Intracellular vascular muscle Ca2+ modulation in genetic hypertension

Abstract

Distribution of intracellular free calcium concentration (Ca2+) was compared in spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat isolated vascular muscle cells at rest and during stimulation by K+ with Ca2+ agonist or antagonist. Ca2+ activity was quantitated at each point within vascular muscle cells loaded with fura-2 at fluorescence excitation wavelengths of 340, 360, and 380 nm, and fluorescence emission at 510 nm (all filters were ± 5 nm) quantitated by a digital photon-counting camera. Measurements of fluorescence intensity ratio in central and subsarcolemmal areas showed that calcium release, in response to 30 or 100 mM K+ with Ca2+ agonist or during spontaneous contractions, was principally from sarcoplasmic reticulum. Addition of the Ca2+ agonist Sdz 202-791, S (+) stereoisomer (SdzS), caused a dose-dependent increase of Ca2+ in both SHR and WKY rats. Intracellular calcium release sites were defined by 'hot spots' of high fluorescence intensity ratio in both central and peripheral regions of the sarcoplasm. The size and intensity of hot spots increased, and there was an initial transient activation of subsarcolemmal calcium pools in response to high K+ with 1 μM Ca2+ agonist. In contrast, treatment of the cells with the R (-) stereoisomer of Sdz 202-791 (SdzR), a Ca2+ antagonist, prevented the increase in Ca2+ and the increase in hot spot size by either K+ alone or with agonist. Antagonist decreased central core Ca2+ release and fragmented the subsarcolemmal hot spots. The difference between the effect of SdzS on cells of SHR versus WKY rats was a greater enhancement by SdzS of intracellular Ca2+ in SHR than WKY rats, especially at the peripheral rim. Furthermore, peripheral Ca2+ activity reached higher levels in SHR than were found in WKY rats, probably indicating a submembrane Ca2+ regulation deficiency. Antagonist (SdzR) reduced total cell intracellular Ca2+ to control levels in both SHR and WKY rats, which were not different. These findings suggest fundamental differences in vascular muscle cells of SHR and WKY rats, which were evident even in single cells isolated from veins of neonatal animals, implicating Ca2+ channels and modulation of release and upake that would be important for both short-term and long-term regulation of Ca2+ activity.