Takayuki Endoh

Pharmacological characterization of inhibitory effects of postsynaptic opioid and cannabinoid receptors on calcium currents in neonatal rat nucleus tractus solitarius

The profile of opioid and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) has been studied using the whole‐cell configuration of the patch clamp technique. Experiments with selective agonists and antagonists of opioid, ORL and cannabinoid receptors indicated that μ‐opioid, κ‐opioid, ORL‐1 and CB1, but not δ‐opioid, receptors inhibit VDCCs in NTS. Application of [D‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO; μ‐opioid receptor agonist), Orphanin FQ (ORL‐1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of IBa in a concentration‐dependent manner, with IC50s of 390 nM, 220 nM and 2.2 μM, respectively. Intracellular dialysis of the Gi‐protein antibody attenuated DAMGO‐, Orphanin FQ‐ and WIN55,122‐induced inhibition of IBa. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122‐induced inhibition of IBa but not DAMGO‐ and Orphanin FQ‐induced inhibition. Mainly N‐ and P/Q‐type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L‐type VDCCs were inhibited by WIN55,122. These results suggest that μ‐ and κ‐opioid receptors and ORL‐1 receptor inhibit N‐ and P/Q‐type VDCCs via Gαi‐protein βγ subunits, whereas CB1 receptors inhibit L‐type VDCCs via Gαi‐proteins involving PKA in NTS.

Extracellular ATP-induced calcium channel inhibition mediated by P1/P2Y purinoceptors in hamster submandibular ganglion neurons.

The presence and profile of purinoceptors in neurons of the hamster submandibular ganglion (SMG) have been studied using the whole‐cell configuration of the patch‐clamp technique. Extracellular application of adenosine 5′‐triphosphate (ATP) reversibly inhibited voltage‐dependent Ca2+ channel (VDCC) currents (ICa) via Gi/o‐protein in a voltage‐dependent manner. Extracellular application of uridine 5′‐triphosphate (UTP), 2‐methylthioATP (2‐MeSATP), α,β‐methylene ATP (α,β‐MeATP) and adenosine 5′‐diphosphate (ADP) also inhibited ICa. The rank order of potency was ATP=UTP>ADP>2‐MeSATP=α,β‐MeATP. The P2 purinoceptor antagonists, suramin and pyridoxal‐5‐phosphate‐6‐azophenyl‐2′, 4′‐disulfonic acid (PPADS), partially antagonized the ATP‐induced inhibition of ICa, while coapplication of suramin and the P1 purinoceptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), virtually abolished ICa inhibition. DPCPX alone partially antagonized ICa inhibition. Suramin antagonized the UTP‐induced inhibition of ICa, while DPCPX had no effect. Extracellular application of adenosine (ADO) also inhibited ICa in a voltage‐dependent manner via Gi/o‐protein activation. Mainly N‐ and P/Q‐type VDCCs were inhibited by both ATP and ADO via Gi/o‐protein βγ subunits in seemingly convergence pathways.

THE REGULATING MANNER OF OPIOID RECEPTORS ON DISTINCT TYPES OF CALCIUM CHANNELS IN HAMSTER SUBMANDIBULAR GANGLION CELLS

It is well known that opioids produce inhibitory effects on neuronal activity and on synaptic transmission at most synapses. In this study, we have investigated the effects of opioids on the low voltage- and high voltage-activated calcium channels in acutely dissociated submandibular ganglion (SMG) neurons, using the whole-cell configuration of the patch-clamp technique. The kappa-opioid-receptor agonist U-50488H, the delta-opioid-receptor agonist [D-Pen 2,5]-enkephalin and the mu-opioid-receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin inhibited L-, N- and P/Q-type calcium-current components in a dose-dependent manner at 10 nM-1 microM, respectively, but not the T-type calcium current. These inhibitory effects were antagonized by naloxone (1 microM). The results showed that three types of opioid receptors regulate the L-, N- and P/Q-types of calcium channels, respectively, but not the T-type, in SMG neurones.

Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractus solitarius by angiotensin II.

