| The Na~+-K~+ pump (also known as sodium pump or Na+, K+-ATPase) is a member of P-ATPase family and present in all animal cells. The Na~+-K~+ pump uses an ATP to transport 3 Na+ out of cells and 2 K+ into the cell and provides energy for several essential cellular functions (control of cell volume, pH homeostasis, reabsorption of nutrition). In excitable tissues (heart and nerve, etc), the Na~+-K~+ pump function is crucial for maintaining and establishing the normal electrical activity.It has been demonstrated that the Na~+-K~+ pump function is subjected to regulation by a variety of hormones and biochemical changes associated with pathological conditions. For instance, catecholamines, insulin, angiotensin, and thyroid hormone mainly modulate the activity of Na~+-K~+ pump through interaction with receptors located in the plasma membrane and stimulation of downstream signaling pathways. In addition, myocardial ischemia, a leading cause of death and disability in deveoleped contries, leads to a marked inhibition of Na~+-K~+ pump. Thus, delineating detailed modulation of Na~+-K~+ pump by endogenous signaling molecules and biochemical changes is important for better understanding the exact and precise role of Na~+-K~+ pump play in various physiological and pathological conditions.Many endogenous signaling molecules (bradykinin, adenosine, noradrenaline, etc) have been found to confer some degree of protection against myocardial ischemia. Of these, adenosine is perhaps the single most widely studied"cardioprotectnant". Adenosine is present at low concentration and can be released as a result of metabolic stress (ischemia and hypoxia, etc). Adenosine exerts its effects through adenosine A1, A2A, A2B, and A3 receptor subtypes (A1R, A2AR, A2BR, and A3R), which are all expressed in myocardial and vascular cells, and coupled to G proteins to trigger a range of responses, with ultimate effects of limitation of cellular death and dysfunction. The nucleoside mediates tissue protection by different mechanisms including increased oxygen supply/demand, ischemic preconditioning and stimulation of angiogenesis, and also as a paracrine inhibitor of inflammation with effects in the lung, heart and brain. However, as an active transporter maintaining the essential cellular functions, it is not known whether Na~+-K~+ pump is subjected to modulation by endogenous adenosine. In addition, the possible involvement of Na~+-K~+ pump in the cardioprotection of adenosine during ischemia is also not known.Therefore, using the whole-cell patch-clamp technique, the present study aimed to address the following questions: (1) whether there lies a possible modulatory effect of adenosine on the Na~+-K~+ pump under normal conditions; (2) whether adenosine could abolish the inhibition of Na~+-K~+ pump induced by ischemia; (3) the exact and presice mechaniam of ischemia-mediated Na~+-K~+ pump injury.Part 1 The regulation of Na~+-K~+ pump current by adenosine in normal guinea pig ventricular myocytesObjective: To study the effect of adenosine on Na~+-K~+ pump in ventricular myocytes acutely isolated from guinea pigs and to further elucidate the possible mechanisms involved.Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes: guinea pig heart was digested by aorta retrograde perfusion with Collegnase NB8 (from Serva Chemical Co) using Langendorff apparatus. The temperature, pH and water quality should be properly controlled in the process of digestion. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip): the Na~+-K~+ pump exchanges 3 intracellular Na+ for 2 external K+ across the cell membrane during each active transport process. This excess positive charge movement generates a net outward current (Ip). With selected external and pipette solutions, membrane currents through K+ channel, Ca2+ channel, and Na+-Ca2+ exchanger were minimized. Under the experimental conditions, Ip was measured as the DHO-blocked current. Guinea pig ventricular myocytes coexpress two distinct Na~+-K~+ pumpαisoform,α1 andα2, which correspond to the low- and high-affinity isoforms for cardiac glycosides (dissociation constants for DHO are 72μM and 0.75μM, respectively). Thus, total Ip is the sum of current contributed by theα1-isoform (Il) plus that by theα2-isoform (Ih): Ip=Il+Ih. Based on the different affinities for