INTRODUCTION

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CARDIAC ATP-SENSITIVE K⁺ (K_ATP) channels have been suggested to play an important role in changes in electrical activity of the ischemic heart (24, 41). Openings of the channel reduce the myocardial damage induced by metabolic inhibition as a consequence of the shortening of action potential duration, which decreases Ca²⁺ influx, increases Ca²⁺ efflux, and thereby minimizes Ca²⁺ overload in myocytes. This concept has been widely accepted as a major pathophysiological mechanism in K_ATP channels since its discovery in the heart (27, 41).

Phosphatidylinositol 4,5-bisphosphate (PIP₂) was recently reported to alter the ATP sensitivity of K_ATP channels (1, 10, 15, 30). An increase in PIP₂ levels at the cytoplasmic face of the sarcolemma lowers sensitivity of the channel to ATP. PIP₂ reactivates run-down K_ATP channels, which are brought about by exposure to high concentrations of Ca²⁺ (39). However, PIP₂ did not reduce ATP sensitivity in K_ATP channels reactivated by PIP₂ treatment from the run-down state (28). PIP₂ treatment impaired sensitivity of K_ATP channels to the sulfonylurea tolbutamide (22). However, the sustained increase in cytosolic Ca²⁺ concentration decreased tolbutamide sensitivity of the channel, whereas PIP₂ and ATP cooperatively protected the channel from the Ca²⁺-induced impairment of tolbutamide sensitivity in pancreatic -cells (21). These findings suggest that membrane PIP₂ is a maintenance modulator of channel activity and sensitivity to sulfonylureas as well as ATP.

ATP acts as an extracellular signal in various types of tissues involved regulation of vascular tone, muscle contraction, pain, or neuronal communication (6). Under hypoxic or ischemic conditions, the source of extracellular ATP is reportedly parenchymal cells (31). Many biological responses to ATP are mediated via the P₂ purinoceptor family, which are classified into two structurally and functionally distinct group of receptors: ligand-gated nonselective cation channels (P_2X-type receptors) and GTP-binding protein (G protein)-coupled receptors linked to the phospholipase C (PLC) signaling cascade (P_2Y-type receptors; Refs. 5 and 6). Binding of extracellular ATP to P_2Y purinoceptors stimulates PLC to cleave PIP₂ into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP₃) (2). We therefore postulated that stimulation of these pathways may change the activity of K_ATP channels by altering the sarcolemmal PIP₂ concentration. In the present study, we attempted to clarify whether extracellular ATP modulates activity of the K_ATP channels, and the underlying mechanisms were explored in ventricular myocytes isolated from guinea pig hearts.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Studies were performed in single ventricular myocytes enzymatically isolated from guinea pig hearts as described previously (34). Briefly, male guinea pigs weighing 210-300 g were anesthetized with pentobarbital sodium (26 mg/kg) by peritoneal injection and were artificially ventilated. After thoracotomy, the heart was cannulated into the aorta, quickly excised, mounted on a Langendorff apparatus, and perfused at 37°C with oxygenated Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.5 MgCl₂, 0.33 NaH₂PO₄, and 5.5 glucose (pH 7.4 with NaOH). The perfusate was switched to a nominally Ca²⁺-free oxygenated Tyrode solution (100 ml), and Ca²⁺-free Tyrode solution containing 0.04% collagenase (Worthington type II) was perfused for 15 min. The heart was then perfused to wash out collagenase with 100 ml of Kraftbrühe (KB) solution (18) containing (in mM) 40 KCl, 20 taurine, 50 glutamic acid, 20 KH₂PO₄, 3 MgCl₂, 10 glucose, 0.5 EGTA, and 10 HEPES (pH 7.4 with KOH). The left ventricle was removed and cut into small pieces (~5 mm³) in KB solution and dispersed mechanically into single cells. The dispersed myocytes were stored in KB solution at 4°C for at least 1 h before use. Single ventricular myocytes were then placed in a recording chamber (500 µl), which was filled with Tyrode solution. Experiments were started after the cells had settled to the bottom of the chamber.

