Role and regulation of TRP channels in neutrophil granulocytes
Section snippets
Relevance and induction of calcium signaling in neutrophil granulocytes
Important cellular responses in neutrophil granulocytes that are mediated or essentially regulated by the intracellular free Ca2+ concentration [Ca2+]i include production and release of arachidonic acid products [1], degranulation [2], [3], and the respiratory burst (release of superoxide anions and other free radicals) [4], [5]. Furthermore, Ca2+ may be important for the chemotaxis, in particular for activation of β2-integrins [6] allowing the firm adhesion to the endothelial layer of blood
Expression of TRP channels in neutrophil granulocytes
To elucidate if store-operated or store-independent channels of the transient receptor potential (TRP) family may have a role in Ca2+ regulation of neutrophil granulocytes, we have looked for the expression pattern of TRP channels [17]. Our approach was based on RT-PCR using isolated mRNA of neutrophil granulocytes and primer nucleotides with sequences each specific for one member of the TRP family. Table 1 lists the TRP family members for which we found consistent RT-PCR signals. Noteworthily,
TRPM2 and its function as a non-selective cation channel activated by ADPR, H2O2 and NAD
Among the TRP channels that gave positive PCR signals in neutrophil granulocytes (Fig. 1), there is one family member that displays unique and characteristic properties that let us hope to obtain functional evidence for its expression in these cells. This is TRPM2, originally dubbed TRPC7 [28] (although it does not belong to the subfamily of “short” TRPs) and later named LTRPC2. The structure of the TRPM2 protein is schematically shown in Fig. 2. TRPM2 contains as characteristic structural
H2O2 and its relevance as a physiological stimulus of endogenous TRPM2 channels in neutrophil granulocytes
When we started our study exploring the stimulation of TRPM2 by ADPR and by H2O2, we reasoned that H2O2 may act through the generation of ADPR. This assumption proved to be wrong, as we learned when we studied several splice variants of TRPM2 (Fig. 2). When a splice variant with a deletion in the C-terminus (Δ1292–1325 of a total of 1503 amino acids) was tested, we were surprised to find that H2O2 was effective on this as on the full-length variant but that ADPR was virtually inactive [35].
A possible role for ADPR as a second messenger activating TRPM2 in neutrophil granulocytes
Whereas H2O2 appears a stimulus of TRPM2 with physiological relevance, the physiological significance of ADPR is less certain. Formerly, ADPR was mostly considered a degradation product of NAD as well as of cyclic ADP ribose (cADPR). An enzyme (or, more likely, a class of enzymes) named ADP ribosyl cyclase catalyzes the conversion of NAD to cADPR; at the same time, it hydrolyzes NAD and cADPR to ADPR [38]. In granulocytes, there is a transmembrane glycoprotein CD38 that works as ADP ribosyl
Proposed modes of TRPM2 activation in neutrophil granulocytes
What could the biological role of TRPM2 be in neutrophil granulocytes? It is a sound assumption (although not yet experimentally confirmed) that all reported stimuli of TRPM2, i.e. ADPR, H2O2, and NAD, may act in these cells, as proposed in Fig. 3. The relevance of H2O2 has already been discussed. The concentrations of NAD are expected to rise in the presence of the oxidant H2O2; they may contribute to if not even mediate the H2O2 effects. Such a process takes place during the oxidative burst
Stimuli of VR1 and TRPC6 are without effect on currents in neutrophil granulocytes
For the other TRP family members for which RT-PCR signal indicate an expression in neutrophil granulocytes, no functional evidence of their expression can be presented at present. The vanilloid receptor (VR1 or TRPV1) is a ligand-gated cation channel most notorious for its stimulation by capsaicin, the characteristic ingredient of chilly peppers and a mediator of pain reception [43]. Capsaicin failed to increase [Ca2+]i or cation currents in neutrophil granulocytes in our hand (unpublished).
Store-operated calcium entry in neutrophil granulocytes
The expression pattern of TRP channels in neutrophil granulocytes leaves TRPV6 and TRPV5 as candidates for channels mediating SOCE. In HL-60 cells, TRPC1 and TRPC3 are further candidates. Evidence for SOCE has been proposed in neutrophil granulocytes on the basis of measurements of [Ca2+]i with fura-2 [50]. Electrophysiological evidence for store-operated currents, however, has not yet been provided. We have looked for ICRAC-like currents in granulocytes but could not demonstrate them. This may
Conclusions
Among the members of the TRP family, TRPM2 is the only one for which presently characteristic currents can be demonstrated in neutrophil granulocytes. These currents carried by Na+ and Ca2+ can be evoked by ADPR-ribose and by NAD. Their physiological role is probably the activation of the cells in response to chemoattractants; moreover, the currents may serve as a positive feed-back signal during the oxidative burst. The significance of other TRP members for which RT-PCR signals have been
Acknowledgments
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Project B5 of SFB542).
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