Two-photon imaging of endothelin-1-mediated intracellular Ca 2+ handling in smooth muscle cells of rat renal resistance arteries

Abstract

Aims Endothelin-1 (ET-1) is a potent vasoconstrictor which regulates the physiology of cardiorenal system. The aim of this study was to evaluate ET-1-mediated elevation of intracellular Ca2+ in smooth muscle cells (SMC) of renal resistance arteries. Main methods In in vitro studies of primary SMC, which were isolated from rat renal microvessels, the levels of intracellular Ca2+ were calculated from the ratio of emissions at 340 and 380 nm after loading cells with Fura 2-AM dye. In ex vivo studies we used two-photon imaging of intracellular Ca2+ handling in renal resistance arteries excised from rat kidneys and loaded with fluorescent Ca2+ indicator Fluo-4 AM. Key findings The two-photon imaging demonstrates that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of intracellular Ca2+ concentration in smooth muscle vasculature of these vessels. These ex vivo observations are in accordance with in vitro findings indicating that ET-1 mediates activation of TRPC channels and increases the level of intracellular Ca2+ in cultured SMC to 510±83 nM. Significance ET-1-mediated elevation of intracellular Ca2+ is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of SMC. The two-photon imaging of intracellular Ca2+ in intact SMC of rat renal resistance arteries is a powerful technique which allows the detailed ex vivo analysis of intracellular Ca2+ handling by ET-1, an important player in hypertension-related kidney diseases.

P66 Shc regulates renal vascular tone in hypertension-induced nephropathy

“…We have shown that ET-1 induces p66Shc Ser36 phosphorylation and mediates protein-protein interactions of p66Shc in renal glomerular cells (7,41,42). Our recent experiments using 2-photon imaging also revealed that treatment of isolated rat renal resistance arteries with ET-1 caused a rapid increase of [Ca 2+ ] i in smooth muscle vasculature of these vessels (43).…”

Endothelin receptor A and p66Shc regulate spontaneous Ca2+oscillations in smooth muscle cells controlling renal arterial spontaneous motion

“…Ex vivo imaging of [Ca 2+ ] i in SMCs of isolated renal interlobar arteries was carried out as previously described (15). Dissected arteries (150-200 mm inner diameter) were loaded with lowcalcium (0.1 mM Ca 2+ ) physiologic saline solution (PSS) supplemented with 5 mM Fluo-4 AM and 0.05% pluronic acid and stored for 1 h on a rotating shaker at room temperature in the dark.…”

“…The isolated intact vascular tree was prepared for 2-photon imaging microscopy as previously described (15). Renal microvessels (diameter, 100-150 mm; identified as interlobular arteries) were loaded with Fluo4 AM calcium indicator to monitor fluctuations in [Ca 2+ ] i .…”

“…1A). The resolution of 2-photon microscopy can avoid nonspecific autofluorescence of collagen and elastin fibers as well as Fluo4-loaded fibroblasts, adipocytes, and nerve cells of the tunica externa (15). Effective sensitivity of 2-photon microscopy allowed us to observe and quantitate Ca 2+ oscillations in SMCs (Fig.…”

“…We have established a novel technique that allows the detailed ex vivo analysis of intracellular Ca 2+ concentration ([Ca 2+ ] i ) in freshly isolated vessels (15). This approach was applied to determine assemblage of individual Ca 2+ events in SMCs of rat renal resistance arteries in either wild-type or genetically modified rats with the mutated Shc1 gene encoding p66Shc.…”