Patch pipettes were pulled from hard glass tubing (Sutter Instrument, Novato, CA), coated with silicon resin to reduce electrical capacitance, and fire polished immediately before use. Pipette resistances were between 2 and 3 M for whole cell experiments and were ~5-10 M for experiments of outside-out or inside-out membrane patches. All experiments were performed at 23-26°C.

Whole cell and single K_ATP channel currents were recorded from ventricular myocytes with the conventional patch-clamp technique (12). Data were recorded with an amplifier (Axopatch 200B, Axon Instruments, Foster City, CA) and stored by means of a PCM digital data recorder (RD-101T; TEAC, Tokyo, Japan). Replayed recordings were then low-pass filtered (24 dB/octave, E-3211A; NF, Tokyo, Japan) at the cut-off frequency indicated in Figs. 1, 4, 5, and 7 and digitized by pCLAMP6 or -7 software (Axon Instruments) on an IBM computer. In some experiments, on-line recordings of the whole cell current were performed.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of extracellular ATP on ATP-sensitive K+ (KATP) channel currents. A: chart recordings of the KATP channel current at the beginning of whole cell recording during voltage ramps (a). Decreasing the cytoplasmic ATP concentration through the pipette solution containing the lower ATP concentration (0.5 mM) and exposure of the cell to 30 µM pinacidil caused gradual increases in the outward current. In current tracings at point b the maximal current level reached several nanoamperes as illustrated in B. The top of the current tracings was cut in the chart recorder. ATP transiently inhibited KATP channel currents with minimal levels (c). The current was inhibited by 10 µM glibenclamide (d). Data were filtered at the cut-off frequency of 800 Hz. B: current-voltage relations for KATP channel currents (a to d) recorded at the times indicated in A are illustrated. C: the subtracted current-voltage relations of b  d and b  c are illustrated. D: concentration-response relations of %inhibition of the amplitudes of the KATP channel currents at +20 mV for extracellular ATP. Values are means ± SE of 3-10 results.

The ventricular myocytes were voltage-clamped at the holding potential of 70 mV, and voltage ramps were applied every 5 or 10 s from 100 mV to +50 mV at 75 mV/s and, subsequently, ramps to 70 mV at 120 mV/s. The current-voltage relations were calculated from the measurements at the down slope of the voltage ramps. To compare the effects of extracellular nucleotides or chemicals on the activity of K_ATP channels, amplitudes of K_ATP channel currents were measured at +20 mV. The percent inhibition of the channel current was obtained from the following equation (see Fig. 1): %inhibition = 100(I_max  I_test)/(I_max  I_glib), where I_max is the current amplitude maximally activated by reduction in the ATP concentration in the pipette to 0.5 mM and exposure to 30 µM pinacidil, a K_ATP channel opener; I_test is current amplitude during exposure to test solutions; and I_glib is current amplitude during exposure to 10 µM glibenclamide. The concentration-response relations of K_ATP channel currents for ATP concentrations were fitted by the Hill equation: %inhibition = 100/[1 + (IC₅₀/[ATP])^n_H], where IC₅₀ is the concentration for half-maximal channel inhibition, [ATP] is ATP concentration, and n_H is the Hill coefficient.

Pipette solutions contained (in mM) 50 KCl, 90 aspartic acid, 1 KH₂PO₄, 1 MgCl₂, 5 HEPES, 5 EGTA, 0.5 Na₂ATP, and 0.1 GTP (pH adjusted to 7.2 with KOH). Na₂ATP, Na₂ADP, and adenosine were obtained from Boehringer (Mannheim, Germany). Na₂GTP, suramin, and wortmannin were obtained from Nacalai (Kyoto, Japan). Compound 48/80 was obtained from Biomol (Plymouth Meeting, PA). Glibenclamide, staurosporine, pinacidil, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were purchased from Sigma (St. Louis, MO). Glibenclamide was dissolved in DMSO to be stocked and used at the indicated concentration. PIP₂ was dissolved in 0.1% DMSO and diluted by the pipette solution by sonication on ice for 30 min.

