In the present study, we demonstrated that the TRPV1–4 genes are expressed in human leukocytes. While the rates of expression were not influenced by gender, we found significant differences for TRPV1 between healthy subjects and patients hyposensitive to painful thermal and capsaicin stimulation.
The genes belonging to the transient receptor potential (TRP) family, and in particular the members of the TRPV subfamily, are known to be involved in cell responses to internal and external environmental factors, including osmolarity, pressure, heat, cold, hormones and inflammatory mediators . Blood is of particular interest among the non-nervous biological material because of its involvement in immune and inflammatory processes and its role in the distribution of circulating signals (cytokines, hormones). A recent study demonstrating TRPV1–2 gene expression in human peripheral blood cells by means of qRT-PCR and immunocytochemical techniques , stressed the importance of further studies to clarify the physiological and pathophysiological role and regulation of TRPV channels in human mononuclear cells. In this context, our work constitutes an additional effort to investigate the presence and relative amounts of TRPV1–4 gene transcripts in human leukocytes.
For this purpose, we decided to test and validate our Sybr Green qRT-PCR assay based on the relative quantification of TRPV1–4 gene transcripts. While absolute quantification (based on external standards) is the method of choice for nucleic acid quantification, relative quantification is based on the expression levels of one or more target genes versus one or more reference genes and, in many experiments, is adequate for investigating physiological changes in gene expression levels [17, 24]. The normalization procedure should be considered mandatory in qRT-PCR studies and the importance of the choice of the most stably expressed HKGs is too often underestimated during qRT-PCR assays, easily leading to misinterpretation and low reproducibility of the final results. Therefore, we decided to validate, up to their expression stability, four of the most widely used house-keeping genes in human leukocyte samples from the 30 healthy donors, equally divided between males and females. Amplicon lengths and specificity, and PCR amplification efficiencies and reproducibility, were accurately checked, and the stability of the selected control gene expressions among samples was analysed with two widely used softwares, namely geNorm and NormFinder. All four house-keeping genes considered in this study (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed stable expression in human leukocytes samples. We recommend the use of these genes for similar qRT-PCR studies and we hope that our outcomes will help to draw up a preliminary "universal protocol" for a better comparison of qRT-PCR results from different studies on human blood cells.
In addition to proving the presence of TRPV1–4 cDNAs in human leukocytes, our qRT-PCR assay revealed a lack of significant differences in TRPV1–4 expression between healthy males (n = 15) and females (n = 15), and between females sampled during the luteal and follicular phases of the menstrual cycle. This partially contrasts with experimental data concerning the effect of estrogens on the expression of the TRPV genes. For instance, the TRPV1 expression in viscera was found to be increased by estradiol administration in female rats , a pattern that seems not to be confirmed in our experiments on human leukocyte samples. Therefore, the physiological hormonal differences between women in the two menstrual phases, as well as the hormonal differences between the sexes, failed to affect TRPV1–4 gene expression, suggesting a negligible influence of gonadal hormones on the haematic expression of the TRPV subfamily genes, at least in healthy human subjects.
Additionally, the qRT-PCR assays allowed us to determine the relative expression of TRPV1–4 genes in human leukocytes. While the presence of TRPV1–2 transcripts in human blood cells was recently reported, our study is the first to demonstrate TRPV3–4 expression in human leukocytes. According to the results for the 30 healthy donors, we can conclude that TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2 (Fig. 1). Compared to TRPV3, the TRPV4, TRPV1 and TRPV2 genes showed a physiological normalized fold expression of 2.2×, 4.8× and 89.3×, respectively. The outcomes of our analysis (Fig. 2) are in partial agreement with the data reported by , where a ≈150-fold over-expression of TRPV2 compared to TRPV1 is shown. Although substantially reducing the gap between the expression of the two genes, our results confirm this trend: the TRPV1 cDNA levels are 18.4-fold lower than those of TRPV2.
