Calcium channel distribution in the arterial vascular tree and its relation to function.

Clinical evidence in microvascular disease suggests that T-type Ca⁺⁺ channel blockers (CCBs) have benefits over conventional L-type CCBs, however the basis for this remains largely unknown. The objective of this study was to examine vascular reactivity utilising both pharmacological and molecular techniques. This thesis is composed of three sections including (A) an Introduction, (B) Functional Vascular Studies and (C) Molecular Vascular Studies. Section A summarised fundamental principles of the vasculature including an outline of the vascular system, vascular physiology, vascular cell biology, regulation of cytosolic Ca⁺⁺ and vascular pathophysiology. Section B utilised isolated vessels and wire myography to determine the effect of pre-treatment with L-type CCBs (verapamil and nifedipine) and combined L- and T-type CCBs (mibefradil and efonidipine) on endothelin-1 (Et-1) and K⁺-mediated contractile responses in large (rat aorta) and small (rat mesenteric and human subcutaneous) vessels. All four CCBs inhibited both Et-1 and K⁺-mediated contractile responses to a similar extent in large rat vessels, however in rat microvessels the combined L- and T-channel blockers produced significantly greater inhibition of contraction than L-channel blockers alone. The significance of this differential T-channel effect in microvessels was further supported by: (1) demonstration of divergent CCB responses in human microvessels, (2) incremental inhibition of constrictor responses with a combined L- and T-CCB despite maximal L-channel blockade, (3) utilisation of structurally diverse CCBs with varied affinity for L- and T-channels, (6) use of pharmacodynamically and therapeutically appropriate CCB concentrations, (7) confirmation of contractile agonist independent responses, (8) consistent results even in the presence of an altered microvascular physiology in the form of chronic Et-1 activation and (9) exclusion of an endothelium-dependent mechanism. Section C utilised the molecular techniques of quantitative polymerase chain reaction (PCR) and ratiometric western blotting to examine the distribution of the pore-forming subunits Caᵥ1.2, Caᵥ3.1 and 3.2 in both large (rat aorta) and small (rat mesenteric) vessels. The PCR data was equivocal with no difference noted in the distribution of the L- and T-channels between large and small vessels. In contrast to this, quantitative western blot analysis revealed that while there is a similar distribution of the three subunits in the large vessel, there is a significantly increased expression of both T-channel pore-forming subunits in microvessels (Caᵥ3.1: 112 ± 38%*, Caᵥ3.2: 168 ± 48%* relative to L-channel expression, *p