|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,200,000 (Direct Cost : ¥2,200,000)
To elucidate the functional role of gap junction, I investigated physiological and pharmacological properties of hemi-gap junctional channels expressed on a neuron enzymatically isolated from retinae of the lower vertebrates, by using the whole-cell voltage-clamp technique.
Horizontal cells isolated from the retina of catfish (Ictalurus punctatus) were voltage-clamped at +20mV and superfused by CaィイD12+ィエD1-free saline. This induced a gradually increasing outward current. The current was suppressed by saturated halothane, 1-octanol (4.6mM), dopamine (2μM) and 8-bromo-cAMP (2mM), indicating that is was produced by opening of hemi-gap junctional channels. The current was increased by H-89 (2μM ; an inhibitor of protein kinase), and decreased by okadaic acid (10μM ; an inhibitor of protein phosphatase). This observation suggests that phosphorylation and dephosphorylation of gap junctional protein(s) is the main mechanism that regulates the activity of gap junction. Furthermore, the current
was increases by an elevation of not only intracellular pH but also extracellular pH, and decreased by a decline of either of the pHs. This observation suggests that the proton is an important factor that modulates the activity of gap junction. When L-glutamate (0.5mM) was added to CaィイD12+ィエD1 -free saline in the presence of kynurenic acid (5mM ; an antagonist at ionotropic glutamate receptor), any change in the current was not observed. This observation suggests that an increase of cGMP produced by the activation of metabotropic glutamate receptor does not influence the activity of gap junction.
Another types of neurons than horizontal cells isolated from the retina of newt (Cynops pyrrhogaster), salamander (Ambystoma tigrinum) and frog (Rana catesbeiana) were voltage-clamped at -60mV, -20mV and +20mV, and superfused by CaィイD12+ィエD1 -free saline. No significant current was evoked in all types of neurons examined. The observation suggests that gap junction's between these neurons exist mainly on the dendrite, but not on the cell body.