Elevated cAMP Suppresses Muscarinic Inhibition of L-Type Calcium Current in Guinea Pig Ventricular Myocytes

Abstract

We investigated the effect of carbachol (CCh) on L-type Ca2+ current (ICa(L)) enhanced by dialyzed adenosine 3',5'-cyclic monophosphate (cAMP) and/or bath-applied 3-isobutyl-1-methylxanthine (IBMX) in guinea pig isolated ventricular myocytes. At pipette concentrations ([cAMP]pip) from 30 microM to 1 mM, cAMP increased ICa(L) to 25.8 +/- 0.9 microA/cm2 (682 +/- 24.8% increase above control). CCh (100 microM) did not inhibit ICa(L) at any [cAMP]pip. IBMX, a nonselective phosphodiesterase (PDE) inhibitor, increased ICa(L) maximally at 300 microM IBMX (17.9 +/- 0.7 microA/cm2; 449 +/- 20% increase). CCh (100 microM) inhibited ICa(L) by 92 +/- 9.5% at 30 microM IBMX and 78 +/- 4.6% at 100 microM IBMX; this effect was reduced or absent at higher IBMX concentrations (300 and 1,000 microM). Coadministration of cAMP and IBMX also progressively suppressed inhibition by CCh. CCh had a negligible effect on ICa(L) at 750 microM IBMX in the absence of pipette cAMP and at 50 microM IBMX in the presence of 100 microM [cAMP]pip. ACh-activated K+ current (IK(ACh)) was unchanged in atrial myocytes dialyzed with 100 microM cAMP; this excludes a phosphorylation-dependent desensitization of the muscarinic receptor (mAChR) or Gi by cAMP. LY83583 (100 microM), an inhibitor of cyclic guanosine monophosphate (cGMP) production, attenuated inhibition of ICa(L) by CCh in the presence of IBMX. 8-Bromo-cGMP (8-Br-cGMP), an activator of cGMP-dependent protein kinase (PKG), mimicked CCh in its actions on ICa(L) raised by both cAMP (no significant change) and IBMX (49 +/- 5.1% inhibition). Okadaic acid, an inhibitor of type 1 and 2A phosphatases, blocked inhibition of IBMX-stimulated ICa(L) by either CCh or 8-Br-cGMP. Thus the ability of CCh to inhibit ICa(L) appears caused by cGMP/PKG activation of an okadaic acid-sensitive protein phosphatase, and elevated levels of cAMP protect against this action.

Signal Transduction and Ca 2+ Signaling in Contractile Regulation Induced by Crosstalk Between Endothelin-1 and Norepinephrine in Dog Ventricular Myocardium

“…44 -47 The muscarinic inhibition could occur without a concomitant decrease in the cAMP content or in the PKA activity. 45,46 Our observation that an inhibitor of PP, cantharidin, abolished the NIE of ET-1 is consistent with the results of previous investigations described above. In this context, it is noteworthy that the NIE of ET-1 is more susceptible to cantharidin than the NIE of carbachol and the PIE of NE mediated by ␤-adrenoceptors, 48 an indication that the differential effect of cantharidin on various signaling processes is exerted in dog ventricular myocardium.…”

On the Role of Phosphatase in Regulation of Cardiac L-Type Calcium Current by Cyclic GMP

“…This is in accordance with the evidence that muscarinic agonist inhibits I Ca(L) by activating okadaic acid-sensitive PP and that cGMP is the signal molecule for this action (Imai et al, 2001), presumably by phosphorylating PP (Sakai et al, 1999). Similarly, ACh opposed the action of ISO or dibutyryl-cAMP on the inwardly rectifying K ϩ current by activation of okadaic acid-sensitive PP in guinea pig ventricular myocytes (Koumi et al, 1995).…”

“…Okadaic acid inhibits PP1 and PP2A with the latter being more sen- sitive (Herzig and Neumann, 2000). Okadaic acid (300 nM in pipette solution) prevented inhibition of I Ca(L) by 8-Br-cGMP (Sakai et al, 1999). That okadaic acid inhibits membrane bound PP1 and PP2A activities in guinea pig ventricular myocytes (Neumann et al, 1993) indicates that neither enzyme can be excluded from participating in I Ca(L) inhibition.…”

“…Okadaic acid, an inhibitor of PP, prevented dephosphorylation of proteins (Gupta et al, 1994) and inhibition of isoproterenol (ISO)-stimulated I Ca(L) by ACh . The phosphatase hypothesis was strengthened by evidence that inhibition of I Ca(L) by carbachol (CCh) or 8-Br-cGMP was antagonized by dialysis with 300 nM okadaic acid (Sakai et al, 1999). As such, the results resemble those seen in GH 4 C 1 cells where atrial natriuretic peptide increased a maxi-K channel current by activating a phosphatase through a cGMP/PKG pathway (White et al, 1993).…”

The Many Faces of H89: A Review

“…Myocardial ischemia is characterized by accumulation of cAMP and activation of PKA (65), which in turn leads to increased opening of the L-type calcium channels and resultant increase in intracellular calcium (54). This chain of events plays a role in eliciting reperfusion dysrhythmias and contributes to necrosis (so-called reperfusion injury).…”