Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells

Abstract

Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.

Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation

“…18 Because mesangial cells express several P2 receptors, 31 they may be activated by ATP released from the macula densa. Whether Cx40 primarily serves to connect mesangial cells, smooth muscle cells, or both 9,17,33 or acts as hemichannel to release further ATP from mesangial cells, as observed in cell culture, 34 warrants further investigation. In contrast, the myogenic component of the autoregulatory response is not different in Cx40-deficient and wt kidneys.…”

Purinergic signalling in the kidney in health and disease

“…β-Blockers induce relaxation of the glomerular microvasculature by releasing ATP, which acts via P2Y receptors on endothelial cells to produce NO, resulting in vasodilation [180]. Connexin (Cx) 40 hemichannels and extracellular ATP are the key molecular elements of the glomerular endothelial calcium wave [371]. A review that discusses the roles of ATP and adenosine in tubuloglomerular feedback (TGF) regulation of glomerular filtration is available [46].…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…It has been shown that propagation of calcium waves in confluent monolayers of bovine corneal endothelial cells occurs in response to mechanical stimulation in which a glass micropipette is used to mechanically perturb the plasma membrane of a cell [125]. In these studies, ATP to the system [124]. Altogether, these different studies suggest that these endothelial cells express undocked connexin hemichannels at the plasma membrane, which are capable of releasing ATP in response to mechanical stimulation.…”

“…To date, about 20 mammalian connexin isoforms have be identified, and the role of these proteins in gap junctional communication has been extensively characterized. However, there have been reports of undocked connexons or hemichannels at the surface of a number of cell types including astrocytes and glia in the central nervous system [119][120][121], circulating polymorphonuclear granulocytes [79] and monocytes [122], vascular endothelial cells [22,[123][124][125] and vascular smooth muscle cells [126]. It has been proposed that these hemichannels may function as conduits for the transport of molecules, including ATP, between the cytosol of a cell and the extracellular space [127,128].…”

“…It has been proposed that these hemichannels may function as conduits for the transport of molecules, including ATP, between the cytosol of a cell and the extracellular space [127,128]. Under resting physiological conditions, connexin hemichannels remain in a closed impermeable state but it has been suggested that stimuli including decreases in extracellular Ca 2+ ([Ca 2+ ] e ) [129,130], strong membrane depolarizations [131,132], mechanical stimulation [124,125] and metabolic inhibition [133] and carbenoxolone [134]. In addition, cultured astrocytes have been reported to release ATP through connexin hemichannels inducing calcium wave propagation [127].…”