Values are expressed as means ± SE. Student's unpaired t-test or one-way ANOVA test was used to evaluate the difference between groups using Prism software (GraphPad Software). The time constants were obtained by fitting the data to a single exponential function with Prism software.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Inhibition of K_ATP channel currents by extracellular ATP. Because K_ATP channels are known to be independent of membrane voltage (19), the voltage ramp protocol was used to obtain current-voltage relations of K_ATP channels. The membrane potential was stepped to 100 mV from the holding potential of 70 mV, and voltage ramps to +50 mV and then to 70 mV were applied to evoke currents. The corresponding current traces are illustrated in Fig. 1A. The large inward current and small outward current through the inwardly rectifying K⁺ channel current (I_K1) with a negative slope conductance were recorded at point a as shown in Fig. 1, A and B. In Fig. 1A, a gradual increase in the outward current was observed with a reduction in cytosolic ATP concentration by dialysis through the pipette solution containing 0.5 mM ATP and exposure to 30 µM pinacidil (Fig. 1A, point b). The inward current was also reduced before the increase in the outward current because amplitudes of mean I_K1 have been reported to be inhibited by decreasing intracellular ATP concentration (19) and subsequently appeared to be increased in association with the increase in the outward current at point b. This small increase in the inward current may reflect activation of K_ATP channels. After exposure to 0.1 mM ATP, both the outward and inward currents were transiently reduced (point c). Glibenclamide, a specific blocker of K_ATP channels, substantially inhibited these currents at 10 µM (point d). This suggested that the current increased during the reduction in the cytosolic ATP concentration and inhibited by exposure to extracellular ATP must be that via the K_ATP channels (11). In Fig. 1B, the current-voltage relations obtained at the time indicated in Fig. 1A were plotted. The current-voltage relations (point a) showed a negative slope conductance with a small current level at the positive membrane potentials. These are properties of I_K1 currents. The large increase in the outward current in association with the reduction in the cytoplasmic ATP levels was characterized by the increase in the current level at positive potentials with a reduced negative slope (point b). At points c and d, current-voltage relations similar to those at point a were depicted during superfusion with 0.1 mM ATP or 10 µM glibenclamide. The current-voltage relations of d subtracted from b indicated the glibenclamide-sensitive component and the difference between b and c was the extracellular ATP-inhibited currents (Fig. 1C). Because both curves showed similar relations against membrane potentials with the same reversal potential and a weak inwardly rectifying property, the current inhibited by ATP was that via the K_ATP channels (19). In Fig. 1D, concentration-response relations of the K_ATP channel currents for extracellular ATP measured at +20 mV are plotted. IC₅₀ was calculated to be 4.2 µM.

Comparison of relative inhibitory potency of nucleotides and nucleosides on K_ATP channel currents. Both P₁ and P₂ purinoceptors were found in guinea pig atrial and ventricular myocytes (7, 8, 25, 38). Various types of nucleotides were used to test the inhibitory efficacy on the K_ATP channel currents elicited by exposure to 30 µM pinacidil and 0.5 mM ATP in the pipette (Fig. 2). The magnitude of the activated K_ATP channel current and the degree of inhibition of the negative slope were variable from cell to cell. This may have been caused by differences in cell size and in the capability of diffusion of intracellular ATP through the whole cell pipettes. Thus amplitudes of K_ATP channel currents were measured at +20 mV, and the percent inhibition of the current by nucleotides compared with controls are illustrated in Fig. 2D. The effectiveness of ADP and AMP on channel inhibition was less than that of ATP. ADP at 1 mM reduced the channel current by 42.8 ± 9.3% and 1 mM AMP reduced the current by 9.4 ± 4.8% (Fig. 2, A, B, and D). Adenosine had little effect on K_ATP channel currents, even at 10 mM (Fig. 2, C and D). P₂ purinoceptors have been classified into two structurally and functionally distinct families, the P_2X and P_2Y types. Exposure of cells to 0.1 mM ,-methylene ATP, a P_2X-selective agonist, caused only a small reduction in the K_ATP channel currents with an inhibition of 28.3 ± 121% (n = 5). The order of the inhibitory efficacy on the activity of the K_ATP channels was therefore ATP > ,-methylene ATP = ADP > AMP > adenosine.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of various types of nucleotides and nucleosides on current-voltage relations of the KATP channel current evoked by a reduction in ATP in the pipette. The current-voltage relations in the absence (control) and presence of 1 mM ADP (A), 1 mM AMP (B), and 10 mM adenosine (C) are illustrated. In each trace, 10 µM glibenclamide was added at the end of the recordings to find the glibenclamide-sensitive amplitudes of KATP channel currents. D: %inhibition during exposure to each test solution measured at +20 mV and plotted. The 1-way ANOVA test was used to evaluate the difference between groups. (*P < 0.05; **P < 0.01). mATP, ,-methylene ATP.

