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Gap junction internalisation and the effect of hypoxia in isolated adult rat ventricular myocytes
J Mol Cell CardiolZO(SupplementV)(1988)
55
STEADY STATE AND DYNAMIC FURA- HEASURABLB INTRACELLULAR FREE CALCIBN LEVELS IN CARDIGHYOCYTBSUNDER NORHAL AND PATHOPRYSIOLOGICAL CONDITIONS. L. Ver Donck. R. Nuyens, R. Nuydens, H. Geerts. Janssen Research Foundation, B-2340 Beerse, Belgium. Under pathological conditions, cellular damage (e.g. ischemia-reperfusion) is resulting known to be related to a loss of intracellular Ca2+, (Ca +)-homeostasis, cardiomyocytes in Ca2+-overload. We. therefore, exposed isolated rat CaH+-tolerant to pathological stimuli (20 mN extracellular Ca2+, veratrine). Cells underwent with a tremendous increase in Ca$+ from 152 to hypercontracture, coinciding (DIP) of Fura- fluorescence. 493-1400 r&I, as measured by di ital image processing Plunarizine (P)-treatment (5.10- 9 B) significantly (p -c 0.025) reduced Ca$+-levels in both control (78 nN1 and challenged cells (177-717 nN). whereas verapamil (10-D FI) The percentage of rod shaped cells correlated well with Cal+-levels had no effect. in metabolically inhibited cells (ANP-treated). Ca$+ remained low at control levels despite hypercontracture development. Video-aided DIP of Ca$+-changes in
electrically
paced
cells
allowed
determination
of
activation
(a-t,
74 msec)
and
recovery times (r-t. 357 msec) of Ca$+ during the contraction cycle. Isoproterenol ~2.10~~ N) significantly (p < 0.025) reduced a-t (48 msec) and r-t (210 f 81 msec), while F-treated cells revealed control values. It is concluded that F reduces Ca$-+ in cardiomyocytes under pathological conditions, but does not affect Ca$+-mediated physiological
processes.
56
TAURINE REDUCES THE INCREASE INEaz. DUE TO PHENYLEPHRIME IN CARDIAC MYOCYTES. F. Franconi", P. Failli, A. Fazzini: L. Polenzani, A. Giotti.*Istituto di Chimica Biologica, Universita di Sassari, ItalyDip. Farmacologia, Universita di Firenze, Italy. It has been shown that taurine antagonizes the positive inotropic effect induced by phenylephrine in guinea-pig ventricular strips (Franconi F. et al., J.Mol.Cell. Cardial., binding to cardiac membranes 18, 461,1986). Moreover it reduces i"r H prazosin Here we reported data obtained in ventricular myocytes isolated (Franconi et al., ib). from adultguinea-pig hearts according to Powell T. et al. (J. Physio1.,302,131, 1980). Cell preparations2containing more than 70% of rod-shaped cells were used to measure intracellular Ca concentration, lEaI., using Quin-2 method according to Sheu S.S. et al. (Cir.Res., 55,830, 1984). In the-&esence of propranolol, phenylephrine dose-dependently increases Ed. ; this increase is antagonized by phentolamine. 20 mM taurine Taurine's efpreloded shifts to th'e right the dose-dependent curve of phenylephrine. showing the importance of fect is dose-dependent and not mimicked by 20 mM)3-alanine, the sulfonic group in the development of taurine's action. Moreover taurine does not modify the increase in r-3 Ca Induced by isoprenaline. These results suggest that taurine is selectively important f& the development of alpha-stimulation.
57
GAP JUNCTION VENTRICULAR
INTERNALISATION AND THE EFFECT OF HYPOXIA MYOCYTES. K. Shovel, N. J. Severs, A. M. Slade,
IN ISO$ATED ADULT RAF T. Powell and V. +W. Twist
Department of Cardiac Medicine, Cardiothoracic Institute, Dovehouse Street, London SW3 and University Laboratory
of Physiology,
Parks Road, Oxford, UK. to stimulate gap junction internalisation in cells in intact tissue. It is widely believed that the internalisation of gap junctions is the first step in a sequence resulting in their degradation and eventual disappearance. The present study set out to investigate the effect of hypoxia on gap junction internalisation and degradation in the dissociated myocyte. Myocytes were isolated according to the method of Poweil (J. Physiol. 302, 131, 1980). Cells from each of four hearts were incubated for 30 minutes at room temperature under either hypoxic conditions (in Hepes buffered solution containing mannitol, gassed with 100% nitrogen and sealed) or aerobic conditions (in a corresponding solution containing glucose and gassed with 100% oxygen) and processed for thin-section electron microscopy. During the dissociation of myocytes the intact junctions remain with one or other of the formerly neighbouring cells. Gap junctions are found in visible continuity with the plasma membrane (surface-located junctions), or within the cytoplasm as apparent vesicles (annular gap junctions). The ratio of surface-located to annular junctions was calculated and the distance distributions of the gap junctions from the nearest visible plasma membrane were plotted. The relative numbers of gap junctions per cell were determined, and ultrastructural evidence for junction degradation was sought. The results indicate that in contrast to other multicellular systems hypoxia does not stimulate gap junction endocytosis in the isolated myocyte, nor is there any detectable inward movement or degradation of gap junction vesicles. (This work was supported by British Heart Foundation grants FIOO and 86/39.)
