- Fibroblasts isolated from adult rat ventricles consistently respond to (5–100 μM) ATP.
- ATP, ADP, and UTP all elicit similar dose-dependent increases in [Ca2+]i.
- ATP also activates an outwardly rectifying Cl− current; in contrast, ADP and UTP have much smaller electrophysiological effects.
- This ATP-dependent Cl− current is markedly reduced when: i) [Ca2+]i is buffered with BAPTA, or ii) the intrinsic phospholipase C activity in these fibroblasts is inhibited.
- PCR analysis suggests that the ATP-induced current is generated by the Ca2+-activated Cl− current transcript TMEM 16F, also denoted ANO6.
- These findings reveal new physiological and pharmacological principles that regulate ventricular fibroblast function.
What is the central question of this study?
Although electrophysiological and biophysical characteristics of heart fibroblasts have been studied in detail, their responses to prominent paracrine agents in the myocardium have not been adequately addressed. Our experiments characterize changes in cellular electrophysiology and intracellular calcium in response to ATP.
What is the main finding and its importance?
In rat ventricular fibroblasts maintained in cell culture we find that ATP activates a specific subset of Ca2+-activated Cl− channels as a consequence of binding to P2Y purinoceptors and then activating phospholipase C. This response is not dependent on [Ca2+]o but requires an increase in [Ca2+]i and is modulated by the type of nucleotide that is the purinergic agonist.
Effects of adenosine 5′-triphosphate (ATP) on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36–72 hrs) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or UTP all produced large increases in intracellular Ca2+ [Ca2+]i. Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1–100 μM) activated an outwardly rectifying current, having a reversal potential very close to the Nernst potential for Cl−. In contrast, ADP was much less effective and UTP produced no detectable current. The nonselective Cl− channel blockers niflumic acid, DIDS and NPPB (each at 100 μM), blocked the responses to 100 μM ATP. 2-MTATP, an agonist for P2Y purinoceptors activated a very similar outwardly rectifying C1− current. The P2Y receptor antagonists, suramin and PPADS (100 μM each), significantly inhibited the Cl− current produced by 100 μM ATP. ATP was able to activate this Cl− current when [Ca2+]o was removed, but not when [Ca2+]i was buffered with BAPTA-AM. In the presence of the phospholipase C (PLC) inhibitor U73122, this Cl− current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors; and also for the Ca2+-activated Cl− channel, TMEM 16F (also denoted ANO-6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a PLC-dependent increase in [Ca2+]i, followed by activation of a Ca2+-dependent Cl− current in rat ventricular fibroblasts.
This article is protected by copyright. All rights reserved
from Physiology via xlomafota13 on Inoreader http://ift.tt/2oGXl0t