Molecular studies of tachykinin and vanilloid receptors : expression and function

Tachylcinin receptors (NK1, NK2 and NK3) and transient receptor potential vanilloid channels (TRPV1 and TRPV2) are widely distributed throughout the central nervous system (CNS) and peripheral tissues of mammals. They play central roles in nociception, cardiovascular and respiratory function. In addition, tachykinin receptors and TRPV1 appear to play vital roles in neurogenic inflammation and in the modulation of the innate immune system through PBMCs and monocytes. This thesis represents a 10 year research journey that documents: 1) the development of an extensive range of molecular biological techniques for the analysis of tachykinin receptor and TRPV expression; 2) the effects of capsaicin administration on tachykinin receptors and TRPV expression in CNS and peripheral tissue of rats, and 3) the expression and potential function of TRPV1 in human peripheral blood mononuclear cells (PBMC), na‚àövòve monocytes and the human monocytic, THP-1 cell line. In the rat studies, quantitation of tachykinin receptor and TRPV1 expression in both CNS and peripheral tissues, including PBMC, provided some interesting fmdings. The mRNA expression of cFOS as a marker of FOS mediated transcription activity was also quantified as an early measure of cellular transcription levels. Overall, cFOS was the most highly expressed gene in all tissues studied with expression levels >1 x 106 copies mRNA transcript/106 copies GAPDH. In CNS tissues, NK3 expression was consistently higher than NK1 and NK2 expression, which is supported by later published observations based on gene chip data. NK2 expression was very low in all CNS tissues studied, particularly the brain stem which was in agreement with autoradiographic mapping of NIC2 receptors using [ 125 I]-NKA in our laboratory. In general, the expression profile of tachykinin receptors in CNS tissue was NK3>NK1>NK2. In the peripheral tissues studied, the expression profiles differed markedly, though as a general rule they agreed with previously published radioligand binding data. Systemic capsaicin administration produced changes in tachykinin receptor mRNA expression in specific tissues. There was no effect on tachykinin receptor mRNA expression in the cerebral cortex or brain stem in response to capsaicin (data which agreed with our initial pilot study). However, in the cervical spinal cord of capsaicinised rats, NK1 and NK3 mRNA transcripts were significantly lower compared to vehicle controls indicating a significant down-regulation, whilst NK2 and TRPV1 message were significantly higher. Surprisingly, up-regulation of cFOS expression was not observed with the significant changes in receptor expression in the cervical spinal cord. In peripheral tissues, capsaicin administration significantly down-regulated NK1 transcription in the urinary bladder This finding corresponds well with previous data showing a decrease of NK1 receptor radioligand binding in the urinary bladder in response to capsaicin. The most marked changes in receptor expression levels were seen in PBMC where, although initial receptor expression was low there were significant changes in cF0S, NK1, NK2 and NK3 mRNA expression in response to capsaicin. Thus, stimulation of TRPV1‚ÄövÑvÆexpressing neurons by capsaicin in both the CNS and peripheral tissues leads to quite dramatic changes in the mRNA expression of tachylcinin receptors. Collectively, this series of experiments showed that modern molecular techniques could be used to identify quantitative changes in pharmacological receptor transcript following specific interventions. In the human studies, both TRPV1 and TRPV2 expression was demonstrated in PBMC and isolated na‚àövòve monocytes for the first time. The mRNA expression of both TRPVI and TRPV2 was much greater in PBMC (>90% lymphocytes) compared to isolated na‚àövòve monocytes, whilst TRPV2 expression was significantly higher than TRPV1 expression. TRPV1 mRNA and protein (measured by Western blot) was also highly expressed by monocytic THP-1 cells. In PBMC, naive monocytes and THP-1 cells, low concentrations of capsaicin dose-dependently increased cell metabolic activity. This effect occurred immediately and was maintained for at least 1 hour, on exposure to capsaicin concentrations that had previously been shown to cause cell death. The capsaicin-induced increase in metabolic activity was abolished by prior exposure to the TRPVI antagonist, 5'-iodoresiniferatoxin, indicating a TRPV I mediated mechanism. In addition to stimulating metabolic activity, capsaicin was also shown to dramatically modify mitogeninduced inflammatory cytokine release from THP-1 cells. Both phytohaemagglutinin A (PHA) and lipopolysaccharide (LPS) stimulated the release of IL-1I3, IL-6, TNF-a and MCP-1 from THP-1 cells. Capsaicin pretreatment significantly reduced PHAsimulated release of all four cytokines, an effect which concentration-dependent. In contrast, capsaicin pretreatment significantly increased LPS-stimulated IL-6 and TNF-a secretion. Thus, prior exposure of cells to capsaicin, at levels that would theoretically equate to those present after a high dose chilli meal, resulted in significant reductions in PHA-stimulated inflammatory cytokine release. The overall anti-inflammatory effects of capsaicin reported here may partly explain the apparent protective role of TRPV1 in atherosclerosis and diabetes. Thus, this series of experiments identified TRPV expression on na‚àövòve monocytes, and validated THP-I cells as a model system for the study of TRPV1 message, protein and function monocytes. In conclusion, the studies described in this thesis demonstrate a number of important features of tachykinin receptor and TRPV expression in rat and human cells. In the rat brain stem, there is no change in mRNA expression of the tachykinin receptors in response to the systemic administration of capsaicin. This suggests that the modulation of respiratory function identified in previous studies in this laboratory may not have been due to changes in genetic expression but some other dynamic regulatory process (although the capsaicin treatment regimens were different). Significant and surprising changes in cFOS and tachykinin receptor expression were seen in whole blood suggesting an immunological aspect to tachykinin functions. For the first time, TRPV 1 and 2 expression profiles of PBMC (>90% lymphocytes) and na‚àövòve monocytes were compared and shown to be substantially different. Moreover, a (THP-1) cell culture system for the study of TRPV1 function in monocytes was established and revealed that capsaicin not only produces a novel metabolic response in monocytes but may also have significant immunomodulatory effects by dramatically altering mitogen-stimulated cytolcine release.