Hiroshi Higuchi

Age-Related Bidirectional Changes in Neuropeptide Y Peptides in Rat Adrenal Glands, Brain, and Blood

Abstract: Age‐related changes in neuropeptide Y (NPY) regulation were studied in rat adrenal glands, brains, and blood by radioimmunoassay and biochemical characterization using reversed phase HPLC and gel filtration chromatography. NPY immunoreactivity (pmol/g tissue ± SEM) in rat adrenal glands increased from 7 ± 1 (6 weeks old) to 1,500 ± 580 (69 weeks old). Biochemical characterization by HPLC showed that this increase was due to those of NPY and methionine sulfoxide NPY. In contrast, in rat brain, NPY content decreased in an age‐dependent manner specifically in striatum, hippocampus, medulla oblongata, and spinal cord and the sulfoxide form was not detected. In rat blood, the circulating level of NPY was high (3–5 pmol/ml plasma ± SEM) but did not change significantly with age or by adrenal demedullation. Only a small increase of the sulfoxide form of NPY was observed in aged rat plasma. The age‐dependent changes in regulation and modification of NPY in adrenal glands and in specific brain areas may have physiological relevance in the regulation of catecholamine release from adrenal glands and some brain functions during aging.

Central glucoprivation evoked by administration of 2-deoxy-D-glucose induces expression of the c-fos gene in a subpopulation of neuropeptide Y neurons in the rat hypothalamus.

Central glucoprivation evoked by the intracerebroventricular administration of 2-deoxy-D-glucose (2DG) induces eating and suppresses growth hormone (GH) secretion in rats. To elucidate the hypothalamic mechanism of these phenomena, the induction of c-fos gene expression was examined by in situ hybridization using rats with centrally administered 2DG. Autoradiography on X-ray film showed that c-fos gene expression was transiently induced in discrete hypothalamic regions; namely the paraventricular nucleus, arcuate nucleus (ARC), the surrounding regions of the third ventricle dorsal to the ARC, and the periventricular nucleus (PeV). The time course of the expression was different in these nuclei. Double-label in situ hybridization for c-fos mRNA and neuropeptide Y (NPY) or somatostatin mRNAs revealed that 20% of the NPY neurons in the ARC expressed the c-fos gene, while a small population of somatostatin neurons (6.1% in the ARC and 2.6% in the PeV) expressed the c-fos gene following 2DG administration. Since NPY is an orexigenic neuropeptide and has an inhibitory effect on GH secretion, the data suggest that the activation of a subpopulation of NPY neurons in the ARC contributes, in part, to the increased food intake and suppression of GH secretion after central glucoprivation evoked by 2DG.

Expression and Functional Analysis of a Novel Isoform of Gicerin, an Immunoglobulin Superfamily Cell Adhesion Molecule

We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.

Molecular mechanism of the effects of guanine nucleotide and sulfhydryl reagent on muscarinic receptors in smooth muscles studied by radiation inactivation

The molecular sizes of the units concerned in 3-quinuclidinyl benzilate (QNB) binding and in the effects of guanine nucleotide and sulfhydryl reagent on the inhibition of QNB binding by carbachol in smooth muscle of guinea pig ileum were determined to be 76,000, 179,000 and 107,000, respectively by the radiation inactivation method. One or more subunits (GTP subunit) other than the receptor subunit in a muscarinic receptor appeared to be involved in the effect of guanine nucleotide. When guanine nucleotide was present, the receptor subunit seemed to be dissociated from the GTP subunit.

Age-Dependent Increase in Neuropeptide Y Gene Expression in Rat Adrenal Gland and Specific Brain Areas

Age‐dependent changes in the expression of neuropeptide Y (NPY) peptides and prepro‐NPY mRNA (NPY mRNA) were studied in rat adrenal gland and brain areas by means of radioimmunoassay, immunohistochemistry, and northern blot analysis. In the adrenal gland, NPY immunoreactívity (NPY‐I) increased by 80‐fold, mainly in the chromaffin cells, during aging (from 7 to 33 weeks old). The increase in NPY‐I was accompanied by a concomitant increase in the content of NPY mRNA (800 bases in size, by 16‐fold) and putative NPY pre‐mRNA, a result suggesting that this increase results from that in NPY gene expression, probably at the level of transcription. In contrast, in some brain areas, such as striatum and medulla oblongata plus pons, NPY‐I decreased in an age‐dependent manner, whereas NPY mRNA abundances in these areas increased by twofold with age (from 7 to 33 weeks old). The opposite changes between NPY and NPY mRNA content in specific brain areas suggested the accelerated turnover/degradation of NPY peptide in the brain areas. Furthermore, β‐actin mRNA abundance did not change in rat adrenal gland and brain areas during aging. Thus, the characteristic age‐related increase in NPY gene expression in rat adrenal gland and some brain areas seems to be important for physiological regulation of some neuronal functions, such as blood pressure, in aged animals.

BDNF increases the expression of neuropeptide Y mRNA and promotes differentiation/maturation of neuropeptide Y-positive cultured cortical neurons from embryonic and postnatal rats.

