Volume-sensitive chloride current in human cardiac fibroblastsThe current with outward rectification shown in Fig. 1D was insensitive to inhibition of K+ channel blockers including 5 mM tetraethylammonium (TEA), 5 mM 4-AP, or 0.5 mM Ba2+ (n = 4−6), suggesting that the outwardly-rectifying current is not carried by K+ ion. We then employed the Cl− channel inhibitor DIDS to determine whether the current is carried by chloride ions. Figure 5A shows the current traces recorded in a representative cell with the protocol shown in the inset; DIDS (150 µM) suppressed the current. The I-V relationship (Fig. 5B) of the DIDS-sensitive current obtained by subtracting control currents by the current recorded after DIDS application displayed outward rectification and had a reversal potential at −35 mV, which is close to Cl− equilibrium potential (ECl, −46.8 mV). Similar results were obtained in a total of 6 cells. This result suggests that the recorded current under isotonic conditions is carried by Cl− ions.
To investigate whether the Cl− channel is volume sensitive in human cardiac fibroblasts, we employed a 0.7T tonic solution and recorded membrane current using a K+-free pipette solution, symmetrical Cl− ion in pipette and bath medium as described in the Methods section. The membrane conductance was remarkably enhanced by exposure to 0.7T (20 min), and the increased current was highly suppressed by the Cl− channel blocker NPPB (Fig. 5C). The I-V relationship of 0.7T-induced Cl− current is linear under symmetrical Cl− conditions Fig. 5D), similar to the previous report . These results indicate that volume-sensitive Cl− channel (ICl.vol) is present in human cardiac fibroblasts
Angiotensin II, EGF receptors, and reactive oxygen speciesStretch of cardiac myocytes has long been known to release angiotensin II (AngII) , which acts on myocytes in an autocrine-paracrine loop and signals via Src and EGFR kinase in a multistep pathway ultimately leading to the formation of reactive oxygen species (ROS) [47, 90, 99]. AngII and ROS are implicated in cardiac remodelling and the development of heart failure, where ICl,swell is chronically activated [22, 23], and in hypertrophy of vascular smooth muscle [5, 14, 47, 90, 91, 99].
This raised a question: Does activation of the AngII signalling cascade modulate ICl,swell?
The role of AngII signalling was investigated for ICl,swell elicited both by stretching β1 integrins [10, 12] and by osmotic swelling [75, 76].
Block of AT1 receptors by losartan prevents activation of ICl,swell by either stimulus, and exogenous AngII activates a comparable Cl- current. Thus, activation of ICl,swell appears likely to result from the autocrine-paracrine action of AngII. AngII release may not be required, however. Native cardiac and heterologously expressed AT1 receptors are activated by stretch both in the presence of AngII neutralizing antibodies that block responses to exogenous AngII and in the absence of AngII in an expression system . While AT1 receptors are not activated by AngII in these studies, stretch-induced signalling still is blocked by candesartan, an AT1 antagonist.
Other components of the AngII signalling cascade [14, 47, 90, 91] are also implicated in the upregulation of ICl,swell in response to both stretch and swelling [10, 12, 33, 75, 76], and the signalling scheme is shown in Figure 4.
Simplified diagram of autocrine-paracrine signalling cascade that controls ICl,swell. Swelling and stretch initiate the cascade via integrins and one or more downstream molecules. Exogenous AngII and EGF also active ICl,swell. Src family PTK and ROS are likely to interact at additional sites. It is unclear whether ROS activates ICl,swell directly or via intermediates.
AT1 receptor activation causes transactivation of EGF receptors, which possess intrinsic EGFR kinase activity. ICl,swell is blocked by EGFR kinase inhibitors in human atria and rabbit ventricle [12, 33, 75, 76] and is activated by exogenous EGF . EGFR kinase signals via PI-3K, and PI-3K inhibitors wortmannin and LY294002 block ICl,swell. Ultimately this signalling cascade stimulates sarcolemmal NAD(P)H oxidase, which produces ·O2-, and ·O2- rapidly undergoes dismutation to H2O2 a longer-lived, membrane permeant ROS.
Cardiac muscle expresses two NAD(P)H oxidase isoforms, NOX2, the classical phagocyte isoform, and NOX4 [13, 52, 106]. As expected, ICl,swell is blocked by organic inhibitors of NAD(P)H oxidase, including DPI (diphenylene iodinium),