In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors

Abstract

In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels

“…First, the relaxation was mimicked by P2Y receptor agonists (UTP and ATPγS) and was inhibited by NEM, which is known to uncouple G proteins from several membrane receptors. NEM findings are in agreement with our previous results [9], which indicated that P2Y receptors are coupled to G q/11 in airway smooth muscle. Therefore, when stimulated, P2Y receptors promote IP 3 , initiating the signal pathway for [Ca 2+ ]i increase, and this induces relaxation [9].…”

“…NEM findings are in agreement with our previous results [9], which indicated that P2Y receptors are coupled to G q/11 in airway smooth muscle. Therefore, when stimulated, P2Y receptors promote IP 3 , initiating the signal pathway for [Ca 2+ ]i increase, and this induces relaxation [9]. Second, this relaxation was only inhibited by co-application of suramin and RB2, the former is known to antagonize all P2Y receptors but not P2Y 4 and the latter inhibits all P2Y receptors but has no activity on P2Y 2 [7,30,31,33].…”

“…Altogether, these data point out that in airway smooth muscle, ATP activates P2Y 2 and P2Y 4 receptors, inducing [Ca 2+ ]i increase as reported previously [9] and favoring PGE 2 release from this tissue, which in turn activates cAMP-dependent K + channels to produce relaxation.…”

“…In this manuscript, we corroborated that the ATP induces relaxation in tracheal rings is a prostaglandin-dependent since it was prevented by indomethacin treatment, but independent from the epithelium. Recently, we have shown that ATP-induced contraction is also prevented by the same treatment or by epithelium removal [9]. The relaxation however, persisted after the removal of the epithelium, indicating that the muscle cells are producing prostaglandins themselves, probably PGE 2 .…”

“…The Ca 2+ -dependent PLA 2 is activated by physiologic increments in [Ca 2+ ] [18]. In a previous study, we showed that ATP, through P2 receptors, can raise [Ca 2+ ]i in airway smooth muscle cells [9], and therefore, it is likely that this effect increases prostaglandins production (PGE 2 ) by stimulation of Ca 2+ -dependent PLA 2 . Alternatively, activation of purinoceptors in this smooth muscle might modulate the Ca 2+ independent PLA 2 through an unknown mechanism.…”