The comparison of TRPV1–4 gene expression between healthy subjects (n = 30) and the hyposensitive group (n = 5) highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9× normalized fold induction) in the latter group (Table 3). TRPV1 is by far the best understood TRP channel  and both in vivo and in vitro studies have repeatedly shown that these receptors (TRPV1–4) are involved in the transmission of pain and/or thermal stimuli and capsaicin sensation [26, 27]. In particular, TPRV1 gene transcription produces an mRNA coding for a protein devoted to the vanilloid receptor 1; it is recognized as a molecular integrator of inflammatory mediators and is thought to mediate peripheral sensitization, involving a reduced threshold of activation and an increased responsiveness of peripheral nociceptors [28–31].
On the basis of these findings, the higher TRPV1 expression, related to the influence of TRPV genes in the immune response of lymphocytes, might be caused by the more frequent inflammatory and infectious processes in these patients due to their hyposensitive status. Under pathological conditions, the up-regulation of TRPV1 could be an indicator of inflammation at a secondary site related to the influence of TRPV1 on the release of neuropeptides that can up-regulate the expression of adhesion molecules in endothelial cells and consequently activate T-cells .
The up-regulation of TRPV1 in pain-insensitive subjects may also be linked to its polymorphism [32, 33] or to some functional anomalies of this gene  that may be associated with pain perception.
Two recent publications focused on the functional effects of nonsynonymous polymorphism in the human TRPV1 gene and on the genetic influence on variability in human pain sensitivity [32, 33]. TRPV1 presents five nonsynonymous polymorphisms expected to result in nonconservative amino acid substituitions . While TRPV1WT and its variant forms show similar EC50 for capsaicin, HEK293 cells transfected with TRPV1P91S and TRPV1I315M variants exhibit enhanced responsiveness to a second TRPV1 agonist, named anandamide . An additional sixth single nucleotide polymorphism, predicted to reside within membrane-spanning helix five, seems to be responsible for a conservative amino acid substitution (TRPV1I585V) that could alter receptor structure/function during cold perception , increasing sensitivity to cold-induced pain in American females subjects. This observation is apparently in contrast with the previously reported in vitro normal functional response to agonists of the TRPV1I585V variant [33, 35]. In the heterologous expression model described in , the two allelic variants TRPV1I315M and TRPV1P91S resulted in markedly increased abundance of the variant TRPV1 protein, although with a very modest increment in the mRNA levels; the authors suggest that disease state is mediated through altered expression of a normal protein and that much of the intersubject phenotypic variation will be encoded by polymorphisms that influence levels of gene expression.
A TRPV1 splice variant lacking Exon 7 (named TRPV1b) was previously cloned from human dorsal root ganglia , showing that recombinant TRPV1b was not activated by capsaicin (1 microM), protons (pH 5.0) or heat (50 degrees C). When co-expressed with TRPV1, TRPV1b formed complexes with TRPV1, and dose-dependently inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids. These data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1 and that mutual regulation between TRPV1 and TRPV1b might take place in the same neuronal cell in vivo. Therefore, in hyposensitive subjects, the up-regulation of TRPV1b could lead to pathological inhibition of TRPV1 activity, with a consequent loss of the natural ability to perceive thermal, pain and capsaicin stimulation. Other authors  did not exclude the possibility that TRPV1b may be up-regulated under physiological or pathological conditions. Since the qRT-PCR method described in this study does not allow discrimination between cDNAs transcribed from the TRPV1 gene or the recombinant TRPV1b splice variant, further studies are required to clarify the potential role of the TRPV1b splice variant and to quantify its expression in tissues of hyposensitive subjects.
It is evident that the presence of the TRPV1 splice variant or of genetic polymorphism associated with this gene, may provide another mechanism for down-regulating channel activity, and the more we learn about fundamental TRP channel physiology and the potential role of TRPs in disease, the closer we will come to the development of novel therapies for various disease states.