cardiac glycosides (DHO at 1 mM blocked roughly 96% of total Ip, and that 5μM DHO blocked 89% of Ih while blocking only 9% of Il), we used 1 mM and 5μM DHO to identify Il and Ih, respectively. (3) Measuring of the adenosine effect on Ip: when we studied the adenosine effect on Ip, the standard protocol was to measure pump current in control and test conditions in the same cell, and then to calculate the ratio of test to control current and average this ratio from at least five cells. The detailed method is described as following: after whole-cell recording was initiated, a period of at least 5 min was required for the pipette and intracellualr solutions to come to steady state. When steady state was achieved, DHO was superfused to record the DHO-induced changes in holding current, which was reversed upon washout of DHO and was considered to reflect control Ip (Ip(Con)). Once the current had again stabilized, adenosine was applied to the myocytes. This caused a shift in holding current. After a new steady state was achieved, in the continous presence of adenosine, DHO was again applied and this inward shift reflect the changed Ip by adenosine (Ip(Ado)). Finally, Ip(Ado) was normalized to Ip(Con) to obtain the averaged ratio. This procedure uses each cell as its own control and removes the uncertainty due to cell to cell variation in size and pump density. (4) Mechanisms mediating the effect of adenosine on Ip: to clarify the cellular mechanisms responsible for the adenosine effect on Ip, specific adenosine A1R, A2AR, and A3R agonists (CCPA, CGS21680, Cl-IB-MECA, respectively), antagonists (DPCPX, SCH58261, MRS1191, respectively), PKC inhibitors(Staurosporine and bisindolylmaleimide I), and PKA inhibitor(H89)were used.Results: (1) Adenosine specifically inhibits Ih. These results show that a physiological concentration of adenosine (1 nM) decreased Ih by 39±0.04%. The decrease in Ip was not due to pump"rundown"because the adenosine effect was reversible upon washout. Adenosine did not change Il significantly; Il remained unchanged at 93±0.05% of control values (P>0.05). Also, increasing adenosine to 10μM, adenosine had no effect on Il, suggesting that adenosine specifically inhibits Ih. (2) Adenosine concentration-dependently inhibites Ih. Perfusion of adenosine from 10-11-10-5 M induced an appreciable (8% to 47%) concentration-dependent inhibition of Ih, and the inhibition was maximal at 10-8 M. (3) The adenosine-induced inhibition of Ih is voltage independent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used in some experiments. The relationships were normalized to the Ih recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was not statistically significant (P>0.05; two-way ANOVA). Thus, adenosine-induced Ih inhibition is voltage independent. (4) Adenosine binding to A1R triggers the inhibition of Ih. The specific A1R antangonist DPCPX (10 nM) abrogated the adenosine effect on Ih; Ih returned to 96±0.06% of control values (P>0.05). In a parallel study, we further tested A1R specificity by treating the cells with CCPA, a selective agonist for A1R. Perfusion of 10 nM CCPA produced a marked inhibition of Ih. Ih was inhibited by 50±0.04% in the presence of CCPA (P<0.05). Furthermore, treatment of cells with specific A2AR and A3R agonists, CGS21680 (0.2μM) and Cl-IB-MECA (0.5μM), respectively, did not affect Ih; Ih remained constant at 101±0.07% and 98±0.05% of control values (P>0.05), respectively. The specific A2AR and A3R antagonists, SCH58261 and MRS1191 (0.1μM each), did not alter the adenosine effect on Ih. Ih was decreased by 53±0.06% (P<0.05), which was not statistically different from 47±0.03% inhibition when compared to its absence (P>0.05). These data together suggest that the inhibitory effect of adenosine on Ih is mediated by adenosine A1R. (5) The activation of adenosine A1R stimulates the PKC pathway, thereby inhibiting Ih. The inhibitory effect of adenosine on Ih was abolished by PKC inhibitor staurosporine (St, 1.5μM). Current amplitudes returned to 95±0.05% of the initial control values (P>0.05). Again, 1μM Bis-I, a highly specific PKC blocker, clearly abrogated the inhibition of Ih by adenosine. The currents returned to 91±0.04% of control values after perfusion of Bis-I (P>0.05). In contrast, PKA inhibitor H89 did not affect the adenosine effect on Ih. Adenosine still induced 47±0.03% inhibition of Ih (P<0.05). Collectively, these results suggest that adenosine inhibits Ih through a PKC-dependent mechanism. (6) The activation of PKC-δinhibits Ih. Using the classical PKC isoforms (PKC-α,β,γ) inhibitor G?