Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium

Abstract

Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). UTP-prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). Inhibitors of either protein kinase C (PKC) (D-erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl(-) secretion and inhibits basal Na(+) absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y(2) receptors and an increase in [Ca(2+)](in), but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.

Figure 1

Effect of ATP and UTP on I_SC across tracheal epithelium. Paired tissues were treated with either UTP (100 μM mucosal) or ATP (100 μM mucosal). (A) Shows mean I_SC±s.e.mean of 11 pairs. (B) Shows charge transfer, calculated as area between UTP-induced I_SC and basal I_SC (dotted line in A). Positive charge transfer represents increased mucosal negativity. ^*Indicates significant difference from effect of UTP (Paired Student's t-test, P<0.05).

Figure 2

Dose dependency of the increase (Peak) and decrease below basal after 10 min (after 10 min) induced by UTP. Trachea were treated with either 10⁻⁷, 10⁻⁶, 10⁻⁵, 5×10⁻⁵, 10⁻⁴ or 5×10⁻⁴ M mucosal UTP. Responses are represented as a fraction of the response of paired tissues to 100 μM UTP. Data are mean±s.e.mean (n=6 (10⁻⁶, 10⁻⁵), 7 (10⁻⁷, 5×10⁻⁵) or 3 (5×10⁻⁴ M)).

Figure 3

Cross desensitization of ATP and UTP. Paired tissues were pretreated with either UTP (100 μM mucosal; (A), or ATP (100 μM mucosal; (B), before addition of UTP (100 μM mucosal). Data are mean±s.e.mean (n=5).

Figure 4

Effects of submucosal ATP and UTP. Paired tissues were treated with either mucosal or submucosal UTP (A) or ATP (B) before addition of the same nucleotide to the contralateral side. Data are mean±s.e.mean (n=5).

Figure 5

Effect of ion transport blockers on response to UTP (100 μM mucosal). (A) Shows effect on paired tissues either pretreated with bumetanide (100 μM submucosal; bumetanide) or left without pretreatment (control) (n=6). (B) Shows effect on paired tissues either pretreated with amiloride (10 μM mucosal; amiloride) or left without pretreatment (control) (n=6). Data are mean±s.e.mean.