Signaling pathway for channel inhibition by ATP. Binding of extracellular ATP to P_2Y purinoceptors coupled to G protein has been reported to stimulate PLC to cleave PIP₂ into DG and IP₃ (2). We tested the effects of ATP in the presence of 0.2 mM suramin, which binds to P₂ purinoceptors competitively with ATP (17). Suramin prevented ATP-induced channel inhibition, and the inhibition of the channel current was reduced to 14.6 ± 4.5% (n = 5; Fig. 3). Involvement of G proteins or PLC in ATP-induced K_ATP channel inhibition was also tested with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDPS; 0.2 mM), a nonhydrolyzable GDP analog (16), or compound 48/80, a PLC inhibitor (4). The inhibition of the channel current by ATP was reduced to 31.3 ± 9.9% (n = 5) by GDPS and to 15.9 ± 6.4% (n = 6) by compound 48/80. These findings suggested that extracellular ATP inhibited the activity of the K_ATP channels via the pathway including P_2Y purinoceptor, G protein, and PLC.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effects of various types of compounds, inhibitors, or chelators of key elements in the subcellular pathway from P2Y purinoceptors on 0.1 mM ATP-induced KATP channel inhibition. The 1-way ANOVA test was used to evaluate the difference between groups. (**P < 0.01 vs. 0.1 mM ATP). GDPS, guanosine 5'-O-(2-thiodiphosphate); BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

Effects of factors distal to PLC on K_ATP channels. Stimulation of P_2Y purinoceptors causes cleavage of PIP₂ to DG and IP₃. These metabolites consequently activate protein kinase (PKC) and increase the cytosolic Ca²⁺ concentration (2, 6). Staurosporine is known to block the activity of PKC by direct interaction (33). In the presence of 0.1 µM staurosporine, K_ATP channel currents were inhibited by 0.1 mM ATP to an extent similar to the controls. Likewise, when 10 mM BAPTA, a potent Ca²⁺ chelator, was substituted for EGTA in the pipette solution, the inhibitory efficacy of the channels by ATP did not change. The inhibition by ATP was 79.1 ± 12.9% with staurosporine (n = 5) and 91.2 ± 6.3% with BAPTA (n = 5). These findings suggest that neither PKC nor change in cytosolic Ca²⁺ concentration was involved in ATP-induced K_ATP channel inhibition.

