P2Y₁ receptors mediate inhibitory purinergic neuromuscular transmission in the human colon

Abstract

Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2Y(1) receptors. We found that 1) ATP (1 mM) and adenosine 5'-beta-2-thiodiphosphate (ADPbetaS) (10 microM), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 microM) and apamin (1 microM) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited (IC(50) approximately 1 microM) by MRS 2179; 4) ADPbetaS reduced the IJP probably by a desensitization mechanism; 5) apamin (1 microM) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) P2Y(1) receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a P2Y(1) receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. P2Y(1) receptors can be an important pharmacological target to modulate smooth muscle excitability.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca2+ release and SK channel activation

“…Intriguingly, MRS2211 applied alone led to the induction of increased relaxation by 2-Me-S-ADP, for as yet unknown reasons. In contrast, the contraction induced by 2-Me-S-ADP was not affected by any of the P2Y receptor antagonists used in this study and was thus mediated by purinergic receptors other than P2Y 1 , P2Y 6 , P2Y 12 or P2Y 13 . Morphological data on P2 receptor distribution in the gut are scarce.…”

“…Thus, we conclude that α,β-Me-ATP-induced relaxation and contraction were mediated by the activation of the P2Y 1 and P2Y 13 receptors, but the experiments using MRS2500 point to P2Y 1 receptors having the predominant role.…”

“…Furthermore, MRS2578 partially but significantly reduced the α,β-Me-ATP-induced relaxation. These data suggest that the α,β-Me-ATP-induced relaxation is preferentially mediated by the P2Y 1 and P2Y 13 receptors but may also involve other receptors, while the 2-Me-S-ADP-induced relaxation is mediated by P2Y 6 receptor activation ( Figure 6B). P2 receptors are involved in purinergic motility of the rat ileum The purinergic agonist α,β-Me-ATP was introduced as an inhibitor of ionotropic P2X receptors [24,29] .…”

“…This was indeed the case for both the relaxation (0±0 mN, n=8, Figure 6B; see asterisk in Figure 6Aa) and the contraction (2.2±0.2 mN, n=8, Figure 6C). However, the 2-Me-S-ADP-induced relaxation was not affected by co-application of MRS2179 and MRS2211 (-5.0±1.7 mN, n=8, Figure 6B), indicating that 2-Me-S-ADP probably involves receptors other than P2Y 1 and P2Y 13 .…”

“…Immunohistochemical data have demonstrated that ionotropic P2X 2 /P2X 3 and metabotropic P2Y 1 /P2Y 2 receptors are expressed on pre-junctional enteric neurons [7,12] , while smooth muscle cells are predominantly immunoreactive for P2Y 1 receptors [7] . Moreover, there is a great consensus that the purinergic inhibitory junction potential and the corresponding relaxation are highly sensitive to MRS2179 and MRS2500, showing that the P2Y 1 is the receptor type involved in purinergic nerve-mediated relaxation [13][14][15] . This finding has also been confirmed in P2Y 1 knock-out (KO) mice [16][17][18] .…”