ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation

Abstract

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'_adenyl-imidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'_O-(3-thiotriphosphate), ATPãS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca¥. 5. A broad spectrum inhibitor of endogenous serineÏthreonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

Cardiac purinergic signalling in health and disease

“…ATP triggers arrhythmias in electrically stimulated rat cardiac myocytes [703]. ATP prevents the run-down of gap junctional communication between ventricular myocytes in newborn rats by promoting protein phosphorylation [704]. In a later paper, P2X1R were shown to be closely associated with connexin 43 in gap junctions in the human ventricular myocardium [286] and the possibility that decreased expression of P2X1R is related to arrhythmias in the heart in Chagas disease and diabetes was considered [705].…”

RhoA GTPase and F-actin Dynamically Regulate the Permeability of Cx43-made Channels in Rat Cardiac Myocytes

“…This loss of channel activity observed in cardiac myocytes when the activity of PKs is impeded (e.g. in ATPdeprived conditions) was interpreted as being due to the unbalanced activity of endogenous protein phosphatase(s) (3) ascribed to the activity of PP1 (4). When ATP was replaced by GTP (5 mM) in the pipette filling solution, G j also remained relatively stable with time, whereas it rapidly declined when ATP was absent (Fig.…”

“…In the latter configuration, a channel rundown, as for gap junctional channels, was observed when cells were exposed to metabolic inhibitors or dialyzed with ATP concentrations Ͻ2 mM or when ATP was replaced by its non-hydrolyzable analogue AMP-PNP. For both gap junctional and inwardly rectifying K ϩ currents, the channel activity remained sustained when ATP was replaced by GTP in the pipette solution (59) and when the activity of endogenous phosphatases was inhibited in ATP-deprived conditions (3,58) and was lowered when the endogenous phosphatase activity was enhanced (4,58). But the behavior of channels differed when cells were exposed to actin-depolarizing agents: cytochalasin B had no effect on the activity of inwardly rectifying K ϩ channels (58), whereas in the present study, cytochalasin D rapidly reduced gap junctional conductance.…”

Reversible Connexin 43 Dephosphorylation During Hypoxia and Reoxygenation Is Linked to Cellular ATP Levels

“…[37][38][39][40] Surprisingly, metabolic dephosphorylation of Cx43 did not require PP1 because it was insensitive to calyculin A and okadaic acid in our system, although calyculin A and higher concentrations of okadaic acid reduced the relative amount of dephosphoCx43 in control cells. We also considered the possibility that AMP kinase, an ATP sensor, could be involved in this regulatory dephosphorylation indirectly via activation of unknown phosphatases; however, direct activation of AMP kinase by AICAR did not induce Cx43 dephosphorylation (data not shown).…”