4. Whole-cell patch-clamp experiments revealed that ATP activated an ionic current that had a linear current-voltage relationship with a reversal potential near 0 mV. Quinidine, a putative P2 purinergic receptor blocker, abolished the ATPactivated current. The ATP-activated current was Mg2+ dependent.5. Associated with the ATP-activated current was cellular depolarization. In a physiological solution, ATP depolarized cells to the threshold for the firing of action potentials. In the presence of the voltage-activated ion channel blockers tetrodotoxin, 4-aminopyridine, caesium and nitrendipine, ATP depolarized cells to -44 + 6 mV from a resting potential of -66 + 4 mV (n = 11).6. Sodium dodecyl sulphate (SDS) polyacrylamine gel electrophoresis and autoradiography demonstrated that extracellular ATP stimulated the phosphorylation of several extracellular membrane-bound proteins. The phosphorylation of these proteins was concentration, time and Mg2+ dependent. Pre-treatment of cells with the slowly hydrolysable ATP analogues inhibited the ATP-induced phosphorylation. Adenosine 5'-0-3-thiotriphosphate (ATPyS) thiophosphorylated proteins with the same apparent molecular weight as the proteins phosphorylated by ATP.7. These results suggest that the ATP-induced increase of [Ca2+]i is a result of the activation, possibly by protein phosphorylation, of a novel ion channel carrying * Present address:

ATP transiently increases the intracellular Ca2+ concentration in cardiac myocyte suspensions. Pretreatment with norepinephrine (NE) greatly potentiates the ATP response. We performed experiments on adult rat myocyte suspensions loaded with fura-2 to investigate the mechanism of NE potentiation. We found that forskolin (an activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase), and permeative adenosine 3',5'-cyclic monophosphate (cAMP) analogues potentiate the increase in cytosolic Ca2+ concentration induced by ATP. NE, forskolin, and 8-(4-chlorophenylthio)-cAMP all increase Vmax of the Ca2+ response curve of ATP. Measurement of cAMP by radioimmunoassay confirmed that the changes in the ATP response were accompanied by an increase in cAMP. These results suggest that the noradrenergic potentiation of the ATP-induced Ca2+ mobilization involves cAMP as a second messenger. Patch-clamp studies of isolated myocytes showed that neither NE nor forskolin alters the inward current elicited by ATP, but rather they increase the duration of secondary slow action potentials elicited by ATP. NE also increases the Ca2+ current through L-type Ca2+ channels in the myocytes. We conclude that NE potentiates the ATP-induced Ca2+ transient by increasing cAMP levels and that one of the early events is the increase of the inward Ca2+ current during the action potential.

Activation of protein kinase C (PKC) modulates the mobilization of intracellular Ca2+ induced by extracellular ATP in rat ventricular myocytes. Pretreatment of myocytes with PKC activators attenuated both the ATP-induced Ca2+ transient and the noradrenergic potentiation of the Ca2+ response. Various PKC activators decreased both the basal cAMP level and the cAMP levels that had been elevated by norepinephrine, forskolin, or 3-isobutyl-1-methylxanthine. The inhibitory effects of PKC activators were reversed by the PKC inhibitor staurosporine. The ATP-induced Ca2+ response is an integrated response resulting from ATP eliciting an inward cation current (IATP), cellular depolarization, Ca2+ influx through Ca2+ channels, and Ca2+ release from the sarcoplasmic reticulum. We used the whole cell voltage-clamp technique to investigate which steps of this integrated response are affected by PKC. PKC activators did not significantly affect the IATP. In contrast, PKC activators decreased the basal Ca2+ current (ICa) or Ba2+ current and the beta-adrenergic-stimulated ICa. These results suggest that PKC-induced suppression of the ATP-induced Ca2+ response and the beta-adrenergic-potentiated Ca2+ response is achieved at least partially by decreasing the intracellular cAMP level and ICa.

The whole cell voltage-clamp technique was used to study the electrophysiological effects induced by ATP in isolated ventricular myocytes. ATP or 2-methylthio-ATP evoked a transient inward current (IATP) when the transmembrane potential (Vm) was held at -70 mV and increased the Ca2+ current (ICa) when Vm was depolarized to 0 mV. The time course of IATP was fitted by a single exponential equation with a brief time constant (165 ms), whereas the time course of enhancement of ICa by ATP was also fitted by a single exponential equation with a much longer time constant (14 s). IATP was much less pronounced when extracellular Mg2+ was absent, and it was insensitive to dihydropyridines. In contrast, the enhancement of ICa by ATP was not affected by removing extracellular Mg2+, but it was suppressed by Ca2+ channel blockers. Both IATP and ICa were decreased by extracellular Cd2+. Internally applied guanosine 5'-O-(2-thiodiphosphate), which prevents the activation of G proteins, abolished the ATP-enhanced rise in ICa but did not inhibit IATP. These data suggest that ATP elicits IATP and increases ICa through two different mechanisms. IATP appears to be generated via receptor-operated channels that are activated by ATP. The ATP-induced increase of ICa appears to be mediated by G proteins via pathways that are independent of adenosine 3',5'-cyclic monophosphate and phosphoinositide turnover.

Since the original description of the effect of micromolar concentrations of extracellular ATP on the cytosolic calcium concentration ([ Ca2+],) of ventricular cells,' other groups have reported similar findings?' A common finding was that ATP stimulates calcium entry into cells from the extracellular solution primarily through dihydropyridine-sensitive calcium channels. It was not known, however, whether this was a direct or indirect action of ATP on these calcium channels.The whole-cell configuration of the patch-clamp technique was used to investigate the electrophysiologic effects of extracellular ATP on rat ventricular myocytes. The L-type calcium channel was isolated by depolarizing the cell from a holding potential (V,) of -45 or -40 mV to various testing potentials (VT). Outward potassium currents were inhibited by substituting cesium for potassium in the pipette filling solution and the extracellular solution. ATP (50-500 pM) had no effect on the current-voltage (I-V) relationship (N = 10) of the L-type current or on the peak current elicited by stepping to V, = 0 mV (N = 7). These results suggest that ATP does not directly effect the L-type calcium current.During the recording of the L-type calcium current, there was an increase in the inward holding current when ATP was applied to the cell. This phenomenon was similar to that described in smooth muscle? To record the ATP-activated current, cells were held at V, = -70 mV and the current was continuously monitored. FIGURE 1A is a representative experiment demonstrating the ability of 100 p M to produce an inward current. Soon after the application of ATP, a slowly activating, long-lasting inward current was elicited. The I-V relationship of the current shown in FIGURE 1B was generated by computer subtraction of the ramp (-100 to +30 mV) current before ATP application from the ramp current after ATP application. The current of each cell, at every 10-mV interval, was normalized to the current at -100 mV. The I-V relationship was linear and had a reversal potential of -1.9 2 6.5 mV (N = 12).Associated with the ATP-activated current was membrane depolarization. When cells were bathed in an extracellular solution devoid of voltage-sensitive channel blockers, ATP depolarized cells to the threshold for the firing of action potentials. (FIG. 2A). The first action potential was typical of a fast sodium-dependent action