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'-adenylimidodiphosphate, 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), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine 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).

Figure 1. Recording arrangement for the exchange of the dialysing solution of cell 2

A glass capillary set inside patch pipette 2, 100-300 μm from the tip, allowed, by applying a negative pressure, the exchange of internal solution during the whole-cell recordings of gap junctional conductance between myocytes 1 and 2.

Figure 2. Evolution with time of the gap junctional conductance measured in pairs of ventricular myocytes of neonatal rat in different conditions

Both cells were clamped at -70 mV and one of them was stepped to -80 mV every 30 s while the second cell was maintained at the holding potential. Due to the transjunctional voltage difference, a current crossed the cell-to-cell junction, compensated by an opposite current supplied by the feedback amplifier connected to the cell maintained at -70 mV. Junctional conductances are presented in units of their original value (means ±s.e.m.). A, in perforated patch configuration (without Mg-ATP), the degree of cell-to-cell communication remained stable as long as tight seals were preserved (original G_j, 25·3 ± 7·8 nS; n= 12). B, in whole-cell conditions, when Mg-ATP (5 mM) was present in the patch pipette solution, intercellular electrical coupling was well preserved, even after 30 min (original G_j, 29·4 ± 6·3 nS; n= 25). C, in contrast, in the absence of Mg-ATP in the patch pipette solution, a progressive fading of junctional conductance was observed (original G_j, 31·2 ± 11·3 nS; n= 9). D, a channel rundown was similarly observed in the absence of ATP and presence of MgCl₂ (1 mM) (original G_j, 73·9 ± 11·0 nS; n= 9).

Figure 3. Junctional channel rundown elicited by unilateral ATP removal was partially reversible after ATP reintroduction

When the ATP-containing solution filling patch pipette 2 was replaced by an ATP-free medium, a progressive loss of channel activity was observed, leading to a complete interruption of cell-to-cell communication (•). When the ATP-free solution introduced near the pipette tip was replaced by an ATP-enriched solution (as indicated at the top), the decline in junctional conductance was stopped, then partially reversed (□). Conductances are presented in units of their original values, 28 (•) and 22 nS (□). Similar results were obtained in 6 (•) and 9 (□) myocyte pairs.

Figure 4. A non-hydrolysable ATP analogue (AMP-PNP) failed to preserve cell-to-cell communication

When, in dual whole-cell configuration, ATP was replaced in the pipette filling solution with AMP-PNP (5 mM), a progressive decline of the junctional conductance presented in units of its original value was observed. Original G_j, 35·2 ± 15·8 nS, n= 7.

Figure 5. A dephosphorylating treatment interrupted cell-to-cell communication

When a non-specific dephosphorylating enzyme (alkaline phosphatase, 10 U ml⁻¹) was intracellularly applied, the junctional conductance, presented in units of the original conductance, 28 (•) and 32 nS (▴), progressively declined and led to complete interruption of intercellular communication (▴); in contrast, in the presence of an inhibitor of alkaline phosphatase activity (β-glycerophosphate, 50 mM, •), the main part of the intercellular coupling was preserved. Similar results were obtained in each of 9 (▴) and 8 (•) myocyte pairs.

Figure 6. Examples of quantification of the cell-to-cell dye diffusion in cardiac ventricle myocytes of newborn rat cultured for 3 days

Grey density images of fluorescence intensities (A-C and D-F) were obtained by scanning a group of cells with low intensity light pulses. After a prebleach scan (A and D), 6-CF was photobleached in a selected area (polygon 1) by means of strong illumination. The evolution of the fluorescence levels in the selected cells was compared in the same set of cells, in control conditions (A-C) and after a 60 min exposure to H7 (able to penetrate into intact cells; 1 mM) (D-F), just after photobleaching (B and E) and 8 min later (C and F). Graphs: time courses of the fluorescence emission of the selected cell before (G) and after (H) H7 application. The fluorescence intensity is represented as a percentage of the prebleach emission versus the time after photobleaching. In control conditions (G), the fluorescence emission of the bleached cell increased progressively whereas after H7 treatment (H), the fluorescence level only slightly rose in the bleached cell (continuous lines). In both cases, the light emission of unbleached cell remained stable (dashed lines).

Figure 7. Cell-to-cell communication between ventricular myocytes was progressively and reversibly hindered by H7

A, time-dependent reduction of the relative permeability constant (k) of dye transfer after 15, 30 and 45 min exposures to H7, a non-specific inhibitor of serine/threonine protein kinases. B, recovery of cell-to-cell dye transfer after washing off H7. k was determined in control conditions (a), after exposure to H7 (1 mM for 1 h; b), and then 30 min after washing with control medium (c). Bars indicate k values (min⁻¹) as means and s.e.m., with values of n above.

Figure 8. Time course of the H7-induced reduction in gap junctional conductance

In the presence of H7 (1 mM) and ATP (5 mM) in the patch pipette solution, a progressive decline of junctional conductance (presented in units of its original value) was observed, reaching after approximately 10 min a stable plateau equivalent to about 13 % of its initial level (original G_j, 71·1 ± 10·1 nS; n= 6).

Figure 9. Non-selective inhibition of endogenous protein phosphatases preserved cell-to-cell communication in ATP-deprived conditions

The loss of junctional channel activity, in whole-cell, ATP-free conditions, was interrupted in the presence of a ‘broad spectrum cocktail’ of protein phosphatase inhibitors (KF 50 mM + orthovanadate 1 mM), allowing preservation of a substantial cell-to-cell communication (original G_j, 33·2 ± 15·8 nS; n= 9).