Purinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aorta.

AIM: Exogenous ATP elicits a delayed calcium-independent K(+) current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. METHODS: The patch-clamp technique was used to record ATP-elicited delayed K(+) current in freshly dissociated myocytes. RESULTS: ATP-elicited delayed K(+) current was not inhibited by a 'cocktail' of K(+) channel blockers (4-AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K(+) current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of 'twin-pore potassium channels' (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K(+) current, indicating that P2Y(11) is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13-didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real-time quantitative PCR showed that the genes encoding TASK-1 and TASK-2 are expressed. CONCLUSION: Our results indicate that blocker cocktail-insensitive delayed K(+) current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin-pore channels.

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells.

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.