Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors

Abstract

Mechanical activation of the mucosal lining of the colon by brush stroking elicits an intestinal neural reflex and an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport. We tested whether endogenous nucleotides are physiologic regulators of mucosal reflexes that control ion transport. The brush stroking-evoked Isc response in mucosa and submucosa preparations (M-SMP) of rat colon was reduced by the P2Y1 receptor (R) antagonist 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179) and further blocked by tetrodotoxin (TTX). M-SMP Isc responses to serosal application of the P2Y1 R agonist 2-methylthioadenosine-diphosphate (2MeSADP) or the P2Y2/P2Y4 R agonist 5'uridine-triphosphate (UTP) were reduced but not abolished by TTX. The potency profile of nucleotides for increasing Isc was 5'adenosine-triphosphate (ATP; effective concentration at half maximal response [EC50] 0.65 x 10(4) M) congruent with UTP (EC50 1.0 x 10(-4) M) congruent with 2MeSADP (EC50 = 1.60 x 10(-4) M). Mucosal touch and distention-induced Ca2+ transients in submucous neurons were reduced by apyrase and prevented by blocking the P2Y1 R with MRS 2179 and TTX; denervation of the mucosa. It did not occur by touching a ganglion directly. 2MeSADP Ca2+ responses occurred in subsets of neurons with or without substance P (SP) responses. The potency profile of nucleotides on the neural Ca2+ response was 2MeSADP (5 x 10(-7) M) > UTP (6 x 10(-6) M) > ATP (9 x 10(-5) M). The expression of P2Y R immunoreactivity (ir) in nerve cell bodies was in the order of P2Y1 R > P2Y4 R >> P2Y2 R. P2Y1R ir occurred in the cell somas of more than 90% of neuronal nitric oxide synthase, vasoactive intestinal peptide (VIP), calretinin, or neuropeptide Y (NPY)-ir neurons, 78% of somatostatin neurons, but not in calbindin or SP neurons. P2Y2 R ir was expressed in a minority of SP, VIP, NPY, vesicular acetylcholine transporter, and calcitonin gene-related peptide-ir varicose fibers (5-20%) and those surrounding calbindin (5-20%) neurons. P2Y4 ir occurred mainly in the cell somas of 93% of NPY neurons. Reverse transcriptase polymerase chain reaction of the submucosa demonstrated mRNA for P2Y1R, P2Y2, P2Y4, P2Y6, and P2Y12 Rs. Expression of P2Y1, P2Y2, and P2Y4 protein was confirmed by western blots. In conclusion, endogenous nucleotides acting at P2YRs transduce mechanically evoked reflex chloride ion transport in rat distal colon. Nucleotides evoke reflexes by acting primarily at postsynaptic P2Y1 Rs and P2Y4 R on VIP+/NPY+ secretomotor neurons, at P2Y2 Rs on no more than 2% of VIP+ secretomotor neurons, and 2Y2 Rs mainly of extrinsic varicose fibers surrounding putative intrinsic primary afferent and secretomotor neurons. During mucosal mechanical reflexes, it is postulated that P2Y1 R, P2Y2 R, and P2Y4 R are activated by endogenous ATP, UTP, and 5'uridine-diphosphate.

Purinergic receptors and gastrointestinal secretomotor function

“…Rotational shaking of tissue preparations or the EC/BON cells in culture predominantly activates a Gαq/PLC/inositol triphosphate (IP 3 )-Ca 2+ signaling pathway leading to 5-HT release [59]. A separate Gs/Gi/AC/ PKA (protein kinase A)/cAMP signaling pathway provides a minor contribution to mechanosensitive 5-HT release [18]. 13 receptors had no effect.…”

“…3d); the A 3 effect was restricted to the Ca 2+ signaling pathway. The A 1 receptor also contributes, and an A 1 receptor antagonist (CPT), an A 3 receptor antagonist (MRS1191), or adenosine deaminase could also augment the touch/stretch Ca 2+ response ( [18]. Recent isolation of ileal EC cells [58] may permit further study and comparison of purinergic signaling mechanisms in normal and diseased human ileum.…”

Mediation of neurogenic ion transport by acetylcholine, prostanoids and 5-hydroxytryptamine in porcine ileum

“…As PPADS has some selectivity for P 2 -purinoceptors, we cannot rule out the possibility that electrically-evoked mucosal responses are mediated at least in part by ATP acting through P 1 -purinoceptors. However, active intestinal ion transport has been shown to be modulated predominately by P 2 -purinoceptors (Cressman et al, 1999;Christofi et al, 2004). The lack of effect of thiorphan on this response lends further support for the hypothesis that VIP, substance P and other classes of pro-secretory neuropeptides are not substantially involved in mediating this effect.…”

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

“…It is therefore conceivable that these cells are also important mediators of purinergic relaxation in the rat ileum, which harbors SK3-expressing fibroblast-like cells that form gap junctions with smooth muscle cells [42] , as has recently been shown in the murine colon [43] . Another limitation of our study might be that purinergic receptors are also expressed in epithelial cells and hence are involved in secretory processes [44][45][46] . Therefore, we cannot exclude the possibility that purinoceptor activationmediated secretion could have affected the relaxation and contraction in our preparations.…”