The effects of neurotrophic factor on the expression of neuropeptide Y (NPY) mRNA and on morphology of NPY-immunoreactive neurons were investigated. Brain-derived neurotrophic factor (BDNF) increased the expression of NPY mRNA in cultured cortical neurons from both embryonic and postnatal rats. BDNF also increased the number of NPY neurons. Furthermore, multipolar neurites from NPY neurons were observed in cultures treated with BDNF, whereas only monopolar and bipolar neurites were observed in control cultures. These results suggest that BDNF not only increases the expression of NPY mRNA but also promotes the differentiation/maturation of NPY ergic neurons both in number and morphology. NPY expression was strongly increased by neurotrophin-4/5 similarly to BDNF and neurotrophin-3 evoked a slight increase. In contrast, basic fibroblast growth factor, cilliary neurotrophic factor and interferon-gamma had no effect on NPY expression.

Regulation of muscarinic acetylcholine receptors and contractility of guinea pig vas deferens

Abstract The dynamic relationships between the muscarinic acetylcholine (m-ACh) receptor and the contractility of guinea pig vas deferens were studied by measuring the density of m-ACh receptors and contractile responses to ACh and high K + (40 mM) under various experimental conditions. The guinea pig vas deferens has few spare muscarinic receptots for ACh. Brief treatment with dibenamine, an alkylating agent, decreases both the maximal contractile response to ACh and the number of m-ACh receptors to the same extent. The contractility of vas deferens, cultured for 3 days under a load of 320–1200 mg/muscle, was similar to that before culture. Addition of ACh to the culture medium resulted in a decrease in the sensitivity of the vas deferens to ACh and the number of muscarinic receptors in the muscle but did not affect noradrenaline-sensitivity, the amount of α-adrenergic (α-Ad) receptors or the high K + -stimulated contraction. The decrease in the number of muscarinic receptors was prevented by removing Ca 2+ from the culture medium with EGTA. The smooth muscle relaxant, papaverine, partially antagonized the effect of ACh on the number of receptors. When muscles were transferred to normal medium after treatment with ACh, the amount of m-ACh receptors increased very slowly, and this increase was prevented by cycloheximide (5 mM). Colchicine, an anti-microtubule alkaloid, suppressed both the ACh-induced decrease in the receptor number and increase in the receptor numbers after its decrease. The following suggestions are proposed from these results: 1. 1) The amount of m-ACh receptor determines the contractility of smooth muscle to ACh under physiological conditions. 2. 2) Receptor activation, and/or resultant muscle activity by ACh can regulate the amount of m-ACh receptor, but not α-Ad receptor. 3. 3) Microtubules seem to be involved in metabolic turnover of m-ACh receptor in smooth muscle.

Inhibition of agonist-induced degradation of muscarinic cholinergic receptor by quinacrine and tetracaine—possible involvement of phospholipase A2 in receptor degradation

Muscarinic cholinergic receptors (mAChR) are degraded on the addition of agonists through energy- and temperature-dependent processes, probably with clustering and endocytosis. Pretreatment of guinea-pig vas deferens with 0.5 mM quinacrine or 5 mM tetracaine, phospholipase A2 (PLase A2) inhibitors, inhibited the ACh-induced degradation of mAChR in the smooth muscle and kept mAChR on the surface membrane, while cocaine and procaine were not effective. On pretreatment with quinacrine or tetracaine the PLase A2 activity in the smooth muscle decreased continuously during culture without change in the contractile response of the tissue. Pretreatment with cocaine and procaine which had no significant effect on the down regulation of mAChR did not inhibit PLase A2 activity. However, activation of PLase A2 by long-term culture of the muscle with ACh and formation of endogenous inhibitor of PLase A2 were not observed under our experimental conditions. The participation of PLase A2 in the agonist-induced degradation of mAChR is discussed in the light of these findings.

Ca2+/Calmodulin‐Dependent Transcriptional Activation of Neuropeptide Y Gene Induced by Membrane Depolarization: Determination of Ca2+‐ and Cyclic AMP/Phorbol 12‐Myristate 13‐Acetate‐Responsive Elements

Abstract: Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time‐dependently, without a change in β‐actin mRNA level, in NG108‐15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage‐dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin‐dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization‐induced transactivation was also blocked by CaM kinase inhibitors. The 200‐bp 5′‐upstream region (−344/−145) was localized as a Ca2+/calmodulin‐responsive element (CaMRE), which confers depolarization‐induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca‐responsive elements such as CRE, SRE, NF‐AT, or the C/EBPβ‐binding site and was separated from a 64‐bp cyclic AMP/phorbol 12‐myristate 13‐acetate‐responsive element (−144/−81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L‐type Ca channels.

Receptor-activated and energy-dependent decrease of muscarinic cholinergic receptors in guinea-pig vas deferens

Organ cultures of guinea-pig vasa deferentia were used for studying both the decrease in muscarinic cholinergic receptor (mAChR) concentration induced by muscarinic agonists and the simultaneous decrease in contraction to ACh. After a lag period the decrease followed first-order kinetics and was completely blocked by atropine. The decrease was dependent not only on the extent of occupation of mAChR by ACh, but was also related to the efficacy of agonists as deduced from the contractions. Moreover the amount of mAChR was not affected by other contractile agents, norepinephrine and high K+ even under conditions in which mAChR was occupied by atropine or pilocarpine. These findings indicate that the decrease in the amount of mAChR mediated by muscarinic receptors in vas deferens was due to the extent of both receptor occupation and receptor activation by agonists. Furthermore it was shown that the decrease involved energy- and temperature-dependent processes and that cyclic nucleotides did not regulate the quantitative level of mAChR.