Mitsuhiko Matsumoto

Blocking effect of serotonin on β-adrenoceptor activity in follicle-enclosed Xenopus oocytes

The effects of serotonin (5-HT) on a beta-adrenergic response were studied, using the voltage clamp technique, in Xenopus laevis oocytes surrounded by their follicular cells. noradrenaline induced marked hyperpolarization, with a specific increase in the permeability of the membrane toward K+. Application of 10 microM 5-HT had little effect on the resting membrane, but significantly depressed the response to 0.1 microM noradrenaline. The dose-response curve for noradrenaline showed a parallel shift to the right in the presence of 5-HT, suggesting that 5-HT competes with noradrenaline for common binding sites at the beta-adrenoceptor.

Depressing Effects of Protein Kinases a & c on the Receptor-Induced K+-Current Responses in the Ganglion Cells of Aplysia

Application of either dopamine (DA), acetylcholine (ACh), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the ganglion cells of Aplysia under voltage clamp. We have previously reported that these responses are all mediated by a pertussis toxin (PTX)-sensitive G-protcin Gi or Go. Intracellular application of cAMP, an activator of protein kinase A (PKA), or extracellular application of 30 μM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), markedly suppressed these transmitter-induced K+-current responses. The depressing effect of cAMP was reversible while that of PDBu was irreversible as observed for 1 hour. Intracellular injection of the catalytic subunit of either PKA or PKC mimicked the effects of cAMP or PDBu. Pretreatment of the ganglion cells with 100 μM H-7, an inhibitor of PKC, significantly suppressed the effect of PDBu. Furthermore, an intraccllular injection of okadaic acid (OA), an inhibitor of phosphatases, facilitated the blocking effects of both cAMP and PDBu. The dose-response curve obtained by each transmitter-receptor system shifted downward by application of either cAMP or PDBu without affecting the affinity of the agonist to each receptor. K+-channel opening directly induced by guanosine thiotriphosphate (GTPγS) or by raising the temperature was not depressed by cither cAMP or 100 μM PDBu. From these results, we postulated that the acting sites of both PKA and PKC might be somewhere between the receptors and G-protein, and that the phosphorylation of these sites would impair the functional coupling efficiency between the receptors and G-protein.

The transmitter-induced change in slope conductance of a receptor membrane as a function of the resting potential and equilibrium potential of the ion involved

Change in slope conductance has been widely used to evaluate the ion-channel activation by receptor agonists of various postsynaptic membranes. However, the agonist-induced change in slope conductance (delta G) depends not only on the change in membrane ionic permeabilities (delta P) but also on the changes in resting potential (E) and equilibrium potential (EA) of the ion involved (A). A constant field theory was applied to describe delta GA as a function of delta PA, E and EA, assuming that delta PA is not affected by the change in E or EA. This equation predicts that delta GNa will decrease if the membrane is depolarized or when ENa becomes more positive. Similarly, delta GK is expected to increase if the membrane is depolarized or when EK becomes more negative. Further, the equation describes that delta GCl will increase if the membrane is depolarized or when ECl becomes less negative. These changes in GNa, GK and GCl are well consistent with many data previously obtained from various types of receptor membranes. We conclude that the values of slope conductance measured at different E or EA must be carefully corrected in order to estimate the real voltage dependence of delta P or the ion-channel activation by receptor agonists.

Regulatory roles of calmodulin-dependent protein kinase and phosphatase on the receptor-induced K+-current responses