Effects of wortmannin or PIP₂ on ATP-induced inhibition of K_ATP channel currents. From the findings presented above, we postulated an involvement of change in PIP₂ concentration via modification of lipid metabolism in the sarcolemma on ATP-induced K_ATP channel inhibition. Because PIP₂ is a determinant of the basal activity of K_ATP channels in ATP-free solution (28, 39), the reduction in the PIP₂ concentration in the sarcolemma by P_2Y purinoceptor stimulation may consequently decrease the activity of the K_ATP channels. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase and -4-kinase, inhibits conversion to phosphatidylinositol 3,4,5-trisphosphate (PIP₃) from PIP₂ by lipid phosphorylation and the replenishment of PIP₂ (37). In the presence of 10 µM wortmannin, ATP-induced inhibition of K_ATP channel currents was irreversible (n = 4; Fig. 4A). Because production of PIP₂ is regulated by activity of lipid kinases sensitive to wortmannin, inhibition of these enzymes by wortmannin may be expected to reduce the supply of PIP₂ in the sarcolemma. Under these conditions, stimulation of PLC by extracellular ATP may reduce the levels of PIP₂ in the sarcolemma. Similarly, the reduction of PIP₂ in the sarcolemma by stimulation of P_2Y purinoceptors may have been exaggerated when cytosolic ATP levels were reduced with the pipette solution containing 0 mM ATP. The activity of the K_ATP channels was irreversibly inhibited by 0.1 mM ATP in the absence of intracellular ATP in outside-out membrane patches (n = 6; Fig. 4B). With 0.1 mM PIP₂ in the pipette solution, the K_ATP channel currents were not inhibited by 0.1 mM ATP in any of the membrane patches tested (n = 5; Fig. 4C).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Involvement of phosphatidylinositol 4,5-bisphosphate (PIP2) metabolism in ATP-induced KATP channel current inhibition. A: the whole cell KATP channel current was evoked by 30 µM pinacidil (arrow) with the pipette solution containing 0.5 mM ATP. ATP at 0.1 mM and 10 µM wortmannin was superfused during the period shown above the trace. The inhibition of the KATP channel currents was irreversible even after the washout of ATP and wortmannin. B: a single KATP channel current evoked by depleting intracellular ATP to 0 mM in the outside-out mode is illustrated. The channel inhibition by 0.1 mM ATP was also irreversible. C: outside-out patch recordings for KATP channels with the pipette solution containing 0.1 mM PIP2 are illustrated. ATP at 0.1 mM did not inhibit channel activity, but 10 µM glibenclamide did. The patch membrane potential was clamped at +20 mV in B and C, and data were filtered at the cut-off frequency of 800 Hz.

Enhanced sensitivity of K_ATP channels to cytosolic ATP in presence of extracellular ATP. We examined the effects of extracellular ATP on the concentration-response relations of the K_ATP channel currents for ATP at the cytosolic side in inside-out membrane patches (Fig. 5). The presence of 0.1 mM ATP at the extracellular side of the membrane patches rapidly reduced K_ATP channel activity after formation of the inside-out mode. Thus we superfused 30 µM pinacidil to increase the channel current to detect concentration-dependent channel inhibition by ATP throughout the experiments (Fig. 5, A and B). The IC₅₀ for the channel inhibition by ATP was shifted from 1.12 mM in the controls to 13.8 µM in the presence of 100 µM ATP in the pipette. Thus the reduction of PIP₂ levels in membrane patches may be brought about by stimulation of P_2Y purinoceptors by ATP and may increase the ATP sensitivity of the channels. This may be the underlying mechanism of P_2Y purinoceptor-stimulated K_ATP channel inhibition. These findings were comparable to those previously reported in cells expressed with both P_2Y purinoceptors or muscarinic receptors and K_ATP channels (1, 40).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Shift of ATP sensitivity of KATP channels in the presence of 100 µM ATP at the outside of the membrane patches. A and B: the KATP channel currents were recorded in the absence (A) and presence (B) of 0.1 mM ATP in the pipette in inside-out membrane patches. The membrane excised from the cells was exposed to 30 µM pinacidil to evoke the channel current because channel currents would otherwise be inhibited by extracellular ATP even in the absence of intracellular ATP. The current data were filtered at 800 Hz. The baseline levels are indicated with short bars (designated 0) beside current traces. C: the concentration-response relations of KATP channel activity for ATP concentrations are illustrated. The KATP channel currents were recorded at a patch membrane potential of 60 mV with various concentrations of ATP in the internal solution, and the relative current, normalized to the current obtained in the absence of ATP, is plotted with pipette solution containing 100 µM ATP () or without ATP (). The curves were drawn according to the following equation: relative current = 1/[1 + ([ATP]/IC50)nH], where IC50 is concentration for half-maximal channel inhibition, [ATP] is ATP concentration, and nH is the Hill coefficient.

