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

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.


Introduction
The Na + -K + pump is a ubiquitous plasma membranebound enzyme that transports three Na + for every two K + into the cell by hydrolyzing ATP. Functional Na + -K + pumps contain a catalytic α-subunit and a glycosylated β-subunit. The α-subunit alone binds Na + , K + , ATP, and cardiac glycosides. At present, four isoforms of the α-subunit (α 1 -α 4 ) have been identified, and each has a unique tissue distribu tion [1,2] . In guinea pig (Cavia sp.) ventricular myocytes, only the α 1and α 2 -isoforms, which correspond to the low-and highaffinity isoforms for cardiac glycosides, respectively, are expressed [3,4] .
The Na + -K + pump is subject to regulation by a variety of hormones or transmitters, including catecholamines, aldosterone, insulin, angiotensin, thyroid hormone and adenosine [5][6][7][8][9] . For instance, Alzamora et al [5] demonstrated that aldosterone has a non-genomic effect on the Na + -K + pump of vascular tissue, which is mediated by PKC activation. In patch-clamped guinea pig ventricular myocytes, Gao et al [4] showed that the high DHO-affinity pump current (I h ) is regulated by α-adrenergic agonists via a PKC-dependent pathway, whereas the low DHO-affinity pump current (I l ) is regulated by β-adrenergic agonists via a PKA-dependent pathway.
Adenosine, a purine nucleoside, is widely distributed in all tissues and body fluids. It is well known that adenosine exerts its cardiovascular effects by interacting with four types of G-protein coupled receptors (A 1 R, A 2A R, A 2B R, A 3 R) [10] . Considering that the regulatory effect of adenosine is a receptor-mediated process that involves the activation of PKA and PKC [10][11][12] , one would expect to find a potential regulatory effect of adenosine on the Na + -K + pump. Indeed, several recent studies have been carried out to address this issue. However, these studies have yielded disparate results. Caruso-Neves et al [6] demonstrated that in the Malpighian tubule cells of the blood-sucking bug Rhodnius prolixus, adenosine inhibited the Na + -K + pump through interaction with the A 1 R. Krumschnabel et al [13] have also reported adenosine A 1 R mediated Na + -K + pump inhibition in the hepatocytes of goldfish (Carassius auratus) and trout (Oncorhynchus mykiss). These findings together suggest that the A 1 R may play a role in adenosine-induced Na + -K + pump inhibition. In contrast, Darlington et al [14] found an ultra-filtrate in plasma from mammalian species (dog, rat, calf) that could stimulate the Na + -K + pump activity. They identified the stimulator to be adenosine and suggested that the effect of adenosine on the Na + -K + pump was independent of adenosine receptors.
Given the lack of uniformity of these observations described above, the present study was designed to ascertain the effects of adenosine on the Na + -K + pump in guinea pig ventricular myocytes and to further clarify the potential underlying mechanisms involved.
Electrophysiology Cells were placed in the 0.3 mL superfusion chamber mounted on the stage of an inverted microscope (Nikon TE2000-S), allowed to attach to its glass bottom, and then superfused with the extracellular solution containing (mmol/L): 137.7 NaCl, 2.3 NaOH, 5.4 KCl, 1 MgCl 2 , 5 Hepes, 10 glucose, 2 BaCl 2 and 1 CdCl 2 (pH adjusted to 7.4 with NaOH). The chamber was perfused at a rate of about 2 mL/min and the solution exchange was complete within 2 min. The holding current was recorded using a whole-cell, patch-clamp technique and amplified using an Axopatch 700B amplifier (Axon Instruments). The sampling rate was 200 ms/point, and the data were low-pass filtered at 2 Hz. Patch electrodes were pulled with a Flaming/Brown micropipette puller (Sutter Instruments) and fire-polished to a final resistance of 1-3 MΩ when filled with the standard pipette solution, which contained (mmol/L) 50 sodium aspartic acid, 20 potassium aspartic acid, 30 CsOH, 20 TEACl, 5 MgSO 4 , 5 Hepes, 11 EGTA, 10 glucose, 5 Na 2 -ATP, 1 CaCl 2 (pH adjusted to 7.2 with CsOH). Solutions were designed to minimize all other components of membrane current (K + currents were blocked by replacing pipette K + with Cs + and TEA + and adding Ba 2+ to the extracellular solution; Ca 2+ channel and Na + -Ca 2+ exchanger currents were inhibited by including 1 mmol/L CdCl 2 in the extracellular solution). Under these conditions, I p was defined as the difference in currents before and after the addition of DHO, a specific and reversible inhibitor of the Na + -K + pump. We voltage-clamped the myocytes to 0 mV, a saturating voltage for the Na + -K + pump. For each experiment, both control and test I p were obtained from the same cell to avoid cell-tocell variability. For measurement of the voltage dependence of I p , a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used in some experiments. This protocol was applied to each myocyte at least three times: (1) under control conditions, (2) during the adenosine effect on I p and (3) after the Na + -K + pump was blocked with DHO. The I p values were obtained by digital subtraction of the membrane current in the presence of DHO from that in its absence at each test potential. All recordings were carried out at room temperature (22-25 o C) and data acquisition was achieved using pClamp 9.0 software.
