Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4 -1 M and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100 -200 g͞kg doses of sialorphin required the activation of -and ␦-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

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We have recently described a mAb (2.11) that recognizes the Vgamma1-Jgamma4-Cgamma4 chain. With this mAb and an anti-delta mAb we separated gammadelta+ 2.11(+) and gammadelta+ 2.11(-) intraepithelial lymphocytes (i-IEL) by FACS. Transcripts of rearranged TCR Vgamma1 and Vdelta2 genes in both i-IEL populations were analyzed by PCR followed by sequence analysis of cDNA spanning the junction of variable (V) and joining (J) genes. Roughly the same number of Vgamma1 and Vgamma2 transcripts were found in the 2.11(+) population while >90% of the transcripts in the 2.11(-)population contained a Vgamma gene sequence. Furthermore, gamma transcripts in the 2.11+ population were functional, while only 30-40% of the Vgamma transcripts in either population contained an in-frame sequence. The observed frequency of transcripts is what would be expected from cell populations that have not gone through cellular selection mediated by the RCR. Expression of Vgamma mRNA in RCRalphabeta and TCRgammadelta thymocytes was studied by a technique that analyzed populations of transcripts of rearranged genes. In both T cell population similar levels of Vgamma transcripts were found and about two out of three transcripts were out-of-frame. During the cloning and sequence analysis, we indentified a clone that expresses the Vgamma segment rearranged to the Jgamma3-Cgamma region in C57BL/6 mice. Together with the PCR cloning and sequencing of the complete Cgamma region in C57BL/6 mice these data demonstrate that the Jgamma-Cgamma gene is functional in this strain. Taken together, these studies revealed that: (i) cells expressing the Vgamma1 chain are an important subset of the gammadelta i-IEL population and that they show extensive junctional diversity; (ii) there is no correlation between expression of in-frame Vgamma transcripts and expression of Vgamma2 chains at the cell surface; and (iii) cells expressing the Vgamma3 chain might be a minor subset of the gammadelta T cell population in C57BL/6 mice.