Hypoxia is reported
s.37
55
STEADY STATE AND DYNAMIC FURA- HEASURABLB INTRACELLULAR FREE CALCIBN LEVELS IN CARDIGHYOCYTBSUNDER NORHAL AND PATHOPRYSIOLOGICAL CONDITIONS. L. Ver Donck. R. Nuyens, R. Nuydens, H. Geerts. Janssen Research Foundation, B-2340 Beerse, Belgium. Under pathological conditions, cellular damage (e.g. ischemia-reperfusion) is resulting known to be related to a loss of intracellular Ca2+, (Ca +)-homeostasis, cardiomyocytes in Ca2+-overload. We. therefore, exposed isolated rat CaH+-tolerant to pathological stimuli (20 mN extracellular Ca2+, veratrine). Cells underwent with a tremendous increase in Ca$+ from 152 to hypercontracture, coinciding (DIP) of Fura- fluorescence. 493-1400 r&I, as measured by di ital image processing Plunarizine (P)-treatment (5.10- 9 B) significantly (p -c 0.025) reduced Ca$+-levels in both control (78 nN1 and challenged cells (177-717 nN). whereas verapamil (10-D FI) The percentage of rod shaped cells correlated well with Cal+-levels had no effect. in metabolically inhibited cells (ANP-treated). Ca$+ remained low at control levels despite hypercontracture development. Video-aided DIP of Ca$+-changes in
electrically
paced
cells
allowed
determination
of
activation
(a-t,
74 msec)
and
recovery times (r-t. 357 msec) of Ca$+ during the contraction cycle. Isoproterenol ~2.10~~ N) significantly (p < 0.025) reduced a-t (48 msec) and r-t (210 f 81 msec), while F-treated cells revealed control values. It is concluded that F reduces Ca$-+ in cardiomyocytes under pathological conditions, but does not affect Ca$+-mediated physiological
processes.
56
TAURINE REDUCES THE INCREASE INEaz. DUE TO PHENYLEPHRIME IN CARDIAC MYOCYTES. F. Franconi", P. Failli, A. Fazzini: L. Polenzani, A. Giotti.*Istituto di Chimica Biologica, Universita di Sassari, ItalyDip. Farmacologia, Universita di Firenze, Italy. It has been shown that taurine antagonizes the positive inotropic effect induced by phenylephrine in guinea-pig ventricular strips (Franconi F. et al., J.Mol.Cell. Cardial., binding to cardiac membranes 18, 461,1986). Moreover it reduces i"r H prazosin Here we reported data obtained in ventricular myocytes isolated (Franconi et al., ib). from adultguinea-pig hearts according to Powell T. et al. (J. Physio1.,302,131, 1980). Cell preparations2containing more than 70% of rod-shaped cells were used to measure intracellular Ca concentration, lEaI., using Quin-2 method according to Sheu S.S. et al. (Cir.Res., 55,830, 1984). In the-&esence of propranolol, phenylephrine dose-dependently increases Ed. ; this increase is antagonized by phentolamine. 20 mM taurine Taurine's efpreloded shifts to th'e right the dose-dependent curve of phenylephrine. showing the importance of fect is dose-dependent and not mimicked by 20 mM)3-alanine, the sulfonic group in the development of taurine's action. Moreover taurine does not modify the increase in r-3 Ca Induced by isoprenaline. These results suggest that taurine is selectively important f& the development of alpha-stimulation.
57
GAP JUNCTION VENTRICULAR
INTERNALISATION AND THE EFFECT OF HYPOXIA MYOCYTES. K. Shovel, N. J. Severs, A. M. Slade,
IN ISO$ATED ADULT RAF T. Powell and V. +W. Twist
Department of Cardiac Medicine, Cardiothoracic Institute, Dovehouse Street, London SW3 and University Laboratory
of Physiology,
Parks Road, Oxford, UK. to stimulate gap junction internalisation in cells in intact tissue. It is widely believed that the internalisation of gap junctions is the first step in a sequence resulting in their degradation and eventual disappearance. The present study set out to investigate the effect of hypoxia on gap junction internalisation and degradation in the dissociated myocyte. Myocytes were isolated according to the method of Poweil (J. Physiol. 302, 131, 1980). Cells from each of four hearts were incubated for 30 minutes at room temperature under either hypoxic conditions (in Hepes buffered solution containing mannitol, gassed with 100% nitrogen and sealed) or aerobic conditions (in a corresponding solution containing glucose and gassed with 100% oxygen) and processed for thin-section electron microscopy. During the dissociation of myocytes the intact junctions remain with one or other of the formerly neighbouring cells. Gap junctions are found in visible continuity with the plasma membrane (surface-located junctions), or within the cytoplasm as apparent vesicles (annular gap junctions). The ratio of surface-located to annular junctions was calculated and the distance distributions of the gap junctions from the nearest visible plasma membrane were plotted. The relative numbers of gap junctions per cell were determined, and ultrastructural evidence for junction degradation was sought. The results indicate that in contrast to other multicellular systems hypoxia does not stimulate gap junction endocytosis in the isolated myocyte, nor is there any detectable inward movement or degradation of gap junction vesicles. (This work was supported by British Heart Foundation grants FIOO and 86/39.)
Hypoxia is reported
s.37