Isoform-specific regulation of the Na+ -K+ pump by adenosine in guinea pig ventricular myocytes

Abstract

Aim: The present study investigated the effect of adenosine on Na(+)-K(+) pumps in acutely isolated guinea pig (Cavia sp.) ventricular myocytes.

Methods: The whole-cell, patch-clamp technique was used to record the Na(+)-K(+) pump current (I(p)) in acutely isolated guinea pig ventricular myocytes.

Results: Adenosine inhibited the high DHO-affinity pump current (I(h)) in a concentration-dependent manner, which was blocked by the selective adenosine A(1) receptor antagonist DPCPX and the general protein kinase C (PKC) antagonists staurosporine, GF 109203X or the specific delta isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A(1) receptor agonist CCPA and a specific activator peptide of PKC-delta, PP114. In contrast, the selective A(2A) receptor agonist CGS21680 and A(3) receptor agonist Cl-IB-MECA did not affect I(h). Application of the selective A(2A) receptor antagonist SCH58261 and A(3) receptor antagonist MRS1191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A (PKA) antagonist, did not exert any effect on adenosine-induced I(h) inhibition.

Conclusion: The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of I(h) via adenosine A(1) receptors and the PKC-delta isoform.

Figure 1

Adenosine inhibits I_h in guinea pig ventricular myocytes. (A) Upper panel: a typical trace showing the effect of 1 nmol/L adenosine on I_h. The lower and upper horizontal lines indicate the application of 5 μmol/L DHO and an adenosine-containing solution, respectively. The vertical bar illustrates the measured I_h amplitude. Lower panel: a typical trace showing that the adenosine effects on I_h were not due to pump “run-down”. (B) Representative traces of the effect of adenosine (1×10⁻¹¹−1×10⁻⁵ mol/L) on I_h. (C) The percentage inhibition of I_h was plotted for each concentration of adenosine used. The error bars indicate means±SEM.

Figure 2

Adenosine has no effect on I_l in guinea pig ventricular myocytes. (A) A typical trace showing the effect of 1 nmol/L adenosine on I_l. The vertical bar illustrates the measured I_l amplitude. (B) A typical trace showing the effect of 10 μmol/L adenosine on I_l.

Figure 3

The voltage dependence of I_h is not shifted by adenosine. (A) Voltage-ramp protocol applied to myocytes. (B) Normalized current-voltage relationship of I_h in the presence (•) and absence (▪) of adenosine. The error bars indicate means±SEM. The lower panel graphs the ratio of I_{h (Ado)}/I_{h (Con)}.

Figure 4

Adenosine-induced inhibition of I_h is mediated by adenosine A₁R, but not by A_2AR or A₃R. (A) Upper panel: a typical trace showing the effect of the A₁R selective antagonist DPCPX (10 nmol/L) alone on I_h. Lower panel: a typical trace in the left panel showing the effect of adenosine on I_h in the presence and absence of DPCPX (10 nmol/L). The right panel shows a summary of the results. (B) A typical trace in the left panel showing the effect of the A₁R selective agonist CCPA (10 nmol/L) on I_h. The right panel shows a summary of the results. (C, D) Typical traces in the left panels showing the effects of the A_2AR and A₃R selective agonists CGS21680 (0.2 μmol/L) and Cl-IB-MECA (0.5 μmol/L), respectively, on I_h. The right panels show summaries of the results. (E) A typical trace in the left panel showing the effect of adenosine on I_h in the presence and absence of the A_2AR and A₃R selective antagonists SCH58261 and MRS1191 (0.1 μmol/L each). The right panel shows a summary of the results. The error bars indicate means±SEM. ^bP<0.05 vs controls.

Figure 5

PKC-δ is primarily involved in the inhibitory effect of adenosine on I_h, whereas PKA is not involved. (A) Typical traces in the left panels showing the effects of adenosine on I_h in the presence and absence of the general PKC antagonists St (1.5 μmol/L, upper panel) or GF 109203X (1 μmol/L, lower panel). The right panels show summaries of the results. (B) Upper panel: a typical trace in the left panel showing the effect of adenosine on I_h in the presence and absence of the PKC-α and β inhibitor Gö-6976 (100 nmol/L). The right panel shows a summary of the results. Middle panel: a typical trace in the left panel showing the effect of adenosine on I_h in the presence and absence of the PKC-δ inhibitor rottlerin (10 μmol/L). The right panel shows a summary of the results. Lower panel: a typical trace in the left panel showing the effect of the PKC-δ activator PP114 (200 nmol/L) on I_h. The right panel shows a summary of the results. (C) A typical trace in the left panel showing the effect of adenosine on I_h in the presence and absence of the PKA antagonist H89 (1 μmol/L). The right panel shows a summary of the results. The error bars indicate means±SEM. ^bP<0.05 vs controls.