-6976 (100 nM), we show that the inhibitory effect of adenosine on Ih was not affected. Ih was still inhibited by 53±0.05% (P<0.05) in the presence of G?-6976. In addition, we tested the role of the novel PKC isoformδusing specific inhibitor rottlerin. Adenosine failed to inhibit Ih in the presence of 10μM rottlerin; Ih returned to 91±0.05% of control values (P>0.05), indicating that PKC-δis required for the adenosine effect on Ih. Like adenosine, PP114, a specific activator peptide of PKC-δ, also inhibited Ih. Ih was decreased by 52±0.04% on application of PP114 (P<0.05). Taken together, these results indicate that PKC-δplays a crucial role in the inhibition of Ih by adenosine.Conclusion: Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.Part 2 The regulation of Na~+-K~+ pump current by adenosine in ischemic guinea pig ventricular myocytesObjective: To delineate the Na~+-K~+ pump current during myocardial ischemia and to determine whether adenosine treatment could prevent the inhibition of Na~+-K~+ pump current during myocardial ischemia.Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors: metabolic inhibition (MI) is a prominent feature of myocardial ischemia. Thus, we used metabolic inhibitors FCCP and 2-DG to produce combined inhibition of oxidative and glycolytic metabolism.Results: (1) MI time-dependently inhibits Ip. MI reduced Ip in a time-dependent manner; its amplitudes were reduced by 30±0.05%, 49±0.02%, 62±0.04% (r=0.82, P<0.05) following 2.5, 4, and 6 min of MI, respectively. Increasing the time of MI to 10 min, however, did not produce any further reduction in Ip (67±0.02%, P>0.05), suggesting that a maximal effect was achieved at a MI time of 6 min. (2) Antimycin A inhibits Ip. To gain further evidence that the effect of FCCP and 2-DG was because of metabolic inhibition, we tested another metabolic inhibitor, antimycin A. After exposure of myocytes to 10μM antimycin A for 6 min, there was a 68±0.02% reduction in Ip (P<0.05). (3) MI specifically inhibits Il. After exposure of myocytes to MI for 6 min, Ih remained unchanged at 97±0.04% of control values (P>0.05). These results seem to suggest that only theα1-isoform of the Na~+-K~+ pump is involved in the inhibition of Ip induced by MI. We then proceeded to directly examine the effect of MI on Il. MI inhibited Il by 60±0.03% (P<0.05), which was not significantly different from 62±0.04% inhibition of total Ip produced by MI (P>0.05). Thus, MI-induced inhibition of Ip isα1-isoform specific. (4) Inhibition of Ip by MI is voltage-dependent. For measurement of the voltage dependence of Ip a voltage-ramp protocol going from +20 to -100 mV in a 4-s period was used. The relationships were normalized to the Ip recorded at 0 mV to facilitate comparison of their slopes. The difference between these slopes was statistically significant (P<0.05; two-way ANOVA). MI shifted the voltage dependence of Ip towards more positive potentials by approximately 20 mV. The V1/2 values in the absence and presence of MI were -107 and -87 mV, respectively. (5) Adenosine does not influence the inhibitory effect of MI on Ip. MI still reduced Ip by 66±0.05% (P<0.05) in the presence of 10μM adenosine and this was statistically insignificant when compared to its absence (P>0.05). Adenosine at 100μM, a therapeutic dose for ischemic myocardial injury, also did not affect MI-induced Ip inhibition. These results suggest that Na~+-K~+ pump was not involved in the cardioprotection of adenosine against myocardial ischemia.Conclusion: Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.Part 3 The mechanism of myocardial ischemia-induced inhibition of Na~+-K~+ pump currentObjective:To delineate the characteristic of Na~+-K~+ pump current during myocardial ischemia and to further clarify the underlying mechanism involved.Methods:(1) Enzymatic isolation of guinea pig ventricular myocytes. (2) Whole-cell patch-clamp technique was performed to record the Na~+-K~+ pump current (Ip). (3) Simulation of myocardial ischemia using metabolic inhibitors. (4) Measuring of intracellular pH (pHi): The pHi of single isolated myocytes was measured using carboxy-SNARF-1, a dual-emission pH-sensitive fluoroprobe. The myocytes were loaded with the membrane-permeable acetoxy-methylester form of carboxy-SNARF-1 for 10 min and then pHi was measured using laser confocol microscope before and after MI superfusion. The fluorophore was excited by light at 514 nm, and the emitted fluorescence signals were measured simultaneously at 580 nm and 640 nm. The 580 nm/640 nm emission ratio was converted to a pHi value using the nigericin calibration techniqueResults:(1) Myocardial ischemia-induced intracellular acidification inhibits Ip. No inhibition of Ip was observed when cells were exposed to the extracellular solution with anoxia (bubbling with 100% N2) and glucose deprivation but normal pHo (7.4); Ip was only decreased by 7.5±0.04% and this decrease was statistically insignificant (P>0.05). However, when treatment of myocytes with the extracellular solution containing normal oxygen and glucose but acidosis (pHo 6.0), Ip was markedly decreased by 50±0.02% (P<0.05) as under MI, demonstrating that the change in pHo itself was sufficient to inhibit Ip. Since extracellular acidosis leads to a rapid radification of the cytosol, and there is evidence suggest that Na~+-K~+ pump is not sensitive to pHo, we propose that pHi may be the key component triggering Ip inhibition during myocardial ischemia. To address this point, we increased the pHi buffering capability by elevating the concentration of HEPES in the pipette solution to 20 mM. Under these conditions, extracellular acidosis did not induce a significant inhibition of Ip; Ip returned to 94±0.07% (P>0.05) of control values. By contrast, perfusing myocytes with acidosic agent could mimic the effect of extracellular acidosis on Ip. Briefly perfusing myocytes with NH4Cl followed by washout, which is a common protocol that was used to induce an intracellular acidification. NH4Cl withdrawal drastically reduced Ip by 60±0.03% (P<0.05). Also, this inhibition was completely abolished with 20 mM HEPES in the patch pipette; Ip returned to 96±0.04% (P>0.05) of control values. (2) MI induces a rapid acidification of the cytosol, thus inhibiting Ip. With 20 mM HEPES in the pipette solution, the inhibitory effect of MI on Ip was markedly prevented; Ip return to 81±0.02% (P<0.05) of control values, which is not significantly different from that with 5 mM HEPES in the pipette solution (62±0.04%, P<0.05). We further monitored pHi with SNARF-1 under MI. It was found that resting pHi in these cells was 7.28±0.07. Perfusion of MI resulted in a sustained intracellular acidification. The mean pHi values corresponding to MI time of 2.5, 4, and 6 min were 7.02±0.06, 6.81±0.06, and 6.65±0.08, respectively. Therefore, MI leads to Ip inhibition in a pH-dependent manner in guinea pig ventricular myocytes. (3) ROS, PKA, and PKC are not involved in MI inhibition of Ip. Inclusion of 1 mM MPG in the patch pipette failed to block the MI-induced decrease in Ip and this was statistically insignificant when compared to its absence (68±0.05% for MI+MPG, P>0.05 vs. 62±0.04% for MI). In addition, the specific PKA and PKC inhibitors, H89 and Bis-I, respectively, did not significantly affect MI inhibition of Ip. Conclusion: Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.SUMMARY1 Adenosine specifically inhibits theα2-isoform of Na~+-K~+ pump, but not theα1-isoform of Na~+-K~+ pump. The effect of adenosine on Na~+-K~+ pump is mediated by adenosine A1R and PKC pathway. However, the adenosine A2AR, A2BR, A3R, and PKA pathway are not involved.2 Simulated myocardial ischemia markedly inhibits the Na~+-K~+ pump function in a time-dependent manner. Simulated myocardial ischemia specifically inhibits theα1-isoform of Na~+-K~+ pump, which is voltage-dependent. Adenosine does not affect the inhibition of Na~+-K~+ pump induced by myocardial ischemia, suggesting that the cardioprotection of adenosine against myocardial ischemia does not involves the Na~+-K~+ pump.3 Simulated myocardial ischemia leads to a rapid acidification of the cytosol, thus causing the inhibition of Na~+-K~+ pump. However, oxidative stress, PKA, and PKC are less likely to be involved in the inhibition of Na~+-K~+ pump by myocardial ischemia.
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