Haruhiro Higashida

CD38 is critical for social behaviour by regulating oxytocin secretion

CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists

M-type (KCNQ2/3) potassium channels are suppressed by activation of Gq/11-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(ΔA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(ΔA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells: primary structure and functional expression from cDNAs

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.The complete amino acid sequences of two potassium channel proteins from NG108‐15 neuroblastoma‐glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.

Two genetic variants of CD38 in subjects with autism spectrum disorder and controls.

The neurobiological basis of autism spectrum disorder (ASD) remains poorly understood. Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play a role in the etiology of ASD. Here, we first examined the immunohistochemical expression of CD38 in the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalized with OT in secretory neurons. In studies of the association between CD38 and autism, we analyzed 10 single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly from a case-control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource Exchange (AGRE). The SNPs of CD38, rs6449197 (p<0.040) and rs3796863 (p<0.005) showed significant associations with a subset of ASD (IQ>70; designated as high-functioning autism (HFA)) in the U.S. 104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan to replace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6-4.6% of the Japanese population and was associated with ASD in the smaller case-control study. The SNP was clustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.

Sodium channel mRNAs I, II and III in the CNS: cell-specific expression

The cellular localization of rat brain sodium channel alpha-subunit mRNAs I, II and III in the central nervous system (CNS) was examined by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes, one set recognizing sodium channel isoforms in the coding region and the other in the non-coding region of the sodium channel messages. The independent sets of probes demonstrated qualitatively similar patterns of sodium channel mRNA expression. In the hippocampus, sodium channel mRNA I was very weakly expressed in the pyramidal layer and in the granular layer of the dentate gyrus; in contrast, sodium channel mRNA II was strongly expressed by neurons in these regions. Sodium channel mRNA III exhibited low-to-moderate expression in some neurons of the pyramidal layer of the hippocampus and granular layer of the dentate gyrus, and was not detectable in others. In the cerebellum, sodium channel mRNA I was moderately expressed in some Purkinje cells, weakly expressed in scattered cells in the molecular layer and negligibly expressed in the granular layer. Sodium channel mRNA II was strongly expressed in Purkinje and granule cells, and was moderately expressed in some cells in the molecular layer. Sodium channel mRNA III was generally not detectable in the cerebellum. In the spinal cord, motor neurons and scattered neurons throughout the gray matter exhibited moderate-to-strong expression of both sodium channel mRNA I and II. A population of cells in the spinal zone of Lissauer showed heavy expression of mRNA II, but not mRNA I. Sodium channel mRNA III was not detectable in spinal cord neurons. These observations are consistent with a general regional distribution of sodium channel message isoforms, with mRNA II being preferentially expressed in rostral regions of the CNS and mRNA I in caudal regions. However, the results also indicate that different cell types, within a given region, display different patterns of sodium channel mRNA expression. Moreover, these data suggest that individual neurons may express multiple forms of sodium channel mRNA.

Inositol 1,4,5-trisphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastoma x rat glioma hybrid cells.

