Nitric oxide and carbon monoxide: Novel neurotransmitters in the enteric nervous system

The gases nitric oxide (NO) and carbon monoxide (CO) function as novel signaling molecules in the central and peripheral nervous systems. Their role in the regulation of physiology and pathophysiology of disease extends to vital functions such as learning and memory, stroke, respiration and erections. In this series of studies, we ascertain the role of NO and CO as co-neurotransmitters in the enteric nervous system. We determine that nNOS dysregulation contributes to diabetic gastroparesis and other diabetic enteropathies using streptozotocin treated (STZ) and non-obese (NOD) diabetic mouse models. Diabetic mice develop enteric dysfunction exhibited by delayed gastric emptying, abnormal intestinal transit and defects in NANC neurotransmission in organ bath preparations. Monitoring nNOS expression using western and Northern blots, immunohistochemistry and in situ hybridization techniques in diabetic mice, we establish that insulin regulates nNOS expression and function. Using genetically altered strains of mice lacking the synthetic enzymes for CO and NO, heme oxygenase 2 (HO2) and neuronal nitric oxide synthase, (nNOS) respectively, we determine that NO and CO are important in the control of gastric emptying and intestinal transit in whole animal models. We define the relative contributions of NO and CO to nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission within the ENS using ex-vivo organ bath preparations of mouse pylorus, ileum and internal anal sphincter muscles from wild-type nNOS Δ/Δ , HO2Δ/Δ and nNOS Δ/Δ /HO2Δ/Δ mice. We correlate these functional observations with molecular interactions between CO and NO by measuring enzyme activity and cGMP production in intestinal smooth muscle. We demonstrate that vasoactive intestinal peptide (VIP) causes a HO2 mediated smooth muscle relaxation that is absent in HO2 Δ/Δ mice. We provide evidence for an enhanced VIP mediated relaxation in the nNOS knockouts. We ascertain that CO in the presence of YC-1, a stimulant of sGC, causes NANC relaxation to the same degree as NO alone. We determine that CO is dynamically activated by protein kinase C and casein kinase II to mediate relaxation and acts in an additive or synergistic way with nNOS. These data establish CO as a novel neurotransmitter that also augments the effects of NO as a co-neurotransmitter.