Neuronal nitric oxide inhibits intestinal smooth muscle growth

Abstract

Hyperplasia of smooth muscle contributes to the thickening of the intestinal wall that is characteristic of inflammation, but the mechanisms of growth control are unknown. Nitric oxide (NO) from enteric neurons expressing neuronal NO synthase (nNOS) might normally inhibit intestinal smooth muscle cell (ISMC) growth, and this was tested in vitro. In ISMC from the circular smooth muscle of the adult rat colon, chemical NO donors inhibited [(3)H]thymidine uptake in response to FCS, reducing this to baseline without toxicity. This effect was inhibited by the guanylyl cyclase inhibitor ODQ and potentiated by the phosphodiesterase-5 inhibitor zaprinast. Inhibition was mimicked by 8-bromo (8-Br)-cGMP, and ELISA measurements showed increased levels of cGMP but not cAMP in response to sodium nitroprusside. However, 8-Br-cAMP and cilostamide also showed inhibitory actions, suggesting an additional role for cAMP. Via a coculture model of ISMC and myenteric neurons, immunocytochemistry and image analysis showed that innervation reduced bromodeoxyuridine uptake by ISMC. Specific blockers of nNOS (7-NI, NAAN) significantly increased [(3)H]thymidine uptake in response to a standard stimulus, showing that nNOS activity normally inhibits ISMC growth. In vivo, nNOS axon number was reduced threefold by day 1 of trinitrobenzene sulfonic acid-induced rat colitis, preceding the hyperplasia of ISMC described earlier in this model. We conclude that NO can inhibit ISMC growth primarily via a cGMP-dependent mechanism. Functional evidence that NO derived from nNOS causes inhibition of ISMC growth in vitro predicts that the loss of nNOS expression in colitis contributes to ISMC hyperplasia in vivo.

Mitogenic factors promoting intestinal smooth muscle cell proliferation

“…3). Knowledge of the inhibitory influences on growth of ISMC in vivo is also limited, but there is evidence for an inhibitory intrinsic neural input (4,26,29) as well as extracellular matrix-mediated mechanisms (3). These could give insight into understanding of the shift in the balance toward cell growth that in the well-defined model of TNBScolitis is evident by 48 h and complete by day 6 (25).…”

“…We recently identified a mechanism of neural regulation of CSMC proliferation in vitro and proposed that inflammationinduced damage might increase the responsiveness of CSMC to growth factors in vivo (29). Now we hypothesized that the growth factors PDGF and IGF-1 could be important in the early hyperplastic response in colitis, as it is in other smooth muscle systems, and studied this both in vitro and in the TNBS model of colitis in the rat.…”

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression

“…We used the TNBS-induced model of colitis in mice to show that proliferation of CSMC is also associated with GDNF expression in vivo. Immunocytochemistry of sections of the mid-descending colon showed that labeling for the proliferation marker PCNA (Pelletier et al, 2010) was present in some nuclei of CSMC by Day 2 of colitis, and dual-label immunocytochemistry correlated this with the selective appearance of cytoplasmic labeling by anti-GDNF antibodies (Fig. 3E-G).…”

“…As a homeostatic response, smooth muscle proliferation will promote re-innervation via expression of GDNF. In turn, successful re-innervation will tend to inhibit growth and thus restore contractility (Blennerhassett and Lourenssen, 2000;Pelletier et al, 2010).…”

“…This receptor is normally not expressed in these cells, but appears among control cells shortly after enzymatic isolation and tissue culture, as well as by 24 h after intracolonic TNBS administration in the rat. Further, the normal innervation of ISMC also maintains these cells in a quiescent, differentiated state and disruption of innervation promotes hyperplasia and phenotypic modulation (Blennerhassett and Lourenssen, 2000;Pelletier et al, 2010).…”