Triakontatetraneuropeptide [diazepam-binding inhibitor (DBI) 17–50, TTN] is one of the major endogenous peptides generated through the cleavege of DBI, a neuropeptide also known as acyl-CoA-binding protein 1. DBI and DBI-derived peptides are also called endozepines, after the ability of at least some of them to recognize the diazepam binding site on the GABA_A receptor, and are widely distributed in the CNS and in peripheral organs 23. In particular, immune tissues express DBI 4 and evidence exists that TTN and its related peptides octadecaneuropeptide (DBI 33–50, ODN) and eiksoneuropeptide (DBI 51–70, ENP) can affect the immune response. In particular, TTN and ODN stimulate the production of tumor necrosis factor (TNF) alpha, interleukin (IL)-1 beta, IL-8, granulocyte/macrophage colony-stimulating factor, IL-6 and IL-8 in human monocytes 56, and ODN enhances the LPS-induced secretion of IL-6 in human peripheral blood mononuclear cells 7.

In human polymorphonuclear leukocytes (PMNs) we previously showed that TTN rises intracellular calcium ([Ca⁺⁺]_i) and stimulates chemotaxis, O₂^- generation, phagocytosis and IL-8 production 89. Pharmacological analysis of TTN-induced [Ca⁺⁺]_i changes and IL-8 production suggested that these responses are brought about through a pertussis toxin (PTX) sensitive, G protein-coupled receptor linked to phospholipase C (PLC), which once activated induces both Ca⁺⁺ release from thapsigargine-sensitive intracellular stores and Ca⁺⁺ entry from the extracellular space 9. In those experiments, preliminary evidence also suggested the possible involvement of protein kinase (PK) C, inasmuch as the PKC inhibitor calphostin C significantly affected (although did not completely block) TTN-induced effects.

The present study was therefore undertaken to investigate more in-depth, by use of a pharmacological approach, the role of PKC in the modulation of the response to TTN by human PMNs, and to better characterize TTN-induced Ca⁺⁺ entry through the plasma membrane by testing its sensitivity to different Ca⁺⁺-channel blockers. The results show that the stimulatory effect of TTN is profoundly affected by PKC activation, and suggest that different PKC isoforms may play distinct roles. Evidence is also provided that TTN-induced Ca⁺⁺ entry is specifically sensitive to T-type Ca⁺⁺-channel blockers. The possibility that T-type Ca⁺⁺-channels may represent the target for the effects of these drugs on human PMNs is further supported by the observation that these cells express the mRNAs for T-type Ca⁺⁺-channel α1 subunits.

The endozepine TTN behaves as a chemoattractant factor for human PMNs, resulting in a typical pattern of cell activation, which includes a rise of [Ca⁺⁺]_i, with subsequent IL-8 mRNA expression and release of this proinflammatory chemokine, chemotaxis, induction of oxidative metabolism and phagocytosis 89. In the present study we have further characterized the pharmacological profile of the response to TTN by human PMNs, showing that PKC exerts a complex modulation of TTN-induced [Ca⁺⁺]_i rise and that TTN-induced Ca⁺⁺ entry in these cells is sensitive to T-type Ca⁺⁺ channel blockers. A tentative synopsis of the experimental evidences obtained so far is given in Fig. 5, which should also be taken as a reference frame for the subsequent discussion of the findings of this study.

In our experiments, the PKC activator PMA prevented both TTN-induced [Ca⁺⁺]_i rise and IL-8 mRNA expression. Involvement of PKC was confirmed by the low EC₅₀ value of PMA, by the inactivity of its negative control α-PMA, as well by the ability of two chemically unrelated PKC inhibitors such as Ro 32-0432 and rottlerin to revert this response. These observations stand for the existence of a PKC-operated inhibition of TTN-dependent pathways, a finding in line with the notion that in PMNs activation of PKC inhibits the signals responsible for mobilization of [Ca⁺⁺]_i 11. Interestingly however PMA did not completely prevent TTN-induced [Ca⁺⁺]_i rise, and a residual effect of the peptide (around 20% of the maximal effect) was still evident even in the presence of high concentrations of PMA (see Fig. 1). This is in line with our previous results showing that the PKC inhibitor calphostin C per se significantly reduced the effects of TTN in human PMNs, suggesting that activation of PKC was also part of the signaling process triggered by this peptide in PMNs 9.

