Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 294 (6), R1769-76

Connexin 40 and ATP-dependent Intercellular Calcium Wave in Renal Glomerular Endothelial Cells

Affiliations

Connexin 40 and ATP-dependent Intercellular Calcium Wave in Renal Glomerular Endothelial Cells

Ildikó Toma et al. Am J Physiol Regul Integr Comp Physiol.

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.

Figures

Fig. 1
Fig. 1
Real-time fluorescence imaging of the mechanically induced calcium wave in cultured glomerular endothelial cells (GENCs). Calibrated, pseudocolor Fluo-4/Fura red ratio images are shown at the indicated time points after the stimulation of a single cell in the center of the field (labeled by ×). Scale bar = 20μm.
Fig. 2
Fig. 2
Representative recordings of the elevations in intracellular calcium concentrations, [Ca2+]i, in peripheral cells at various distances from the center during propagation of the calcium wave. [Ca2+]i responses in GENCs (solid line) and MMDD1 cells (dotted line) measured at the same distances are illustrated. The calcium wave showed different characteristics in the two cell types: there were no signs of attenuation in GENCs, but the wave rapidly waned over distance in MMDD1 cells.
Fig. 3
Fig. 3
Summary of the effects of various pharmacological inhibitors on the calcium wave speed (open bars, left scale) and the increases in [Ca2+]i (Δ [Ca2+]i, solid bars, right scale) in peripheral cells at various distances from the center. The voltage-operated calcium channel inhibitor nifedipine, and a calcium-free bathing solution had no effect, while gap junction uncoupling with 18- α-glycyrrhetinic acid (18 α-GA), Cx40 silencing with Cx40 siRNA, scavenging extracellular ATP (apyrase, hexokinase cocktail), and the nonselective P2 receptor antagonist suramin caused significant decreases in the speed and Δ [Ca2+]i of the propagating calcium wave. The effects of ATP scavenging were reversible after enzyme washout (recovery) in paired experiments. Treatment of GENCs with mismatched siRNA sequence (MM) and the void transfection vector (-siRNA) had no influence on calcium wave propagation. n = 6 coverslips in each group, and at least 3 cells were analyzed per coverslip. *P < 0.05, compared with control.
Fig. 4
Fig. 4
Localization of connexin isoforms and silencing of Cx40 in cultured GENCs. AD: immunofluorescence of Cx37 (A), Cx40 (B), Cx43 (C) in wild-type GENCs and Cx40 immunofluorescence labeling in Cx40 siRNA-transfected GENCs (D). No Cx37 and Cx43 labeling was found. Mostly patchy Cx40 labeling (green) was found along the plasma membrane (B). There was no immunologically detectable Cx40 protein in Cx40 silenced cells (D). Cell nuclei are labeled with 4,6-diamino-2-phenylindole. Scale bar = 10 μm. E: RT-PCR detection of Cx37, 40, and 43 mRNA in GENCs. Only Cx40 mRNA was found. βactin served as a loading control (bottom). EF: RT-PCR confirmation of effective Cx40 knockdown in GENCs transfected with Cx40 siRNA. Control (C), no treatment; -siRNA, transfection reagents, no siRNA; MM, mismatch sequence. β-actin served as a loading control.
Fig. 5
Fig. 5
Evidence that an extracellular, humoral substance is mediating the calcium wave. Pseudocolor Fluo-4/Fura red ratio images before (A) and 5 s after (B) the stimulation of a single cell in the center of the field (labeled by ×). Some cells were scraped off along a narrow, about 10- μm-wide, line in the GENC monolayer (dark linear area), eliminating physical contact between cells. Mechanical stimulation of a single cell on one side of the line (×) triggered a calcium wave that propagated in all directions, including noncontiguous cells on the opposite side of the line. Scale bar = 20 μm.
Fig. 6
Fig. 6
Purinergic receptor profile and signaling in cultured GENCs. AB: RT-PCR detection of purinergic receptor mRNA in wild-type (A) and Cx40 siRNA-treated GENCs (B). No changes in receptor expression were found in response to siRNA knockdown of Cx40. C: representative recordings of 100 μM exogenous ATP-induced elevations in [Ca2+]i in wild-type (WT) and Cx40 siRNA-silenced GENCs (Cx40 siRNA). Arrow indicates the time ATP was added to the bathing solution. Responses were nearly identical.
Fig. 7
Fig. 7
Extracellular ATP measurement in situ during the GENC calcium wave using a biosensor technique. A: representative image is shown of the fluorophore-loaded GENC monolayer (pseudocolor) and ATP-sensing PC12 cells atop also loaded with calcium dyes (red). A glass pipette was used for gentle mechanical stimulation of a single GENC. Differential interference contrast (DIC) overlay is shown for additional detail. Scale bar = 50 μm. A full video of the mechanically induced, propagating GENC calcium wave and ATP detection by PC12 cells can be found in a supplemental video file. B: summary of the speed of the GENC calcium wave (open bars, left scale) and simultaneous local, extracellular ATP measurements (solid bars, right scale) using the PC12 cell biosensor assay. siRNA silencing of Cx40 in GENCs significantly reduced the speed of the calcium wave and abolished elevations in bath ATP. Preincubation of PC12 cells with suramin (100 μM), a purinergic receptor inhibitor, blocked biosensor cell calcium responses, even when the GENC calcium wave was intact. n = 12 from four different coverslips in each group. *P < 0.05, compared with control.

Similar articles

See all similar articles

Cited by 30 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback