Inflammatory response has been recognized as a central feature in the development and progression of atherosclerosis, and VSMCs (Vascular Smooth Muscle Cells) - the main cellular component of media, play an important role in this process. Many reports indicate that the biologically active vitamin D metabolite - 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 = calcitriol), besides its well established role in calcium homeostasis, plays an essential role in the regulation of the inflammation process. The aim of this study was to determine the regulatory effects of calcitriol, applied at two supra-physiological doses (10 nM and 100 nM), in VSMC culture. Secretion of the pro-inflammatory cytokines, IL-6 and TNF-α, was significantly attenuated in calcitriol-treated VSMC culture, but the level of anti-inflammatory TGF-β was generally unchanged. Since in advanced atherosclerosis lesions several cell types, including VSMCs, overproduce the HSP70 chaperone protein, we also checked the effects of calcitriol on its synthesis. The presence of 1,25(OH)2D3 did not affect HSP70 synthesis under physiological conditions but the synthesis of HSP70 in VSMCs exposed to heat shock was significantly inhibited by calcitriol (=100 nM). We observed that 1,25(OH)2D3 induced SOD 1 activity, stimulated the expression of IκB-α, and did not influence the level of NF-κB-p65 in VSMCs. The results of our study suggest that 1,25(OH)2D3 may serve as a natural anti-inflammatory agent and may therefore play a beneficial role in the physiology of VSMC in some contexts of atherosclerosis.
Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22°C, open channel conductance was 9 pS with 110 mM Ba²⁺ and 24 pS with 110 mM Ba²⁺ as charge carrier. According to the life time of the open state, 2 ’modes’ of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba²⁺ than with Ca²⁺ as charge carrier. The Ca-agonist Bay K 8644 (0.5 pM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 µM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of Ica by 1 µM D 600 at 3.6 mM [Ca²⁺] and 35°C. At a holding potential of -45 mV, D600 induced an ’initial block’ of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, ’use-dependent’ block developed with time. More negative holding potentials attenuated reduction of Ica by D600, hyperpolarizations to -100 mV had an ’unblocking’ effect. In regard to reduction of Ica, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D890 (membrane impermeable). When applied with the bath, 1 or 10 pM D 600 reduced Ica dose-dependently whereas D 890 was ineffective. When D890 was applied via the patch electrode to the cytosol, it reduced Ica. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.