PKC consists of a family of at least 12 serine/threonine kinases, which are currently divided into three main groups based on their structure and substrate requirements, namely: conventional (Ca⁺⁺-dependent and activated by both phosphatidylserine [PS] and diacylglycerol [DAG], represented by α, βI, βII, and γ), novel (Ca⁺⁺-independent and regulated by PS and DAG, represented by δ, ε, η, and θ), and atypical (Ca⁺⁺-independent and regulated by PS but not by DAG, represented by ζ and ι/λ) 12. Recent studies have investigated the expression and role of PKC isoforms in human PMNs, showing in these cells the presence of conventional (βI, βII, and α, although in lower amounts), as well as novel (δ) PKC 13, and that different isoforms subserve distinct functions: conventional isoforms regulate PMA-stimulated cytoskeletal association and activation of NADPH oxidase, while novel isoforms modulate other responses that involve cytoskeletal components 14.

In our experiments, we have used the phorbol ester compound PMA, which penetrates the cytoplasmic membrane to directly bind and activate PKC 15. PMA however does not show a high degree of selectivity for PKC isoforms, thus resulting in activation of all PKC in the cell. In the present study, we have therefore investigated the ability of the PKC inhibitors Ro 32-0432 and rottlerin to revert the effect of PMA on TTN-induced [Ca⁺⁺]_i rise, as well as to affect per se this response. Rottlerin has been shown to inhibit PKCδ with some selectivity over other PKC isoforms, while Ro 32-0432 is selective for conventional isoforms (reviewed in 12). According to our results, both drugs resulted in the reversion of PMA-induced inhibition of TTN-induced [Ca⁺⁺]_i rise (Table 2), however only rottlerin also inhibited per se the effect of TTN (Fig. 2). The EC₅₀ of rottlerin in this regard was 5.77 μM, which is in good agreement with the reported IC₅₀ for PKCδ (3–6 μM) 12. On the contrary, Ro 32-0432 in the 5–50 nM concentration range (which has been reported to be selective for conventional PKC isoforms 12) had no effect per se on the response of human PMNs to TTN (Fig. 2). These findings, together with the observation that at variance with Ro 32-0432, rottlerin was only partially effective in reverting the effect of PMA (Table 2), may suggest that different isoforms of the enzyme play distinct roles in modulating the responses to TTN. While PKCδ seems to contribute to the rise of [Ca⁺⁺]_i induced by TTN, activation of conventional PKC isoforms (possibly, βI, βII, and/or α) may inhibit this response. When all the PKC isoforms undergo activation, as in the case of treatment with PMA, inhibition prevails over facilitation. In future studies, this hypothesis will be tested by investigating the pattern of activation of different PKC isoforms occurring as a result of TTN stimulation in human PMNs.

Few evidence exists about the possible contribution of membrane Ca⁺⁺ channels to the effects of TTN on [Ca⁺⁺]_i. In rat astrocytes, TTN increases [Ca⁺⁺]_i through a peripheral-type benzodiazepine receptor-mediated opening of Ca⁺⁺ channels which are sensitive to the L-type channel blocker nifedipine but not to the T-type channel blocker mibefradil nor to the N-type channel blocker ω-conotoxin GVIA 16, while in the frog adrenal gland, TTN-induced [Ca⁺⁺]_i rise involves the activation of membrane receptors positively coupled to adenylyl cyclase through a cholera toxin-sensitive G protein, which in turn results in Ca⁺⁺ influx which is inhibited by mibefradil but not by nifedipine or ω-conotoxin GVIA 17. In our experiments, TTN-evoked [Ca⁺⁺]_i rise was significantly reduced by mibefradil and also by flunarizine, another T-type Ca⁺⁺ channel blocker structurally different from mibefradil, whereas nifedipine, verapamil (another L-type channel blocker) and ω-conotoxin GVIA had no effect. Interestingly, the effect of TTN on [Ca⁺⁺]_i was not completely blocked, an evidence consistent with the idea that TTN increases [Ca⁺⁺]_i through both release from intracellular stores and entry through the plasma membrane 8.

