Andrew Y. Zhang

Vascular physiology of a Ca2+ mobilizing second messenger - cyclic ADP - ribose

Cyclic ADP‐ribose (cADPR) is a novel Ca2+ mobilizing second messenger, which is capable of inducing Ca2+ release from the sarcoplasmic reticulum (SR) via activation of ryanodine receptors (RyR) in vascular cells. This signaling nucleotide has also been reported to participate in generation or modulation of intracellular Ca2+ sparks 2+waves or oscillations, Ca2+‐induced Ca2+ release (CICR) and spontaneous transient outward currents (STOCs) in vascular smooth muscle cells (VSMCs). With respect to the role of cADPR‐mediated signaling in mediation of vascular responses to different stimuli, there is accumulating evidence showing that cADPR is importantly involved in the Ca2+ response of vascular endothelial cells (ECs) and VSMCs to various chemical factors such as vasoactive agonists acetylcholine, oxotemorine, endothelin, and physical stimuli such as stretch, electrical depolarization and sheer stress. This cADPR‐RyR‐mediated Ca2+ signaling is now recognized as a fundamental mechanism regulating vascular function. Here we reviewed the literature regarding this cADPR signaling pathway in vascular cells with a major focus on the production of cADPR and its physiological roles in the control of vascular tone and vasomotor response. We also summarized some publish results that unveil the underlying mechanisms mediating the actions of cADPR in vascular cells. Given the importance of Ca2+ in the regulation of vascular function, the results summarized in this brief review will provide new insights into vascular physiology and circulatory regulation.

Protein Methylation Activates Reconstituted Ryanodine Receptor-Ca2+ Release Channels from Coronary Artery Myocytes

Ryanodine receptors (RyR) play an important role in the regulation of intracellular Ca2+ concentration and in the control of vascular tone. However, the mechanism regulating the activity of RyR is poorly understood. The present study determined whether protein methylation participates in the control of RyR activity. Using a planar lipid bilayer clamping system, S-adenosyl-L-methionine (SAM), a methyl donor, significantly increased the activity of a 245-pS reconstituted Ca2+ release channel from coronary arterial smooth muscle (CASM) in a concentration-dependent manner. Addition of the protein methylation blockers, 3-deazaadenosine, S-adenosylhomocysteine or sinefungin into the cis solution markedly attenuated SAM-induced activation of RyR/Ca2+ release channels. By Western blot analysis, arginine N-methyltransferase (PRMT1) and FK506 binding protein (FKBP) were detected in the SR used for reconstitution of RyR. In the presence of anti-PRMT1 antibody (1:100), SAM-induced activation of RyR/Ca2+ channel was completely abolished. In addition, this SAM-induced increase in RyR/Ca2+ channel activity was blocked by 30 µM ryanodine and by FK506 (100 µM), a ligand for the RyR accessory protein. These results suggest that protein methylation activates RyR/Ca2+ release channels and may participate in the control of intracellular Ca2+ mobilization in CASM cells by transferring a methyl group to the arginine moiety of the RyR accessory protein, FKBP 12.

Role of Cyclic ADP-Ribose-Ca2+ Signaling in Mediating Renin Production and Release in As4.1 Cells

The present study was designed to test the hypothesis that cyclic-ADP-ribose (cADPR) serves as a novel second messenger to mediate intracellular Ca2+ concentration in As4.1 cells, a prototype of renal juxtaglomerular cells, and thereby regulates the renin production and release. Western blot analysis showed that CD38, an enzyme responsible for the production of cADPR, was abundant in As4.1 cells. Using cADPR cycling assay, it was found that NaCl stimulated cADPR production in these cells, which was blocked by inhibition of ADP-ribosyl cyclase with nicotinamide. HPLC analysis showed that the conversion rate of β-NGD into cGDPR was dramatically increased by NaCl, which was attenuated by nicotinamide. Using fluorescent microscopic imaging analysis, NaCl (100 mM) was demonstrated to stimulate a rapid Ca2+ increase from the endoplasmic reticulum (ER), which was inhibited by a cADPR antagonist, 8-bromo-cADPR (30 µM), an inhibitor of ADP-ribosyl cyclase, nicotinamide (6 mM), the ryanodine receptors blocker, ryanodine (30 µM), or a Ca2+-induced Ca2+ release inhibitor, tetracaine (10 µM) by 70-90%. Finally, NaCl was found to significantly lower the renin production and release levels in As4.1 cells, which was accompanied by decreases in renin mRNA levels. Pretreatment of these cells with various inhibitors or blockers above significantly blocked the inhibitory effect of NaCl on renin production and release. These results indicate that cADPR-mediated Ca2+ signaling pathway is present in As4.1 cells and that this signaling pathway may play a contributing role in the regulation of renin production and release.