Abstract
AbstractAgonist-stimulated waves of elevated cytoplasmic Ca2+ concentration ([Ca2+]i ) regulate blood vessel tone and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were generated by a combination of inositol 1,4,5-trisphosphate (IP3) and Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR); although, some of the mechanistic details remain uncertain. However, these findings were derived indirectly from observing agonist-induced [Ca2+]i fluctuations in the cytoplasm.Here, for the first time, we have recorded Endothelin-1 (ET-1) induced waves of Ca2+ depletion from the SR lumen in vascular smooth muscle cells (VSMCs) using a calsequestrin-targeted Ca2+ indicator. Our findings show that these waves: (1) are due to regenerative CICR by the receptors for IP3 (IP3R), (2) have a marked latency period, (3) are characterized by a transient increase in SR Ca2+ ([Ca2+]SR ) both at the point of origin and at the wave front, (4) proceed with diminishing velocity, and (5) are arrested by the nuclear envelope. Our quantitative model indicates that the gradual decrease in the velocity of the SR depletion wave, in the absence of external Ca2+, results from continuity of the SR luminal network.
Highlights
For the first time, we have recorded Endothelin-1 (ET-1) induced waves of Ca2+ depletion from the sarcoplasmic reticulum (SR) lumen in vascular smooth muscle cells (VSMCs) using a calsequestrin-targeted Ca2+ indicator
In order to obtain more direct evidence related to regenerative SR Ca2+ release in VSMCs, we transfected cultured rat aortic SMCs with an adenoviral vector expressing a specific [Ca2+]SR indicator that we refer to as D1SR
The D1SR construct consists of enhanced CFP and YFP fluorescent proteins that are joined by a linker containing modified calmodulin (CaM) and M13 sequences [15]
Summary
When removal of extracellular Ca2+ blocks SR refilling and assuming that Ca2+ is free to redistribute along the SR lumen, opening of IP3Rs at the wave front would partially deplete the SR located just ahead of the wave (Fig. 4b) Introduction of such luminal Ca2+ flux into our preliminary quantitative model caused incremental slowing of the simulated Ca2+ depletion wave (Fig. 4c). In this particular case, the shape of wave front becomes oblong instead of circular, as it fails to involve the nuclear envelope (white arrows), and proceeds in the opposite peripheral direction. The indication that the SR depletion waves choose a preferred path by avoiding the nucleus may have physiological implications and relate to our earlier suggestion that vascular dysfunction in aging and disease could be related to loss of vascular smooth muscle Ca2+ waves [36]
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