1. The role of inositol 1,4,5‐trisphosphate (InsP3) and diacylglycerol (DAG) as possible mediators of the membrane current responses of NG108‐15 neuroblastoma x glioma hybrid cells to bradykinin (BK, Brown & Higashida, 1988b) has been tested using intracellular ionophoresis of InsP3 and external application of phorbol dibutyrate (PDBu) and 1‐oleoyl‐2‐acetylglycerol (OAG). 2. Intracellular ionophoresis of InsP3 into cells clamped at ‐30 to ‐50 mV produced (i) a transient outward current, (ii) a transient outward current followed by an inward current, or (iii) an inward current. All currents were accompanied by an increased input conductance. 3. The transient outward current reversed at between ‐80 and ‐90 mV. The reversal potential was shifted to more positive potentials on raising extracellular [K+], suggesting that it resulted from an increased K+ conductance. 4. The outward current was inhibited by apamin (0.4 microM) or d‐tubocurarine (0.2‐0.5 mM); these drugs also inhibit the outward current produced by BK or by intracellular Ca2+ injections (Brown & Higashida, 1988 a, b). The outward current was also slowly reduced in 0 mM [Ca2+] or 0.5 mM [Cd2+] plus 2 mM [Co2+] solution. 5. Ionophoretic injection of inositol 1,3,4‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate, guanosine trisphosphate or inorganic phosphate did not evoke an outward current but produced only an inward current with an increased conductance, reversing at between ‐10 and ‐20 mV. 6. Bath application of PDBu (10 nM‐1 microM) or OAG (1‐10 microM) produced an inward current with a fall in input conductance. The inward current was voltage dependent and was accompanied by an inhibition of the time‐dependent current relaxations associated with activation or deactivation of the voltage‐dependent K+ current, IM. 7. PDBu did not clearly reduce the Ca2+ current or the Ca2+‐dependent K+ current recorded in these cells. During superfusion with PDBu, the outward current produced by intracellular ionophoresis of InsP3 was greatly enhanced. 8. The results support the view that the two membrane current responses to BK might both result from accelerated membrane phosphatidylinositide hydrolysis. One product, InsP3, releases Ca2+ and activates an apamin‐curare‐sensitive outward K+ current; this effect is imitated by intracellular InsP3 ionophoresis. The second product, DAG; activates protein kinase C to inhibit the voltage‐dependent K+ current IM and generate an inward current; this effect is imitated by external application of PDBu or OAG.

Muscarinic Receptor-mediated Dual Regulation of ADP-ribosyl Cyclase in NG108-15 Neuronal Cell Membranes

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma × glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30–52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

The Predominant Contribution of Oligopeptide Transporter PepT1 to Intestinal Absorption of β‐Lactam Antibiotics in the Rat Small Intestine

Although recent evidence suggests that certain β‐lactam antibiotics are absorbed via a specific transport mechanism, its nature is unclear. To confirm whether peptide transport in the rat can be largely ascribed to the intestinal oligopeptide transporter PepT1, the transporter has been functionally characterized and its significance in the intestinal absorption of β‐lactam antibiotics was evaluated.

Voltage‐ and calcium‐activated potassium currents in mouse neuroblastoma x rat glioma hybrid cells.

1. Membrane currents were recorded from voltage‐clamped, microelectrode‐impaled cells of the NG108‐15 mouse neuroblastoma x rat glioma clonal cell line, differentiated with prostaglandin E1. 2. A slow outward tail current reversing at post‐pulse potentials between ‐80 and ‐90 mV was evoked by depolarizing pre‐pulses to near 0 mV. The tail current was inhibited by Cd2+ ions (0.2‐1 mM) and hence attributed to activation of a Ca2+‐dependent K+ current by a priming voltage‐activated Ca2+ current. 3. Two components to this tail current could be distinguished pharmacologically: an early (less than or equal to 50 ms) component inhibited by 1‐5 mM‐tetraethylammonium (TEA), and a late component lasting several hundred milliseconds inhibited by apamin (0.1‐0.4 microM) or d‐tubocurarine (0.1‐0.5 mM). 4. Ionophoretic injection of Ca2+ ions evoked a transient outward current with an apparent reversal potential (from ramped current‐voltage curves) of ‐70 mV. This current was succeeded or sometimes replaced by an inward current with an apparent reversal potential between ‐20 and ‐10 mV. 5. The outward current induced by Ca2+ injections was unaffected or partly inhibited by TEA (1‐5 mM), but was strongly inhibited by apamin or d‐tubocurarine. 6. Hyperpolarizing voltage steps from between ‐30 and ‐40 mV induced inward current relaxations reversing at between ‐80 and ‐90 mV. These were considered to result from deactivation of the voltage‐dependent sustained K+ current, IM. 7. Application of methacholine, muscarine or Ba2+ ions produced an inward current, reduced input conductance and reduced IM deactivation relaxations. 8. It is concluded that differentiated NG108‐15 cells possess several of the K+ currents present in sympathetic neurones, including a delayed rectifier current, two species of Ca2+‐activated K+ current and the M‐current.

Sympathetic Potentiation of Cyclic ADP-ribose Formation in Rat Cardiac Myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [3H]cADP-ribose formation from [3H]NAD+ in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 μm increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nm GTP and 10 μmguanosine 5′-3-O-(thio)triphosphate, whereas 10 μm GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from β-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.