009 EGUIJ,TORY ROLES OF CALMODULIN-DEPENDENT PROTEIN KINASE AND PHOSPHATASE ON THE RECEPTOR-INDUCED K+-CURRENT RESPONSES SATOSHI KAWASAKI’, SHINGO KIMURA’, REIKO FUJITA’, KOICHIRO TAKASHInlA’, MITSUHIKO MATSUMOT02, KAZUHIKO SASAKI’ ‘Dept. of Physiology, School of Medicine, Iwate Medical Univ., Morioka 020-8505, ‘Lab. 01’ Physics, School of Allied Medical Sciences, Hirosaki Univ., Hirosaki 036-8203 Application of dopamine, acetylcholine , or Phe-Met-Arg-Phe-NH, to identified neurons of Aplysia ganglia induces identical K’-current responses which are produced by activation of specific G-proteins of the Gi family. These K’-current responses were depressed by intracellular injection of either KN-9.3, an inhibitor for Ca”-calmodulin dependent protein kinase, W-7, a calmodulin inhibitor, or U-73 122, a phospholipase C inhibitor, irrespective of their receptor tj pes. In contrast, intracellular injection of cypermethrin, an inhibitor of calcineurin, significantly augmented the K’-current responses. Howe\ier, all the above inhibitors did not affect at all the K’-current response elicited by direct activation of G-protein via intracellular apphcation of GTPyS. These results suggest that both Ca”-calmodulin dependent protein kinase and calcineurin are physiologically activated by stimulation of these receptors, regulating the opening process of the receptor-operated Kkhannels, and that the acting site of these enzymes might be localized somewhere between the receptors and G-protein.

221 Inversion of an inward current induced by dopamine after the application of GTP-analog observed in the neurons of Aplysia

Kohji Sate’ , Kazuko Sakata2, Shoichi Shimada2, Masaya Tohyama2 We constructed a cell line which stably expresses glycine transporter 1 (GlyTl) proteins. The cell line showed significant 142C-giycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in>out). Using this cell line, we investigated glycine release mediated by this transporter. The 142C-Glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Lis’ or extracellular Clwith acetatemarkedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synpases, and that the release might be influenced by the extrace

251 Uncoupling between receptor and G-protein by protein kinase A & C in the receptor-induced K+-current responses

KAZUHIKO SASAKI’, SATOSHI KAWASAKI’, SHINGO KIMURA’, REIKO FUJITAl , KOICHIRO TAKASHIMA’, MITSUHIKO MATSUMOT02, MAKOTO SATO’ Application of either acetylcholine, histamine, dopamine, or Phe-Met-Arg-Phe-NH2 induces a G-protein mediated K+-current response in the ganglion cells of Aplysia under voltage clamp. Application of CAMP, an activator of protein kinase A (PKA), or phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), markedly depressed the transmitter-induced responses. Intracellular injection of the catalytic subunit of either PKA or PKC mimicked the effects of CAMP or PDBu. Furthermore, application of okadaic acid, an inhibitor of phosphatase 1 & 2A, facilitated the effects of both CAMP and PDBu. On the other hand, Kf-channel opening of the same cell induced by guanosinethiotriphosphate (GTPgammaS) was not depressed by either CAMP or PDBu. These results suggested that the acting sites of both PKA and PKC might be somewhere between the receptor and G-protein, and that the phosphorylation of these sites would impair the functional coupling between these proteins.

S2-1 G-proteins regulating ionic channel activities: Modulatory effects of various protein kinases on the receptor induced K+-current responses

A decade ago, we published in Nature a hypothesis that K’-channel opening produced by various receptor stimulation is triggered by activation of PTX-sensitive Gproteins regardless of the transmitter or hormone receptors. This time, we identified the neurons of Aplysia ganglion, each of which responded with K+-channel opening to DA, ACh, HA, or FMRF-amide. Intracellular injections of cAMP or catalytic subunit of protein ffinase A depressed all of the receptor-induced c-current responses. Extracellular application of phorbol dibutyrate or intracellular injection of the catalytic subunit of protein Kinase C also depressed all of the receptor-induced p-current responses. Intracellular injection of GTPgS induces irreversible opening of the K+-channel by directly activating PTX-sensitive G-proteins. This c-current response was not depressed by either kinase. Intracellular application of various protein phosphatase inhibitors augmented or depressed depending on the preceding protein phosphorylation by different protein kinases. These results suggested that the receptor-induced K+-current responses are regulated by both intrinsic protein kinase and phosphatase activities.