Atrial myocytes are exposed to shear stress during cardiac cycle and hemodynamic disturbance. It is known that shear stress elicits longitudinal global Ca2+ wave (“L wave”) in atrial myocytes (1). Here, we investigated cellular mechanisms for shear-mediated Ca2+ response in atrial myocytes using confocal Ca2+ imaging (30 Hz). We applied shear stress to single myocytes using pressurized micro flow system. Atrial myocytes were enzymatically isolated from male Sprague-Dawley rats (230-300 g) and from wild-type (WT) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R) knockout (KO) mice (C57/B6, 24-28 g) (anesthesia: pentobarbital sodium, 150 mg kg-1, i.p.). Shear stress of ~16 dyn cm-2 aperiodically induced L wave (79±7.7 μm s-1) 1.2±0.26 times for 8 s-long exposure (n=39), with a delay of 0.2-3 s. Shear-induced L wave was restituted after 3-4-min resting period after the first occurrence. Values are means ± S.E.M. of wave events occurring for 8 s-long shear, compared by student t test. Blockade of ryanodine receptor (RyR) (zero for 1 mM tetracaine vs. 1.0±0 for control, n=5, p<0.0001) or IP3R abolished the L wave occurrence under shear (zero for 3 μM 2-APB vs. 1.4±0.8 for control, n=8, p<0.001). In type 2 IP3R KO cells, shear stress failed to induce L wave (WT, 1.8±0.34, n=13 vs. KO, zero, n=17, p<0.0001). Consistent with these results, inhibition of phospholipase C (PLC) using U73122 (5 μM) removed shear-induced L wave (0.13±0.13 vs. 1.1±0.26 for control, n=8, p<0.01), although its inactive analogue U73343 (5 μM) did not affect it (1.25±0.25 vs. 1.0±0 for control, n=4, p>0.05). These observations indicate that PLC-IP3-IP3R signaling and Ca2+-induced Ca2+ release via RyRs play a role in the generation of L wave under shear. Pre-treating atrial cells with the blockers for stretch-activated channel, TRPM4 or NADPH oxidase did not alter the occurrence of L wave under shear. Suramin (10 μM), the inhibitor of purinergic receptor, suppressed the L wave occurrence under shear stress (zero vs. 1.2±0.50 for control, n=4, p<0.05). Antagonist of P2Y1 receptor MRS2179, but not P2X receptor antagonist (iso-PPADS), eliminated the L wave generation under shear (control, 1.0±0 vs. 200 nM MRS2179, 0.25±0.25, n=4, p<0.05; control, 1.0±0 vs. 10 μM iso-PPADS, 0.83±0.17, n=6, p>0.05). Suppression of connexon that releases ATP using carbenoxolone (50 μM; zero vs. 1.2±0.17 for control, n=6, p<0.001), or extracellular application of apyrase (2 U ml-1) that metabolizes ATP inhibited the occurrence of L wave under shear (zero vs. 1.8±0.58 for control, n=5, p<0.05). Our data suggest that longitudinal Ca2+ wave is triggered by type 2 IP3R-mediated Ca2+ release that is activated by connexon-mediated ATP release and subsequent activation of P2Y1 receptor-PLC signaling in atrial myocytes under shear stress.