We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amino acid sequence data. A casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF ligand family. We examined the phosphorylation state of transmembrane tumour necrosis factor-alpha (mTNF) with [32P]orthophosphate labelling and in vitro kinase assays, in lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR ligation in vivo, and this was less effective if phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase in intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this cytokine family.

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Pelvic ganglia are mixed sympathetic-parasympathetic ganglia and provide the majority of the autonomic innervation to the urogenital organs. Here we describe the structural and histochemical features of the major pelvic ganglion in the male mouse and compare two different mouse strains. The basic structural features of the ganglion are similar to those in the male rat. Almost all pelvic ganglion cells are monopolar and most are cholinergic. All contain either neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP), or both peptides together. The peptide coexistence varies between strains, with C57BL/6 mice having similar proportions of neurons with NPY alone, VIP alone or both peptides. In contrast, virtually all pelvic neurons in the Quackenbush-Swiss (QS) strain express NPY, i.e. the level of VIP/NPY coexistence is much higher. Cholinergic axons provide the major nerve supply to epithelia of reproductive organs, bladder smooth muscle and, as described previously, penile erectile tissue. They also provide a minor component of the smooth muscle innervation of the prostate gland, seminal vesicles and vas deferens. Virtually all non-cholinergic pelvic ganglion cells are noradrenergic and contain NPY. Their major target is smooth muscle of reproductive organs. This study shows that the male mouse pelvic ganglion bears many similarities to that in the rat, but that VIP/NPY colocalisation is much more common in the mouse. We also show that there are differences in peptide expression in parasympathetic pelvic neurons between strains of mice. These studies provide the framework for future investigations on neural regulation of urogenital function, particularly in transgenic and knockout models.