Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42 degrees C) increased ET fluorescence by 24 +/- 4%, whereas in control (37 degrees C), fluorescence decreased by 8 +/- 1% compared with baseline (P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 +/- 7 nmol cytochrome c. g dry wt(-1). ml(-1) were reduced in heat compared with 22 +/- 13 nmol. g(-1). ml(-1) in controls (P < 0.001). SOD decreased cytochrome c reduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.

Adenosine receptors (ADORs) in the enteric nervous system may be of importance in the control of motor and secretomotor functions. Gene expression and distribution of neural adenosine A1, A2a, A2b, or A3 receptors (Rs) in the human intestine was investigated using immunochemical, Western blotting, RT-PCR, and short-circuit current (I(sc)) studies. Adenosine A1R, A2aR, A2bR, or A3R mRNAs were differentially expressed in neural and nonneural layers of the jejunum, ileum, colon, and cecum and in HT-29, T-84, T98G, and Bon cell lines. A1R, A2aR, A2bR, and A3R immunoreactivities (IRs) were differentially expressed in PGP 9.5-immunoreactive neurons. A2bR IR occurs exclusively in 50% of submucosal vasoactive intestinal peptide (VIP) neurons (interneurons, secretomotor or motor neurons) in jejunum, but not colon; A2aR is also found in other neurons. A3R IR occurs in 57% of substance P-positive jejunal submucosal neurons (putative intrinsic primary afferent neurons) and less than 10% of VIP neurons. Western blots revealed bands for A3R at 44 kDa, 52 kDa, and 66 kDa. A2aR and A2bR are coexpressed in enteric neurons and epithelial cells. 5'-N-methylcarboxamidoadenosine or carbachol evoked an increase in I(sc). A2bR IR is more prominent than A2aR IR in myenteric neurons, nerve fibers, or glia. A1R is expressed in jejunal myenteric neurons and colonic submucosal neurons. Regional differences also exist in smooth muscle expression of ADOR IR(s). It is concluded that neural and nonneural A1, A2a, A2b, and A3Rs may participate in the regulation of neural reflexes in the human gut. Clear cell and regional differences exist in ADOR gene expression, distribution, localization, and coexpression.

SUMMARY Mechanisms resulting in abdominal pain include altered neuro-immune interactions in the gastrointestinal tract, but the signaling processes that link immune activation with visceral hypersensitivity are unresolved. We hypothesized that enteric glia link the neural and immune systems of the gut and that communication between enteric glia and immune cells modulates the development of visceral hypersensitivity. To this end, we manipulated a major mechanism of glial intercellular communication that requires connexin-43 and assessed the effects on acute and chronic inflammation, visceral hypersensitivity, and immune responses. Deleting connexin-43 in glia protected against the development of visceral hypersensitivity following chronic colitis. Mechanistically, the protective effects of glial manipulation were mediated by disrupting the glial-mediated activation of macrophages through the macrophage colony-stimulating factor. Collectively, our data identified enteric glia as a critical link between gastrointestinal neural and immune systems that could be harnessed by therapies to ameliorate abdominal pain.

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 (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.

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Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5′-triphosphate (UTP), or uridine diphosphate (UDP) (P2X 1-7 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 12 receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A 1 , A 3 , P2Y 12 ) or excitatory (A 2 , P2Y 1 ) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y 1 , other P2Y, P2X) or inhibitory (A 1 , A 3 ) receptors are involved in mechanically evoked secretory reflexes or "neurogenic diarrhea." Distinct neural (pre-or postsynaptic) and non-neural distribution profiles of P2X 2 , P2X 3 , P2X 5 , P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , or P2Y 12 receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A 2b , P2Y 2 , P2Y 4 , and P2Y 6 receptors are involved in fluid and Cl -, HCO 3 -, K + , or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A 2a or A 3 receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.

Superoxide anion radical (O(2)(*-)) is released from skeletal muscle at rest and is particularly elevated during conditions of heat stress (42 degrees C). Previous studies have shown that in isolated rat diaphragm O(2)(*-) release is not dependent on mitochondrial electron transport, reduced NADP oxidase activity, or the integrity of membrane anion channels. This study hypothesized that O(2)(*-) release, as measured by cytochrome c reduction, is linked to metabolism of arachidonic acid. Phospholipase A(2) inhibition with manoalide significantly decreased O(2)(*-) release. In downstream pathways, neither the blockage of cyclooxygenase with indomethacin nor the inhibition of cytochrome P-450-dependent monooxygenase with SKF-525A decreased O(2)(*-) release. However, lipoxygenase (LOX) inhibition with general LOX blockers 5,8,11,14-eicosatetraynoic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate greatly attenuated the signal. Furthermore, the specific 5-LOX inhibitor diethylcarbamazine also significantly decreased O(2)(*-) release. Immunohistochemistry localized 5- and 12-LOX to the cytosol and sarcolemma of muscle cells. Confocal studies, using the O(2)(*-)-sensitive fluorescent indicator hydroethidine, demonstrated that LOX inhibition had no significant influence on intracellular O(2)(*-) formation. When compared with the cytochrome c results, this indicates that intra- and extracellular O(2)(*-) must arise from different sources. These data show for the first time that arachidonic acid metabolism through LOX activity, is a major source of extracellular O(2)(*-) release in skeletal muscle.

Nucleotides such as ATP and UTP are everywhere. They play a key role in transducing mechanosensory signals via P2Y receptors in the large intestine or colon, leading to secretion of the sensory mediator 5-HT, and act as autocrine, paracrine, or neurocrine mediators in neural reflexes regulating chloride secretion.