It is recognized that brain contains all the components of the renin–angiotensin systems (RAS). The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage‐dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+‐dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of angiotensin II (Ang II) on VDCC currents (ICa) in the NTS using patch‐clamp recording methods. An application of Ang II caused facilitation of L‐type ICa in a concentration‐dependent manner with an EC50 of 167 nm and a Hill coefficient of 1.73. AT1 receptor antagonist losartan antagonized the Ang II‐induced facilitation of ICa. Intracellular dialysis of the Gαi‐protein antibody attenuated the Ang II‐induced facilitation of ICa. Both Src tyrosine kinase inhibitor and mitogen‐activated protein kinase (MAPK) inhibitor attenuated the Ang II‐induced facilitation of ICa. p38 MAPK inhibitor also attenuated the Ang II‐induced facilitation of ICa. These results indicate that Ang II facilitates L‐type VDCCs via Gαi‐proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS.

Muscarinic M2 receptor inhibition of calcium current in rat nucleus tractus solitarius

The cholinergic system in the central nervous system plays an important role in higher brain functions, through muscarinic receptors. The nucleus tractus solitarius is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of acetylcholine (Ach) on VDCC currents (ICa) in the nucleus tractus solitarius using patch-clamp recording methods. In 68 out of 99 neurons, an application of ACh caused inhibition of N-type and P/Q-type IBa in a concentration-dependent manner. Pretreatments with AF-DX116 (muscarinic M2 receptor antagonist) attenuated the ACh-induced inhibition of IBa. Intracellular dialysis of the G&agr;i-protein antibody also attenuated the ACh-induced inhibition of IBa. These results indicate that ACh inhibits N-type and P/Q-type VDCCs via Gi-protein &bgr;&ggr; subunits mediated by M2 receptors in nucleus tractus solitarius.

Multiple signal pathways coupling VIP and PACAP receptors to calcium channels in hamster submandibular ganglion neurons

The Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two novel neuropeptides which produce particular biological effects caused by interaction with G-protein-coupled receptors. We have shown in a previous study where VIP and PACAP 38 inhibit voltage-dependent calcium channel (VDCC) currents (ICa) via G-proteins in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to further characterize the signal transduction pathways of VIP-and PACAP 38-induced modulation of ICa. Application of 1 microM VIP and PACAP 38 inhibited ICa by 33.0 +/- 3.1% and 36.8 +/- 2.6%, respectively (mean +/- S.E.M., n = 8). Application of strong voltage prepulse attenuated PACAP 38-induced inhibition of ICa. Pretreatment of cAMP dependent protein kinase (PKA) activator attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Intracellular dialysis of the PKA inhibitor attenuated the VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of protein kinase C (PKC) activator and inhibitor attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of cholera toxin (CTX) attenuated PACAP 38-induced inhibition of ICa. These findings indicate that there are multiple signaling pathways in VIP and PACAP 38-induced inhibitions of ICa: one pathway would be the VPAC1/VPAC2 receptors-induced inhibition involving both the PKA and PKC, and another one concerns the PAC1 receptor-induced inhibition via Gs-protein betagamma subunits. The VIP-and PACAP 38-induced facilitation of ICa can be observed in the SMG neurons in addition to inhibiting of ICa.

Calcitonin gene-related peptide- and adrenomedullin-induced facilitation of calcium current by different signal pathways in nucleus tractus solitarius

Calcitonin gene-related peptides (CGRP) and adrenomedullin (ADM) belong to the calcitonin family of peptides and are structurally related. Both peptides are found in the neurons of the CNS and play a role in many neuronal functions, including the control of blood pressure. The nucleus tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Recently, hypotension and bradycardia were observed after CGRP and ADM injection in the NTS. Voltage-dependent Ca(2+) channels (VDCCs) serve as crucial mediators of membrane excitability and Ca(2+)-dependent functions, such as neurotransmitter release, enzyme activity, and gene expression. The purpose of this study is to investigate the effects of CGRP and ADM on VDCC currents (I(Ca)) carried by Ba(2+) (I(Ba)) in the NTS, using patch-clamp recording methods. Application of CGRP and ADM caused facilitation of I(Ba) in a concentration-dependent manner. Intracellular dialysis of the anti-Galpha(s)-protein antibody attenuated CGRP-induced facilitation of I(Ba). Intracellular dialysis of the anti-Galpha(i)-protein antibody attenuated ADM-induced facilitation of I(Ba). Pretreatment with SQ22536 (an adenylate cyclase inhibitor) and intracellular dialysis of PKI(5-24) (a protein kinase A inhibitor) attenuated CGRP-induced facilitation of I(Ba). In contrast, pretreatment with PD98,059 (a mitogen-activated protein kinas inhibitor) attenuated ADM-induced facilitation of I(Ba). Mainly L-type VDCCs were facilitated by both CGRP and ADM. These results indicate that CGRP facilitates L-type VDCCs via Galpha(s)-protein involving adenylate cyclase and protein kinase A. In contrast, ADM facilitates L-type VDCCs via Galpha(i)-protein involving mitogen-activated protein kinase in the NTS.

