![]() On isolated renal afferent arterioles, TPCs mediate a i rise in response to ET-1 or to norepinephrine. It was demonstrated that NAADP induces Ca 2+ mobilization in cultured SMCs from pulmonary artery, and in these cells TPCs are involved in endothelin-1- and angiotensin II-induced i rise. , NAADP produces a dual effect in blood vessels: NO-dependent vasorelaxation in intact vessels and vasoconstriction in endothelium-denuded vessels. The signaling pathways mediated by NAADP are involved in differentiation, histamine-activated von Willebrand factor exocytosis in human umbilical vein endothelial cells (HUVECs), secretion, contraction of uterine smooth muscles in response to oxytocin and detrusor muscle by carbachol, autophagy processes, etc. have shown that both compounds stimulate TPC2. ![]() In experiments with isolated lysosomes, it was demonstrated that both TPC1 and TPC2 were activated by PI-3,5-P2 while NAADP was without effect. TPCs are evolutionally ancient proteins that are present not only in animals, but also in plants, fungi and unicellular eukaryotes. The function of these channels as a target of NAADP was described a decade after they were cloned. TPC2 is localized in lysosomes, and TPC1 is co-expressed with lysosome and endosome markers. The molecular target activated by NAADP is two-pore channels (TPCs), which are represented in human and mouse by two forms: TPC1 and TPC2. The Ca 2+-mobilizing action of NAADP was shown for virtually all mammalian cell types examined. This messenger released Ca 2+ from a new calcium store: lysosomes and lysosome-related acidic compartments. Somewhat later, NAADP was found to be another messenger that caused mobilization of calcium ions from intracellular depots. The latter is activated by cyclic ADP Ribose (cADPR). The well-known mechanism of agonist-induced elevation of free cytoplasmic calcium concentration ( i) is its mobilization from endo/sarcoplasmic reticulum via InsP3- and ryanodine-sensitive channels. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE. These data suggest that TPC1 is involved in the NE-stimulated i rise in SMCs. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. ![]() ![]() Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. IC 50 for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. Both cis- or trans-NED 19 inhibited the rise of i in SMCs induced by 100 μM NE by 50–60%. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ( i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca 2+ release via two-pore channels (TPCs) localized in endolysosomal vesicles.
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