Drugs The drugs used in these studies and their abbreviations include CCPA, DPCPX, CGS-21680, Cl-IB-MECA, staurosporine (St), H89, adenosine (Ado), GF 109203X, MRS1191, dihydroouabain (DHO), Gö-6976, rottlerin, PP114, and SCH58261. All chemical reagents were purchased from Sigma Chemical Co (St Louis, MO, USA). Adenosine and DHO were dissolved in deionized water, and all other chemicals were dissolved in dimethylsulfoxide (DMSO) to prepare stock solutions that were stored at -20 o C. The final concentration of DMSO never exceeded 0.1%, which produced no detectable effect on I p .
Data analysis and statistics The data were analyzed with Clampfit 9.0 (Axon Instruments) and Origin 7.0 (Originlab Corporation) software. All values are presented as means±SEM. Statistical analysis of differences between two groups was carried out using Student's paired t-test. Two-way ANOVA was performed to determine significance between the voltage dependence curves. A value of P<0.05 was considered statistically significant.

Results
Adenosine specifically inhibits I h in guinea pig ventri cular myocytes I l (α 1 -isoform related I p ) and I h (α 2 -isoform related I p ) are distinguishable by their different sensitivities to cardiac glycosides, with 5 µmol/L DHO blocking I h and 1 mmol/L DHO blocking I l [3,16] . Experiments were carried out to determine the effects of adenosine on each of these two isoforms.
First, we examined the effect of adenosine on I h . Figure 1A illustrates that a physiological concentration of adenosine (1 nmol/L) [17,18] decreased I h from 16.1±0.3 pA (I h (Con) ) to 9.9±0.7 pA (I h (Ado) ) by 39% (n=8, P<0.05, upper panel). The decrease in I p was not due to pump "rundown" because the adenosine effect was reversible upon washout (lower panel of Figure 1A). Figure 1B shows that adenosine inhibited I h in a concentration-dependent manner from 1×10 -11 to 1×10 -5 mol/L (8% to 47%). Adenosine (1×10 -8 mol/L) caused maximal inhibition. No significant effect was observed at concentrations of adenosine below 1×10 -11 mol/L. The mean data from five to seven  Figure 1C, where the percentage inhibition of I h is plotted against the concentration of adenosine.
Next, we investigated the effect of adenosine on I l . The entire experiment was performed in the presence of 5 µmol/L DHO to block I h , and I l was measured following the application of 1 mmol/L DHO. Figure 2A shows that adenosine did not change I l significantly [113.8±0.9 pA for I l (Ado) , P>0.05 vs 115.4±0.7 pA for I l (Con) , n=7]. In addition, increasing adenosine to 10 µmol/L had no effect on I l [91.2±0.8 pA for I l (Ado) , P>0.05 vs 94.4±0.9 pA for I l (Con) , n=10, Figure 2B]. These results indicate that the inhibitory effect of adenosine on the Na + -K + pump current is specifically mediated via the Na + -K + pump α 2 -isoform, so our subsequent studies were focused primarily on the effect of adenosine on I h .
The adenosineinduced inhibition of I h is voltage independent Because I p in guinea pig ventricular myocytes is voltage dependent [16] , we went further to examine the effect of adenosine on the I h -V m relationship. Figure 3A shows the voltage-ramp protocol applied to the myocytes. The relationships were normalized to the I h recorded at 0 mV to facilitate comparison of their slopes, which are summarized in Figure 3B. The normalized I h was generally lower in the presence of adenosine than that in its absence. The difference between these slopes was not statistically significant (n=6, P>0.05; two-way ANOVA). Thus, adenosine-induced I h inhibition is voltage independent.
