Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells.
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.
Toma
Ildikó
I
Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo St., ZNI 335, Los Angeles, CA 90033, USA.
Bansal
Eric
E
Meer
Elliott J
EJ
Kang
Jung Julie
JJ
Vargas
Sarah L
SL
Peti-Peterdi
János
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004727:Endothelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005919:Glomerular Filtration Rate / Q000502:physiology; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D007606:Juxtaglomerular Apparatus / Q000502:physiology; D007678:Kidney Glomerulus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D034741:RNA, Small Interfering / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism; D012083:Renin / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D000097003:Gap Junction alpha-5 Protein
D000255:Adenosine Triphosphate; D002118:Calcium; D017630:Connexins; D006034:Glycyrrhetinic Acid; D020033:Protein Isoforms; D058919:Purinergic P2 Receptor Antagonists; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D012083:Renin; D013498:Suramin; D000097003:Gap Junction alpha-5 Protein