ATP-induced inhibition of K_ATP channel currents is dependent on cytosolic ATP concentration. In Fig. 6, intracellular ATP dependence of extracellular ATP-induced K_ATP channel inhibition is shown. Extracellular ATP at 0.1 mM inhibited whole cell K_ATP channel currents that were evoked by 30 µM pinacidil in the presence of 3 mM ATP in the pipette. The inhibitory efficacy was 35.5 ± 10.0% (n = 7), and this was less than that in the presence of 0.5 mM ATP in the pipette solution (Fig. 6B; P < 0.01). ATP-induced channel inhibition was transient as illustrated in Fig. 1A. Figure 6C shows the time course of relative amplitudes of K_ATP channel currents in response to extracellular ATP in the presence of intracellular ATP of 3 or 0.5 mM. The time to maximal inhibition of the K_ATP channel current was 245.0 ± 110.0 s in the presence of 3 mM ATP (n = 7) in the pipette and 819 ± 30.0 s at 0.5 mM ATP (n = 8; P = 0.12). The time constants for the inhibitory phase of the K_ATP channel amplitude were 80.1 ± 29.6 s in the presence of 0.5 mM ATP (n = 3) in the pipette and 371.4 ± 101.8 s in the presence of 3 mM ATP (n = 4; P < 0.05). In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPS; 0.2 mM), a nonhydrolyzable GTP analog, in the pipette solution, ATP-induced K_ATP channel inhibition was irreversible despite the presence of 0.5 mM ATP at the cytosol (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Dependence of ATP-induced KATP channel inhibition on intracellular ATP. A: current-voltage relations obtained in the presence of intracellular 3 mM ATP are illustrated in the absence (control) and presence of extracellular 0.1 mM ATP. To determine the amplitudes of the channel currents sensitive to glibenclamide, 10 µM glibenclamide was added at the end of the experiments. B: comparison between the findings obtained with 0.5 mM ATP and 3 mM ATP in the pipette. The currents were measured at +20 mV, and the %inhibition was plotted (Student's unpaired t-test; **P < 0.01). C: time course of the relative amplitude of KATP channel currents during exposure to extracellular 0.1 mM ATP plotted in the presence of 3 mM and 0.5 mM ATP in the pipette. Data were normalized to the amplitudes of KATP channel currents immediately before exposure to extracellular 0.1 mM ATP.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Irreversible inhibition of KATP channel current by 0.1 mM ATP in the presence of 0.2 mM guanosine 5'-O-(3-thiotriphosphate) (GTPS) in the pipette. KATP channel currents were evoked by depletion of the intracellular ATP concentration to 0.5 mM and exposure to 30 µM pinacidil as indicated by an increase in the outward current amplitudes. Data were filtered at the cut-off frequency of 800 Hz.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present study, we demonstrated that extracellular ATP reduced K_ATP channel currents because of the binding of ATP to purinoceptors. These findings revealed a significantly stronger inhibitory efficacy with ATP than with ,-methylene ATP, and the order for blocking effectiveness, ATP > ,-methylene ATP = ADP > AMP > adenosine, suggested that ATP-induced K_ATP channel inhibition was mediated by P_2Y purinoceptors. Cardiac myocytes express different types of ATP receptors that consist of ligand-gated ion channels (P_2X type) and that couple to PLC via heterotrimeric G proteins (P_2Y type) (9). The present observations that extracellular ATP-induced K_ATP channel inhibition involved receptor-mediated stimulation of PLC coupled to G proteins support the idea that P_2Y-type receptors are expressed in guinea pig ventricular myocytes and physiologically regulate K_ATP channels. Although K_ATP channel inhibition mediated by either P_2Y purinoceptors or muscarinic receptors has been reported in cells overexpressed with K_ATP channels and PLC-linked receptors (1, 40), the present findings provide evidence that the breakdown of PIP₂ mediates the activity of the K_ATP channels via PLC-linked receptors under physiological conditions.

In inside-out membrane patches, PIP₂ is reportedly a regulator of K_ATP channels (1, 10, 15, 30). In the present study, we observed that the presence of extracellular ATP in inside-out membrane patches shifted the IC₅₀ toward lower concentrations of ATP (Fig. 5). This suggests that the decrease in PIP₂ levels in the membrane increases the sensitivity of the K_ATP channels to intracellular ATP and thereby reduces the activity of the channels. Ca²⁺ treatment of membrane patches attenuated the activity of the K_ATP channels (rundown of the channels), and subsequent treatment with PIP₂ restored channel activity (28, 39). Rundown of K_ATP channel activity observed during Ca²⁺ treatment (28, 39) or in ATP-free solution (28) may reflect depletion of PIP₂ levels in the membrane. However, the rundown of channel activity may not be simply due to depletion of PIP₂ in the membrane because channel activity restored from the run-down state by PIP₂ treatment did not exhibit less ATP sensitivity (28). Thus a more complicated mechanism regarding modulation of the channel gating by PIP₂ may be required.