The sensitivity of TTN-induced [Ca⁺⁺]_i rise to T-type channel blockers led us to investigate the expression of T-type Ca⁺⁺ channels in human PMNs. Molecular studies have identified at least 3 voltage-gated Ca⁺⁺ channel α1 subunits which share the biophysical and pharmacological properties of T-type channels, namely α1G, α1H, and α1I 18. In the present study, northern blot analysis provided evidence for the expression of the mRNA for all these 3 α1 subunits in human PMNs (Fig. 4). We are not aware of other studies showing the expression of T-type Ca⁺⁺ channels in human PMNs, either at the level of mRNA or of the protein subunits, and the present findings may therefore represent the first evidence that such channels are expressed by these cells. Nonetheless, whether true voltage-dependent T-type Ca⁺⁺ channels occur and play a relevant role in human PMNs remains still largely to be established. Electrophysiological studies of Ca⁺⁺ influx in non-excitable cells such as leukocytes, including PMNs, have provided evidence for the existence of receptor-mediated, non voltage-operated Ca⁺⁺ entry in these cells 19. Interesting observations however regard the possible occurrence, at least in lymphocytes, of non-voltage-operated Ca⁺⁺ channels, with an amino acid sequence which is closely related to classical voltage-operated Ca⁺⁺ channels (reviewed in 20). In human PMNs flunarizine has been shown to inhibit Ca⁺⁺ entry triggered by fMLP or by the Ca⁺⁺ ionophore A23187 21. Available data, including those reported in the present study, seem thus to support the existence in human PMNs of Ca⁺⁺ entry mechanisms which are closely related to T-type Ca⁺⁺ channels, from both the structural and the pharmacological point of view. Such mechanisms are likely to be involved in the response of human PMNs to TTN, and may represent the target of T-type channel blockers such as flunarizine and mibefradil.

In conclusion, the present results together with previous studies 89 suggest that in human PMNs the endozepine TTN activates G protein-coupled membrane receptors, resulting in a signaling cascade which comprises PLC and PKC. This latter enzyme in particular may exert both positive and negative effects on TTN signaling, possibly depending on the specific isoform(s) involved. TTN-activated pathways finally result in increased [Ca⁺⁺]_i, due to both Ca⁺⁺ release from intracellular stores and Ca⁺⁺ entry possibly through T-type Ca⁺⁺ channels. [Ca⁺⁺]_i rise then signals for activation of cell function, including mRNA expression and release of the chemokine IL-8 (Fig. 5). DBI is released from nerve terminals and its fragments can be detected in liquor and peripheral blood 22. Endozepines such as TTN may therefore contribute to the central nervous system-immune system cross-talk. In addition DBI, also called acyl-CoA binding protein, is widely distributed in many peripheral organs such as gut and endocrine cells of the pancreatic islets 2324, liver, kidney 25, adrenals 26, adipose tissue, heart, muscles and mammary gland 27 of different species, and in circulating mononuclear cells 28, in red blood cells 29 and even in neoplastic cell lines 30. High concentrations of DBI and/or of its processing products may therefore occur locally, e.g. as the result of leakage from damaged cells during tissue injury. DBI-derived peptides may thus also add to the multiple agents constituting the local microenvironment in inflamed tissues. Studies are therefore warranted to develop TTN-receptor ligands to assess the relevance of TTN-operated pathways as novel targets for the pharmacological modulation of PMNs during the inflammatory process.

FM and MC designed the study, performed spectrofluorimetric experiments and drafted the manuscript; MF performed RT-PCR experiments; SC and GF collaborated to spectrofluorimetric studies; AMF collaborated to the design of the study and to the analysis and interpretation of the data; SL and GMF collaborated to data interpretation and to the critical discussion of the manuscript. All Authors read and approved the final manuscript.