Inhibition of I h by adenosine is mediated by A 1 recep tors All four adenosine receptor subtypes are expressed in guinea pig ventricular myocytes, among which A 1 R is consid-ered predominant [19,20] . Thus, we tested whether adenosine inhibition of I h was mediated by A 1 R. Figure 4A indicates that DPCPX (10 nmol/L), a selective A 1 R antagonist, had no effect on I h by itself (upper panel), but completely abolished adenosine-induced I h inhibition [7.8±0.5 pA for I h (Ado) , P<0.05 vs 14.7±0.3 pA for I h (Con) , and 14.1±0.6 pA for I h (Ado+DPCPX) , P>0.05 vs 14.7±0.3 pA for I h (Con) , n=8, lower panel]. In addition, CCPA (10 nmol/L), a selective agonist for A 1 R, produced a marked inhibition of I h [7.8± 0.4 pA for I h (CCPA) , P<0.05 vs 15.7±0.3 pA for I h (Con) , n=9, Figure 4B]. In addition, the CCPA effect was absent in the presence of DPCPX (data not shown). These results strongly suggest that the effect of adenosine was the result of stimulation of the adenosine A 1 R.
To investigate the possible participation of other adenosine receptor subtypes, the A 2A R and A 3 R selective agonists CGS21680 and Cl-IB-MECA, respectively, were tested. As shown in Figure 4C Figure  4D]. We also observed that perfusion of adenosine, together with SCH58261 and MRS1191 (0.1 µmol/L each), the A 2A R and A 3 R selective antagonists, respectively, did not alter the adenosine effect on I h [11.8±0.6 pA for I h (Ado) , P<0.05 vs 22.1±0.5 pA for I h (Con) , and 10.3±0.8 pA for I h (Ado+SCH58261+MRS1191) , P<0.05 vs 22.1±0.5 pA for I h (Con) , n=9] ( Figure 4E). It is, therefore, highly unlikely that A 2A R and The activation of PKCδ inhibits I h PKC is stimulated by A 1 R activation in ventricular myocytes [11,21] . Therefore, we examined the effect of adenosine in the presence of staurosporine, a well-characterized PKC inhibitor. Perfusion of 1.5 µmol/L staurosporine alone did not modify I h (data not shown) but completely abolished adenosine-induced I h. inhibition [10.5±0.6 pA for I h (Ado) , P<0.05 vs 19.8±0.5 pA for I h (Con) , and 18.8±0.9 pA for I h (Ado+St) , P>0.05 vs 19.8±0.5 pA for I h (Con) , n=10, upper panel of Figure 5A]. Similar results were also obtained with GF 109203X, a highly specific PKC inhibitor. Adenosine did not inhibit I h in the presence of 1 µmol/L GF 109203X [11.9±0.6 pA for I h (Ado) , P<0.05 vs 22.4±0.8 pA for I h (Con) , and 20.3±0.9 pA for I h (Ado+GF 109203X) , P>0.05 vs 22.4±0.8 pA for I h (Con) , n=8, lower panel of Figure 5A]. This reinforces the view that adenosine inhibits I h through a PKC-dependent mechanism.
Because adult cardiomyocytes express multiple PKC isoforms [22] , we asked which one might be involved. We initially explored the role of the classical PKC isoforms (PKC-α and β) using the inhibitor Gö-6976. The upper panel of Figure 5B shows that adenosine still inhibited I h in the presence of 100 nmol/L Gö-6976 [14.5±0.6 pA for I h (Ado) , P<0.05 vs 27.4±0.8 pA for I h (Con) , and 12.8±0.9 pA for I h (Ado+Gö-6976) , P<0.05 vs 27.4±0.8 pA for I h (Con) , n=8]. This indicates that classical PKC isoforms are not involved. Given the prominent role of PKC-δ during A 1 R activation by adeno sine [23,24] , we next examined the effect of rottlerin, a specific PKC-δ inhibitor, on the adenosine effect. Inhibition of PKC-δ with 10 µmol/L rottlerin completely abolished the effect of adenosine [12.9±0.6 pA for I h (Ado) , P<0.05 vs 24.4±0.8 pA for I h (Con) , and 22.3±0.8 pA for I h (Ado+rottlerin) , P>0.05 vs 24.4±0.8 pA for I h (Con) , n=8, middle panel of Figure 5B], indicating that PKC-δ is required for the adenosine effect on I h . To confirm this, a specific activator peptide of PKC-δ, PP114, was used. Similar to adenosine, PP114 (200 nmol/L) caused a marked decrease in I h [10.8±0.7 pA for I h (PP114) , P<0.05 vs 22.7±0.6 pA for I h (Con) , n=9, lower panel of Figure 5B]. The PP114 effect was also blocked by GF 109203X or rottlerin (data not shown). Taken together, these results indicate that PKC-δ plays a crucial role in the inhibition of I h by adenosine.