PKC or Ca²⁺ mobilization from the intracellular store sites may not be involved in K_ATP channel inhibition by ATP (Fig. 3). We observed irreversible channel inhibition in the presence of wortmannin or the absence of intracellular ATP, whereas staurosporine or BAPTA did not influence extracellular ATP-induced K_ATP channel inhibition (Fig. 4, A and B). The levels of PIP₂ in the sarcolemma appeared to be regulated by a balance of replenishment from ATP via wortmannin-sensitive lipid kinases (37), expenditure via stimulation of PLC, and dephosphorylation by intrinsic phosphatase activity. Thus the reduction of sarcolemmal PIP₂ levels by means of stimulation of P_2Y purinoceptors may be a final determinant for the inhibition of activity of K_ATP channels. This was supported by the observation that ATP-induced channel inhibition was prevented when 0.1 mM PIP₂ was present in the outside-out pipette solution. However, the possible involvement of PIP₃, which may be a main mediator of K_ATP channels rather than PIP₂ in diabetic ob/ob mice (23) or the insulinoma cell line (13), is not excluded. We did not find whole cell K_ATP channel current activation with PIP₂ in the pipette up to 1 mM. This may be due to the following reasons. Diffusion of PIP₂ through the pipette may not be adequate to activate K_ATP channels, which has been reported to take several hours (1), and the majority of PIP₂ may be absorbed into the glass wall of the pipette (14). Likewise, injected PIP₂ may be dephosphorylated by intrinsic phosphatase activity.

Extracellular ATP-induced inhibition of the K_ATP channels was transient (Figs. 1A and 6C) and was dependent on the concentration of ATP at the cytoplasmic side (Fig. 6B). Because ATP is a phosphate donor for lipid kinase, increased ATP levels in the cytosol may result in greater production of PIP₂ levels in the sarcolemma. Observations that both the magnitude of the reduction and the inhibitory time constant of K_ATP channel currents on simulation of P_2Y purinoceptors were dependent on the cytosolic ATP concentration may suggest that cytoplasmic levels of ATP underneath the sarcolemma may also influence the rate of the reduction of PIP₂ levels in the sarcolemma. The finding that persistent inhibition of the channel currents was observed in the presence of GTPS in the cytosol (Fig. 7) suggests that desensitization of the extracellular ATP-induced reduction of K_ATP channel currents occurred at a site proximal to stimulation of PLC. In a recent study a new insight that hydrolysis of PIP₂ by PLC in the membrane accounts for a new form of desensitization in the GTP-binding protein-gated inwardly rectifying potassium channel current stimulated by acetylcholine has been demonstrated in atrial cells (20).

Takizawa et al. (32) reported that the whole cell K_ATP channel current was inhibited by norepinephrine. We also found that norepinephrine at a concentration of 0.1 µM inhibited the K_ATP channels (data not shown). Thus a similar mechanism may be involved in norepinephrine-induced K_ATP channel inhibition because ₁-adrenoceptors stimulate phosphatidylinositol metabolism (3, 35). The IC₅₀ for ATP-induced K_ATP channel inhibition was 4.8 µM, which may be a physiologically relevant concentration at the extracellular space. Similar effective concentrations for extracellular ATP-induced modulation of ionic channels have been reported for the delayed-rectifier K⁺ current in the guinea pig heart (26) and for the Ca²⁺ channel current in the ferret heart (29) via P_2Y purinoceptors. In the normal heart, the interstitial ATP concentration is reportedly in the nanomolar range (31), but it may be increased to micromolar levels in the ischemic heart (36). However, the overall effects of extracellular ATP on cardiac cells during ischemia remain to be determined.