Intracellular Ca2+ release in vascular muscle cells by caffeine, ryanodine, norepinephrine, and neuropeptide Y

Abstract

Distribution of intracellular free calcium concentration, (Ca2+)i, in living cells has been measured with submicron resolution in isolated, single vascular muscle cells from neonatal Wistar-Kyoto (WKY) rats. The experiments were carried out on cells loaded with fura-2 using a digital photon-counting camera (PMI VIM) with computer analysis of 16-bit images. Determination of the fluorescence intensity at 360 nm excitation allowed correction for a nonhomogenous intracellular fura-2 dye distribution and revealed areas with high calcium concentrations (hot spots, HS) associated with putative sarcoplasmic reticulum (SR), even in nonactivated cells. Investigation of the effects of caffeine and ryanodine revealed that caffeine activates both centrally and peripherally located SR to release calcium, while ryanodine preferentially acts on peripherally located SR, presumably to lock calcium channels of the SR in an open state. The interaction of calcium release and uptake mechanisms in excitation-contraction coupling is supported by intracellular calcium distributions during spontaneous contraction and relaxation, which suggest a pattern of calcium release from peripherally located SR followed by release from centrally located SR. Additional studies on the effects of neuropeptide Y and norepinephrine virtually rule out any potentiation of norepinephrine by neuropeptide Y via direct modulation of (Ca2+)i in vascular muscle cells.