ATP increases [Ca²⁺]_i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa

Abstract

 Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca²⁺ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca²⁺ ([Ca²⁺]_i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca²⁺]_i in crypt base cells with an EC₅₀ of 4.5 μmol/l (n = 11). This [Ca²⁺]_i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca²⁺]_i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca²⁺]_i (peak and plateau) as compared to basal cells and in surface cells this [Ca²⁺]_i transient was even further reduced. Attempts to identify the relevant P₂-receptor demonstrated the following rank order of potency: 2MeS-ATP > ADP ≥ ATP >> AMP > UTP > AMP-PCP > adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (V _te) and equivalent short-circuit current (I _sc): ΔI _sc = –36.4 ± 5.4 μA/cm², n = 17. Adenosine itself (1 mmol/l) induced an increase of I _sc of similar magnitude to that induced by ATP: ΔI _sc = –55.1 ± 8.4 μA/cm², n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A₁/A₂-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca²⁺]_i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase I _sc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca²⁺]_i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt.