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The function and the mechanism of agonist-induced asynchronous wave-like [Ca²⁺]i oscillations in the in situ smoothe muscle cells of the rabbit inferior vena cava Lee, Cheng-Han

Abstract

Background: [Ca²⁺][sub i] imaging studies of in situ vascular smooth muscle cells (VSMC) in agoniststimulated arterial blood vessels have revealed asynchronous wave- like [Ca²⁺][sub i] oscillations as an important signaling mechanism. However, the functions and the mechanisms of these asynchronous wave-like [Ca²⁺][sub i] oscillations remain poorly defined. Given the importance of [Ca²⁺][sub i] in determining vascular tone, understanding the functions and the mechanisms of this particular type of [Ca²⁺][sub i] oscillations is crucial to our understanding of excitation-contraction coupling in vascular smooth muscle (VSM). Methods: Confocal [Ca²⁺][sub i] imaging of in situ VSMC from endothelium-denuded rabbit inferior vena cava (IVC) was employed to examine the subcellular Ca²⁺ responses to drugs. This was compared at times with whole-vessel spectrophotometric [Ca²⁺][sub i] measurement which assessed the averaged Ca²⁺ response from hundreds of VSMC. Isometric contraction studies were performed in parallel to examine the contractile effects of the same series of drugs. RT-PCR studies were utilized to verify the expression of implicated molecules. Furthermore, whenever applicable, parallel electron microscopy (EM) studies were also performed in parallel to correlate the disruption of VSMC ultrastructure to disruption in Ca²⁺ signaling. Results & Conclusions: The rabbit IVC is a large capacitance vessel that displays typical contractile dose-response curves for caffeine and PE. We observed that both caffeine and phenylephrine (PE) initially elicited Ca²⁺ waves in individual in situ VSMC of the IVC. The [Ca²⁺][sub i] in cells challenged with caffeine subsequently returned to baseline while the [Ca²⁺][sub i] in cells challenged with PE exhibited repetitive asynchronous Ca²⁺ waves. The lack of synchronicity of the wave-like [Ca²⁺][sub i] oscillations between VSMC can explain the observed tonic contraction at the whole-tissue level. The nature of these Ca²⁺ waves was further characterized. For caffeine the amplitude was all-or-none in nature, with individual cells differing in sensitivity, leading to their recruitment at different concentrations of the agonist. This concentration-dependency of recruitment appears to form the basis for the concentration-dependency of caffeine-induced contraction. Furthermore, the speed of Ca²⁺ waves correlated positively with the concentration of caffeine. In the case of PE, we observed the same characteristics with respect to wave speed, amplitude and recruitment. Increasing concentrations of PE enhance the frequency of the [Ca²⁺][sub i] oscillations. We therefore concluded that PE stimulates wholetissue contractility through differential recruitment of VSMCs and enhancement of the 9-tfrequency of asynchronous wave-like [Ca²⁺][sub i] oscillations once the cells are recruited. [the rest of the abstract can be found in the attached PDF file]

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