here we show the effector neurons and signaling mechanisms for the orexigenic action of orexins in rats. Immunohistochemical methods showed that orexin axon terminals contact with neuropeptide Y (NPY)‐ and proopiomelanocortin (POMC)‐positive neurons in the arcuate nucleus (ARC) of the rats. Microinjection of orexins into the ARC markedly increased food intake. Orexins increased cytosolic Ca2+ concentration ([Ca2+]i) in the isolated neurons from the ARC, which were subsequently shown to be immunoreactive for NPY. The increases in [Ca2+]i were inhibited by blockers of phospholipase C (PLC), protein kinase C (PKC) and Ca2+ uptake into endoplasmic reticulum. The stimulation of food intake and increases in [Ca2+]i in NPY neurons were greater with orexin‐A than with orexin‐B, indicative of involvement of the orexin‐1 receptor (OX1R). In contrast, orexin‐A and ‐B equipotently attenuated [Ca2+]i oscillations and decreased [Ca2+]i levels in POMC‐containing neurons. These effects were counteracted by pertussis toxin, suggesting involvement of the orexin‐2 receptor and Gi/Go subtypes of GTP‐binding proteins. Orexins also decreased [Ca2+]i levels in glucose‐responsive neurons in the ventromedial hypothalamus (VMH), a satiety center. Leptin exerted opposite effects on these three classes of neurons. These results demonstrate that orexins directly regulate NPY, POMC and glucose‐responsive neurons in the ARC and VMH, in a manner reciprocal to leptin. Orexin‐A evokes Ca2+ signaling in NPY neurons via OX1R–PLC–PKC and IP3 pathways. These neural pathways and intracellular signaling mechanisms may play key roles in the orexigenic action of orexins.

Immunohistochemical localization of leptin receptor in the rat brain

The distribution of leptin receptor in the rat brain was determined by immunocytochemistry and Western blotting. Strong leptin receptor immunoreactivity was detected in the arcuate, paraventricular and ventromedial nuclei of the hypothalamus, and lateral hypothalamic area. The olfactory bulb, neocortex, cerebellar cortex, dorsal raphe nucleus, inferior olive nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve also showed intense immunoreactivity. Western blotting analysis yielded a 120-kDa major band.

Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area

The orexin–orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10−12–10−8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and ω-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas.

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.

Reciprocal synaptic relationships between orexin- and melanin-concentrating hormone-containing neurons in the rat lateral hypothalamus : a novel circuit implicated in feeding regulation

OBJECTIVE: Both orexin (ORX)- and melanin-concentrating hormone (MCH) are expressed in different neurons in the lateral hypothalamic area (LH), and are considered to have common effects on stimulating food intake. There are no reports to demonstrate neural interactions between them at the ultrastructural level. We observed these neurons in the LH to evaluate the relationships between them.DESIGN: We used two different types of double immunostaining to reveal the ultrastructure of both the ORX- and MCH-containing neurons. A preembedding double immunostaining technique was used to study the synaptic relationships between the two kinds of neuron.RESULTS: The main new findings are as follows: 1) Both ORX- and MCH-containing neurons received other synaptic input and made synaptic input to other neurons; 2) Reciprocal synaptic relationships were observed between the ORX- and MCH-containing neurons.CONCLUSION: The ORX- and MCH-containing neurons in the lateral hypothalamic area may influence food intake through synapse with each other.

Regulation by Orexin of Feeding Behaviour and Locomotor Activity in the Goldfish

Orexin is a hypothalamic neuropeptide that is implicated in the regulation of feeding behaviour and the sleep‐wakefulness cycle in mammals. However, in spite of a growing body of knowledge concerning orexin in mammals, the orexin system and its function have not been well studied in lower vertebrates. In the present study, we first examined the effect of feeding status on the orexin‐like immunoreactivity (orexin‐LI) and the expression of orexin mRNA in the goldfish brain. The number of cells showing orexin‐LI in the hypothalamus of goldfish brain showed a significant increase in fasted fish and a significant decrease in glucose‐injected fish. The expression level of orexin mRNA in the brains of fasted fish increased compared to that of fed fish. We also examined the effect of an i.c.v. injection of orexin or an anti‐orexin serum on food intake and locomotor activity in the goldfish. Administration of orexin by i.c.v. injection induced a significant increase of food intake and locomotor activity, whereas i.p. injection of glucose or i.c.v. injection of anti‐orexin serum decreased food consumption. These results indicate that the orexin functions as an orexigenic factor in the goldfish brain.

Delayed neuronal cell death in the rat hippocampus is mediated by the mitogen-activated protein kinase signal transduction pathway

Transient global ischemia caused by 5 min of cardiac arrest induced delayed neuronal cell death (apoptosis) in the CA1 region of the rat hippocampus. To characterize the molecular mechanisms that regulate apoptosis in vivo, the contributions to cell death of mitogen-activated protein kinase family members were examined in the hippocampal region after brain ischemia-reperfusion. Ischemia-reperfusion led to a strong activation of the JNK/SAPK (c-Jun NH2-terminal protein kinase/stress activated protein kinase), ERK (extracellular signal-regulated kinase), and p38 enzymes. These results with other previous studies suggest that the activation of JNK/SAPK in accordance with p38 contributes to the induction of apoptosis in CA1 neurons.

Distribution, function, and properties of leptin receptors in the brain

Leptin, a peptide hormone, is implicated in the modulation of food intake and maintenance of energy balance in many vertebrates including humans. It is considered to act via its receptor mainly through several hypothalamic nuclei that play critical roles in the regulation of appetite. This article looks mainly at the functional significance of leptin in rat brain by drawing on published reports of morphological and physiological analyses. Our immunohistochemical observations indicate that the leptin receptor is distributed throughout the brain, including the hypothalamus, and interestingly, is found in the hippocampus and neocortex. Physiological experiments with single living cells isolated from fresh rat hypothalamus clearly demonstrate that leptin has a significant effect on feeding-regulating neurons in the hypothalamus. Studies to date support a role for leptin not only in modulating food intake and appetite in rats and humans, but also in relation to learning and memory processes.

Orexinergic innervation of POMC-containing neurons in the rat arcuate nucleus.

A pre-embedding double immunostaining technique was used to study the synaptic relationships between orexin-like immunoreactive axon terminals and preopiomelanocortin (POMC)-like immunoreactive neurons in the rat arcuate nucleus. Most of the synapses were axo-dendritic, while some axo-somatic synapses were also found. Both the axo-somatic and axo-dendritic synapses were symmetrical. In some cases the presynaptic orexin-like immunoreactive axon terminals contained a few large dense-cored vesicles. The results suggest that the orexinergic axon terminals in the arcuate nucleus may play an important role in the regulation of food intake via synapses through POMC neurons.