Najma H. Javed

ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis

&NA; Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)‐(3‐iodobenzyl)‐adenosine‐5‐N‐methyluronamide (IB‐MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine‐based drugs in experimental inflammatory bowel disease. High‐density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS‐treated (7 days), and oral IB‐MECA‐treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin‐trap monitoring of free radicals. Remarkable colitis‐induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis‐related dysregulation. Downregulation occurs in membrane transporter, mitogen‐activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox‐sensitive genes. Oral IB‐MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB‐MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB‐MECA blocked the colitis‐induced upregulation (≤20‐fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real‐time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB‐MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high‐density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine‐based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

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

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 × 104 M) ≅ UTP (EC50 1.0 × 10−4 M) ≅ 2MeSADP (EC50 = 1.60 × 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 × 10−7 M) > UTP (6 × 10−6 M) > ATP (9 × 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. J. Comp. Neurol. 469:16–36, 2004.

Mechanical stimulation releases nucleotides that activate P2Y1 receptors to trigger neural reflex chloride secretion in guinea pig distal colon.

Stroking the mucosal lining of the guinea pig colon with a brush elicits an intestinal neural reflex, and an increase in short‐circuit current (Isc) indicative of chloride secretion. We tested whether endogenous and exogenous nucleotides are physiologic regulators of mucosal reflexes that modulate chloride secretion. The basal Isc was augmented by 6‐N,N‐diethyl‐β,γ‐dibromomethylene‐D‐adenosine‐5′‐triphosphate (ARL67156) inhibition of nucleotide breakdown or adenosine A1 receptor blockade and reduced by apyrase inactivation of nucleotidases, P2 receptor antagonists, tetrodotoxin (TTX), or piroxicam. ARL67156 augmented, and apyrase inhibited, stroking‐evoked Isc responses. TTX and atropine inhibited nucleotide‐evoked Isc responses. The agonist potency profile for Isc, 2‐methylthioadenosine‐diphosphate (2MeSADP) = 2‐methioadenosine‐triphosphate ≫ 5′adenosine‐triphosphate (ATP) ≥ 5′adenosine‐diphosphate > 5′uridine‐triphosphate ≥ 5′uridine‐diphosphate, supports a P2Y1 receptor (R). The P2 receptor antagonists suramin and pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulfonic acid, reduced stroking responses (36%) and their effects were additive. The selective P2Y1 R antagonist, 2′deoxy‐N6‐methyl adenosine 3′,5′‐diphosphate diammonium salt, reduced stroking (54%) and 2MeSADP (70%) responses at P2Y1 Rs. The P2X1/3 R agonist, α,βMeATP, increased Isc. A desensitizing dose of α,βMeATP reduced stroking Isc responses but did not prevent the 2MeSADP‐evoked Isc response. Reverse transcriptase polymerase chain reaction analysis revealed mRNAs for P2Y1 R, P2Y2 R, P2Y4 R, P2Y6 R, and P2Y12 R in submucosa. The expression of P2Y R immunoreactivity (ir) in cell bodies of submucous neurons followed the order of P2Y1 = P2Y2 ≫ P2Y4 R ir; P2Y1 Rs and P2Y2 R ir were abundant (21–50% of neurons). P2Y1 R ir was abundant in cholinergic secretomotor neurons and fewer than 2% of neuropeptide Y (NPY)/choline acetyltransferase secretomotor neurons, and P2Y2 R ir was expressed in virtually all NPY secretomotor neurons and approximately 30% of calbindin/intrinsic primary afferent neurons. P2Y4 R ir was present in NPY‐positive neurons. P2Y ir was rare or absent in varicose nerve fibers. The functional data support the hypothesis that mechanical stimulation with a brush releases nucleotides that act predominantly at P2Y1 Rs and to a lesser extent on P2X1/3 Rs to mediate reflex chloride secretion. A separate P2Y2 R neural circuit pathway exists that is not activated by mechanical forces. Other receptors including P2Y4, P2Y6, P2Y12, or P4 Rs cannot be excluded. J. Comp. Neurol. 469:1–15, 2004.

Autonomic Nervous System and Secretion across the Intestinal Mucosal Surface

Chloride secretion is important because it is the driving force for fluid movement into the intestinal lumen. The flow of accumulated fluid flushes out invading micro-organisms in defense of the host. Chloride secretion is regulated by neurons in the submucosal plexus of the enteric nervous system. Mechanosensitive enterochromaffin cells that release 5-hydroxytryptamine (5-HT) and activate intrinsic afferent neurons in the submucosal plexus and initiate chloride secretion. Mechanical stimulation by distention may also trigger reflexes by a direct action on intrinsic afferent neurons. Dysregulation of 5-HT release or altered activity of intrinsic afferents is likely to occur in states of inflammation and other disorders.

Oral Feeding of Probiotic Bifidobacterium infantis: Colonic Morphological Changes in Rat Model of TNBS-Induced Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology. It has been proposed that modifying the bacterial flora in intestine with probiotics may decrease the inflammatory process and prevent relapses in UC. We investigated the possible protective and therapeutic effects of a single strand of probiotic, Bifidobacterium infantis (BI), on colonic inflammation, in rats with regular feedings. Two groups of Lewis rats were prepared (n = 8). The first group was the control, sham-fed group (n = 4). The other group was the experimental BI-fed group (n = 4). Colitis was induced in both groups by intrarectal administration of TNBS under light anesthesia. The sham-fed colitis induced groups received a daily oral gavage feeding of 1.0 mL distilled water, whereas the B. infantis-fed group received 0.205 g of B. infantis dissolved in 1.0 mL distilled water daily. The change in body weight and food and water intake was recorded over the course of each study and analyzed. The rats were euthanized and tissues from the descending colon were harvested and analyzed microscopically and histologically. Results of our study indicated significant reduction in inflammation, mucosal damage, and preservation of goblet cells, as compared to the control animals. Modulation of gastrointestinal (GI) flora suggests a promising field in developing strategies for prevention and treatment of inflammatory bowel diseases by dietary modifications.

The regulatory effects of Bifidobacterium infantis on the secretomotor activity of the enteric nervous system

Background: Bifidobacterium infantis (BI) and other probiotics are non-pathogenic living organisms that have gained increased attention for their possible therapeutic implications on the health of the digestive tract. The mechanisms by which probiotics exert their effects are largely unknown.Aims: This study explored the protective and regulatory effect of oral BI on the enteric nervous system in the 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis rats. Materials and Methods: Electrical field stimulation and chemical stimulation by 5 hydroxytryptamine or serotonin were used to elicit changes in short-circuit current response of the colonic rat tissue. Results: BI-fed colitis rats expressed trends of higher secretomotor activity and revealed signs of decreased macroscopic inflammatory damage when compared to sham-fed colitis rats, suggesting a protective and preventative role of oral BI. Conclusion: These findings may provide additional insights for understanding the prophylactic and therapeutic value of specific probiotics in intestinal inflammatory disorders, offering the possibility of a non-invasive alternative to toxic and immune-compromising drugs.