= 5). B (Akt) Rabbit Polyclonal to CKI-epsilon signalling (Fulton 1999). Mechanical forces applied to cardiac muscle mass also stimulate NOi production (Pinsky 1997; Prendergast 1997; Vila Petroff 2001). For instance, sustained stretch of stimulated cardiac myocytes requires PI-(3)KCAkt signalling to activate eNOS-dependent NOi production (Vila Petroff 2001). Results from our previous work also show that in cat atrial myocytes, muscarinic (Dedkova 2003), 2-adrenergic (Wang 2002) and 1-adrenergic (Wang 2005) receptor activation requires PI-(3)KCAkt signalling to stimulate NOi production. In contrast to CaM-dependent activation of constitutive NOS, PI-(3)KCAkt signalling is usually Ca2+-impartial (Conus 1998; Dedkova 2003; Boo & Jo, 2003). These findings therefore RV01 raise the question of whether FS of cardiac myocytes stimulates NOi production entirely through a Ca2+-dependent process or whether Ca2+-impartial signalling via PI-(3)KCAkt also contributes to FS-induced NOi production. Therefore, the primary purpose of the present study was to determine whether a Ca2+-impartial PI-(3)KCAkt signalling mechanism activated by contractile activity, and acting in conjunction with Ca2+CCaM signalling, contributes to NOi production induced by electrical FS of ventricular myocytes. Part of this RV01 work has been published in abstract form (Dedkova 2004). Methods Adult cats of either sex were anaesthetized with sodium pentobarbital (50 mg kg?1, i.p.). Once fully anaesthetized, a bilateral thoracotomy was performed, and the heart was rapidly excised and mounted on a Langendorff perfusion apparatus. After enzyme (type II collagenase; Worthington Biochemical) digestion, ventricular myocytes were isolated as previously reported (Rubenstein & Lipsius, 1995). Animal protocols used were approved by the Institutional Animal Care and Use Committee of Loyola University or college of Chicago, Stritch School of Medicine, Maywood, IL, USA. Electrophysiological recordings from myocytes were performed using a perforated-patch (nystatin) whole-cell recording method, as previously explained (Rubenstein & Lipsius, 1995). CsCl (5 mm) was added to all external solutions to block K+ conductances. L-type Ca2+ current (1998; Nakatsubo 1998) as previously explained (Dedkova & Blatter, 2002; Wang 2002, 2005; Dedkova 2003). NOi measurements were performed at room heat. DAF-2 fluorescence was excited at 480 nm and emitted cellular fluorescence was recorded at 540 nm. Changes in cellular DAF-2 fluorescence intensity (= 4). Therefore, these mean values were used to correct [Ca2+]i transient amplitudes at each activation frequency for each drug RV01 tested. To confirm that these time-dependent changes in [Ca2+]i transients were due to photobleaching and/or loss of Ca2+ indication, we performed additional selected experiments with indo-1/AM, a ratiometric dye for which [Ca2+]i measurements are not affected by changes in dye concentration. The results from indo-1 experiments were not different from the corrected fluo-4 results (data not shown), confirming that photobleaching and/or loss of dye were responsible for the time-dependent decreases in [Ca2+]i transients. Cell shortening of myocytes during FS was decided simultaneously from collection scan images. [Ca2+]i transients also were measured using indo-1/AM, as previously explained (Wang 2003). Myocytes were loaded with Ca2+ indication by exposure to 5 m indo-1/AM in 1 ml Tyrode answer made up of 0.001 g ml?1 of Pluronic F-127 for 10 min at room temperature. Cells were washed for 10 min to allow de-esterification of the indication. For spatially averaged single cell [Ca2+]i measurements, indo-1 fluorescence was excited at 357 nm and cellular fluorescence was recorded simultaneously at 405 nm (= 2000). A control adenovirus expressing nuclear-encoded -galactosidase (Adv-gal) was used to control for nonspecific effects of adenoviral contamination (Heidkamp 2001). Adenoviruses.