To exclude the possible involvement of PKA in the adenosine-mediated I h inhibition, the effect of adenosine was examined in the presence of H89, an inhibitor of PKA. Figure 5C shows that inhibition of PKA with 1 µmol/L H89 failed to abolish the effect of adenosine on I h [11.0±0.6 pA for I h (Ado) , P<0.05 vs 20.7±0.4 pA for I h (Con) , and 10.9±0.8 pA for I h (Ado+H89) , P<0.05 vs 20.7±0.4 pA for I h (Con) , n=8]. Thus, PKA appears not to mediate I h inhibition by adenosine.

Discussion
Numerous studies over the years have highlighted the isoform-specific modulation of Na + -K + pumps by transmitters or kinases in many types of cells [4,25,26] . In this study, we found that the α 2 -isoform Na + -K + pumps are specifically inhibited by adenosine. This implies a link among a specific adenosine receptor, its associated kinases, and inhibition of I h in ventricular myocytes. In light of this, the goal of this study was to identify potential mechanisms by which adenosine exerts its effects on I h.
We first examined which type of adenosine receptor is involved in this phenomenon. Considering that the adenos-   ine concentration we used is close to that described for the high-affinity A 1 R (0.5-100 nmol/L) [27] , we speculate that the inhibitory effects of adenosine on the Na + -K + pump are most likely mediated via the A 1 R. Indeed, using selective AR agonists and antagonists, we have demonstrated a specific role for A 1 R in adenosine-mediated I h inhibition, whereas A 2A R and A 3 R are not involved, in accordance with earlier studies using R prolixus and C auratus [6,13] . There are two possible explanations for the above results. First, the A 2A R is coupled to the cAMP-PKA pathway [28,29] , which is targeted to the α 1 -isoform of the Na + -K + pump [4] . Hence, A 2A R activation could not lead to any change in I h . A second possibility that may be pertinent to our results is the absence of functional A 2A R or A 3 R proteins in cardiac myocytes [10,30] . These two lines of evidence completely rule out the involvement of A 2A R and A 3 R in the present study.
We next examined the possible mechanism(s) underlying I h inhibition by A 1 R stimulation. Binding of adenosine to A 1 R inhibits adenyl cyclase and stimulates PKC via activation of the pertussis toxin sensitive G proteins G i and/or G o [28,29] . Using selective antagonists for PKC and PKA, we observed that the adenosine A 1 R triggers the PKC pathway to inhibit I h , but the cAMP-PKA pathway is not involved. Specifically, this inhibition is predominantly mediated by the novel PKC-δ isoform. Our results are consistent with those of Gao et al [4] , who demonstrated that I h was specifically regulated by PKC. However, in their study, I h was increased by α-adrenoceptor stimulation via the PKC pathway, which is not congruent with our result showing a PKCδ-mediated decrease in I h . The most likely explanation for this discrepancy is the stimulation of different PKC isoforms by α-adrenoceptor activation. Indeed, we observed that α-adrenoceptor activation increases I h in a PKC-β dependent manner using the inhibitor LY333531, which substantiates our results (data not shown). Taken together, these observations strongly suggest that adenosine-induced I h inhibition is mediated by the PKC-δ isoform. The exact mechanisms for PKC-δ-mediated inhibition of I h require further study, however, it most likely involves a phosphorylation-dependent process. In this case, PKC-δ may directly phosphorylate the pump protein to induce conformational changes, thus decreasing the turnover rate of each pump [1,31] . In addition, the recent observation that phospholemman (FXYD1) associates with the cardiac Na + -K + pump [32] offers another subunit that may confer sensitivity to PKC-δ.
In conclusion, the major findings are that adenosine inhibits I h via activation of A 1 R and PKC-δ. This finding may have implications for our understanding of the antiarrhythmic effect of adenosine when used clinically. The inhibition of Na + -K + pump prolongs action potential duration and myocardial refractoriness, which is involved in the mechanisms of two other widely used antiarrhythmic agents, bretylium and amiodarone [33,34] . Thus, it is tempting to speculate that the antiarrhythmic effects of adenosine are in part caused by Na + -K + pump inhibition.