The role of constitutive PKA‐mediated phosphorylation in the regulation of basal I_Ca in isolated rat cardiac myocytes

Abstract

1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca 2 þ current (I Ca ) observed in the absence of exogenous b-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I Ca in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 mmol l À1) increased basal I Ca (Po0.05), whereas H-89 inhibited I Ca in a concentration-dependent manner with an IC 50 B5 mmol l À1. H-89 also inhibited the response to 1.0 mmol l À1 isoprenaline, although relatively high concentrations (30 mmol l À1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 mmol l À1 H-89 on time-dependent recovery of I Ca from voltage-dependent inactivation as well as the steady-state gating of I Ca . T 0.5 (time for I Ca to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (Po0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of I Ca were unaffected by 10 mmol l À1 H-89 or 1 mmol l À1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V 0.5 for activation and inactivation became more negative. 5 Data show that basal I Ca is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied b-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to b-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

Reduced density and altered regulation of rat atrial L-type Ca2+current in heart failure

“…Although both PKA and CaMKII have previously been shown to contribute to the basal regulation of ventricular I CaL , particularly at the t-tubule membrane (7)(8)(9)12), basal I CaL in atrial myocytes from either sham control or failing hearts in the present study was insensitive to inhibition of PKA or CaMKII. Notably, the absence of regulation of basal atrial I CaL by PKA in CAL hearts in the present study contrasts markedly with the increased PKA-dependent regulation of basal I CaL at the t-tubule of ventricular myocytes reported recently from the same hearts (9).…”

“…The reduction in atrial I CaL in coronary artery ligation-induced heart failure has been suggested to be due to decreased cAMP-dependent basal regulation of L-type Ca 2ϩ channels (4, 19). However, although the cAMP-dependent protein kinase, PKA, has also been demonstrated to play an important role in the basal regulation of I CaL in ventricular myocytes (7,8), the constitutive regulation of ventricular I CaL by PKA appears to be increased in a coronary artery ligation model of heart failure (9). In addition, the reduction in atrial I CaL in patients with chronic AF has been associated with a reduction in calciumcalmodulin-dependent protein kinase II (CaMKII) activity and an increase in protein phosphatase activity in AF with…”

Complex modulation of L-type Ca2+ current inactivation by sorcin in isolated rabbit cardiomyocytes

“…The molecular mechanism underlying this effect is unclear; di-hydropyridine (DHP) agonists such as Bay-K-8644 cause enhanced inactivation by a shift in single channel gating in favor of longer single channel open times without alteration to channel amplitude (mode two gating) [27,28]. Alternatively, stimulation of the β-adrenergic pathway causes a more negative shift in the current-voltage relationship and alters the CII/VDI in a similar fashion to sorcin [29,29]. This is also thought to be due to a shift in single channel gating to one with longer open times and hence higher open probability [30].…”

Local Glutamate Level Dictates Adenosine A2AReceptor Regulation of Neuroinflammation and Traumatic Brain Injury

“…To elucidate the signaling pathway associated with A 2A R action, 10 M N-[2-[[3-(4- GF109203X) was added to microglial cultures 30 min before glutamate was added. At these doses, H-89 (IC 50 , 5 M) (Bracken et al, 2006) and GF109203X (IC 50 , 1-2 M) (Cobine et al, 2007) can exert the inhibitory effect on protein kinase A (PKA) or protein kinase C (PKC) substrate phosphorylation and related cellular functions, respectively. All reported values were presented as means Ϯ SEM of three independent experiments conducted in triplicate.…”