|Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
Water is in thermodynamic equilibrium across the plasma membrane of animal cells. When internal or external osmolality is altered, rapid transmembrane water flow occurs and the equilibrium is restored. In this research aimed at investigating (I) osmometric and water-transporting properties of rat and guinea-pig heart cells, (ii) regulation of sarcolemmal water transport by protein phosphorylation and (iii) cell volume-dependent changes in cardiac electrophysiological activities, several lines of new findings were obtained :
1. Superfusion with anisosmotic solution (0.5-4 times normal osmolality) caused a rapid and reversible cell swelling or shrinkage. The osmotic water permeability coefficients (P_f) obtained by videomicroscopic analysis of cell surface dimensions (20-30 μm-s^<-1> at 35℃) was 10-20 times smaller than that of specialized water-transporting tissues (e.g., renal tubules, erythrocytes) ; its Arrhenius activation energy, a measure of the energy barrier to water flux, was as
low as the value for free water diffusion (【approximately equal】4 kcal mol^<-1>).
2. Treatment with Hg^<2+>, a sulfhydryl-oxidizing reagent, reduced P_f by 【approximately equal】80%, and the sulfhydryl-reducing reagent dithiothreitol antagonized the inhibitory action of Hg^<2+>. Inhibitors of volume-sensitive ion transports (DIDS, Gd^<3+>, and ouabain) modified the size of osmotic cell swelling but had little effect on P_f.
3. Activation of adenylate cyclase with forskolin (FSK) caused a dose-dependent increase in P_f (EC_<50> 0.5 μM), whereas the adenylate cyclase-inactive analogue 1,9-dideoxy-FSK had no significant effect on P_f.
4. Stimulatory action of FSK was mimicked by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the membrane permeable dibutyryl-cAMP. Treatment with the muscarinic agonist Ach or the PKA inhibitor H-89 antagonized the FSK-induced increase in P_f.
5. These data indicate that osmotically-induced water flux across the plasma membrane of mammalian heart cells occurs mainly through aqueous pores and is regulated via a PKA-mediated mechanism that may involve phosphorylation of the water channel aquaporin. Redistribution of water changes cell volume, thereby diluting/concentrating the cytoplasmic constituents, and contributes to modified electrical (membrane potential and ionic currents) and contractile activities. Less