Galanin inhibits calcium channels via Gαi-protein mediated by GalR1 in rat nucleus tractus solitarius

Galanin (GAL), a 29-amino-acid neuropeptide, is involved in various neuronal functions, including the regulation of food intake, hormone secretion and central cardiovascular regulation. The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca(2+)-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of GAL on VDCCs currents (ICa) carried by Ba2+ (IBa) in the NTS using patch-clamp recording methods. An application of M617 (GalR1 specific agonist), AR-M961 (GAL receptor GalR 1/2 agonist) and GAL caused inhibition of N- and P/Q-types I(Ba). M617, GAL, and AR-M961 caused inhibition of I(Ba) in a concentration-dependent manner, with IC50s of 678 nM, 325 nM and 573 nM, respectively. This inhibition was relieved, albeit incompletely, by a depolarizing prepulse. Pretreatment with M35 (GalR non-specific antagonist) attenuated the M617-induced inhibition of I(Ba). Intracellular dialysis of the Galpha(i)-protein antibody also attenuated the Gal-induced inhibition of IBa. These results indicate that GAL inhibits N- and P/Q-types VDCCs via Galpha(i)-protein betagamma subunits mediated by GalR1 in NTS.

Angiotensin II-induced inhibition of calcium currents in hamster submandibular ganglion neurons.

Angiotensin II (Ang II) is one of the most important vasoconstrictive hormones but is also known to act as a neuromodulator and a neurotransmitter in the central and peripheral nervous systems. In a previous study, we have shown that Ang II, via AT1 receptors, induced depolarization by inhibition of M-type K(+) channels and SK channels in submandibular ganglion (SMG) neurons. In this study, we investigated the effects of Ang II on calcium channel current (I(Ca)) in acutely dissociated SMG neurons by the patch-clamp technique using the whole-cell configuration. Ang II inhibited total I(Ca) by 32.1+/-2.7%. The half-maximum inhibitory concentration (IC(50)) of Ang II for inhibiting I(Ca) was 0.8 microM. In the presence of 1 microM losartan, which is a selective antagonist of AT1 receptors, the effect of Ang II was attenuated (7.6+/-1.5%). Application of a strong depolarizing voltage prepulse did not affect the Ang II-induced inhibition of I(Ca) (32.8+/-2.8%). Intracellular dialysis of GDP-beta-S attenuated the inhibition of I(Ca) (6.8+/-2.1%). The mean percentage inhibitions of L-, N- and P/Q-type VDCCs by Ang II were 29.1+/-1.7, 16.3+/-6.0 and 1.2+/-0.8%, respectively, of the total I(Ca).

Chronic bradykinin treatment alters 1α,25-dihydroxyvitamin D3-induced calcium current modulation in pre-osteoblasts

Bradykinin (BK) is involved in bone resorption in chronic inflammatory diseases. During bone formation, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in the regulation of Ca2+. In osteoblasts, 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through voltage-sensitive Ca2+ channels (VSCCs). Voltage sensitive Ca2+ channels serve as crucial mediators of membrane excitability and many Ca2+-dependent functions, including bone growth, regulation of proliferation, enzyme activity and gene expression. The purpose of this study was to investigate the effects of BK and 1,25(OH)2D3 on VSCC currents carried by Ba2+ (IBa). Application of 1,25(OH)2D3 facilitated IBa in a voltage-dependent manner. Pretreatment with SQ22536 (an adenylate cyclase inhibitor) attenuated 1,25(OH)2D3-induced facilitation of IBa. Bradykinin and BK1 receptor agonist [Lys-des-Arg9]-BK also facilitated IBa. After 24 h or 7 days exposure to BK, that is, under chronic inflammatory conditions, application of 1,25(OH)2D3 inhibited IBa. In addition, pretreatment with PD98,059, a mitogen-activated protein kinase (MAPK) tyrosine kinase inhibitor, attenuated 1,25(OH)2D3-induced inhibition of IBa. These results indicate that, under normal conditions, 1,25(OH)2D3 acts with adenylate cyclase to facilitate VSCCs, whereas under chronic inflammatory conditions it acts with MAPK to inhibit VSCCs in pre-osteoblasts.