Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP.

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3',5'-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). [Formula: see text]-Nitro-L-: arginine methyl ester (L-: NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.

Enteric glia promote functional recovery of CTM reflex after dorsal root transection.

Transected dorsal root axons of adult rats can be induced to regenerate through the normally non-permissive environment of the dorsal root entry zone (DREZ) into the spinal cord by implanting enteric glia (EG) into the DREZ. We have now examined whether the regenerating central axons make functional connections by studying the return of function of a behavioral response, the cutaneous trunci muscle (CTM) reflex. Implantation of EG into the spinal cord DREZ led to functional recovery of the CTM reflex in 82%, 72% and 70% of animals 1, 2 and 3 months, respectively, after injury. In contrast, the CTM reflex did not recover in animals implanted with 3T3 or C6 glioma cells or with vehicle only.

Enteric glia promote regeneration of transected dorsal root axons into spinal cord of adult rats.

After spinal cord injury axonal regeneration is poor, but may be enhanced by the implantation of olfactory ensheathing glia (OEG). Enteric glia (EG) share many properties of OEG. Transected dorsal root axons normally do not regenerate through the central nervous system myelin into the spinal cord. We tested whether EG, like OEG, could promote regeneration in this paradigm. Three weeks after EG implantation, numerous regenerating dorsal root axons reentered the spinal cord. Ingrowth of dorsal root axons was observed using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal gray commissure. No axonal ingrowth was observed in control animals, indicating that transplanted EG enabled regeneration of the injured dorsal root axons into the adult spinal cord. Thus, EG implantation may be beneficial in promoting axonal growth after central nervous system injury.

A method for purifying enteric glia from rat myenteric plexus.

The enteric nervous system is a large and complex division of the peripheral nervous system. The glia associated with it share some characteristics with the olfactory-ensheathing glia, astrocytes and Schwann cells. To facilitate studies of rat enteric glia, we have developed a method for preparing them in large quantities with a high degree of homogeneity. The enteric glia were isolated from the small intestine of Wistar rats by enzymatic digestion with dispase. The cell isolate was added to a mitotically arrested layer of 3T3 cells. Subsequent separation of the enteric glia from the 3T3 cells was done enzymatically, with unavoidable loss of many enteric glia and potential contamination of enteric glia cultures with the 3T3 cells. Therefore, 3T3 cells were cultured in Nunc 0.2-microm tissue culture inserts that could be readily removed from the wells when no longer needed. There was no loss of the enteric glia. The cultures consisted entirely of GFAP-labeled cells, presumptive enteric glia. This method permits the culturing of large numbers of highly purified enteric glia without the use of expensive growth factors and complement-mediated cytolysis.