Search

Recent evidence indicates that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes, is also present in neural and nonneural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria and studied the effects of CNP substrates, 2',3'-cyclic nucleotides, on functional parameters of rat brain mitochondria. 2',3'-cAMP and 2',3'-cNADP stimulated [Ca.sup.2+] overload-induced [Ca.sup.2+] release from mitochondrial matrix. This [Ca.sup.2+] release under threshold [Ca.sup.2+] load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2',3'-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondria-induced cell death. 2',3'-cAMP effects were observed on the functions of rat brain mitochondria only when PT was developed. This demonstrates involvement of 2',3'-cAMP in PT regulation in rat brain mitochondria. We also discovered that, under PT development, the specific enzymatic activity of CNP was reduced. Thus we hypothesize that suppression of CNP activity under threshold [Ca.sup.2+] load leads to elevation of 2',3'-cAMP levels that, in turn, promote PT development in rat brain mitochondria. Similar effects of 2',3'-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knockdown oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for [Ca.sup.2+] overload-induced [Ca.sup.2+] release. Thus our results reveal a new function for CNP and 2',3'-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT. oligodendrocyte mitochondria; 2',3'-cyclic nucleotide 3'-phosphodiesterase; permeability transition; calcium transport

Protease-activated receptors (PARs) are involved in the contribution of airway epithelial cells to the development of inflammation by release of pro-and anti-inflammatory mediators. Here, we evaluated in epithelial cells the influence of LPS and continuous PAR activation on PAR expression level and the release of the proinflammatory chemokine IL-8. We studied primary human small airway epithelial cells and two airway epithelial cell lines, A549 and HBE cells. LPS specifically upregulated expression of PAR-2 but not of PAR-1. Exposure of epithelial cells to PAR-1 or PAR-2 agonists increased the PAR-1 expression level. The PAR-2 agonist exhibited higher potency than PAR-1 activators. However, the combined exposure of epithelial cells to LPS and PAR agonists abrogated the PAR-1 upregulation. The PAR-2 expression level was also upregulated after exposure to PAR- 1 or PAR-2 agonists. This elevation was higher than the effect of PAR agonists on the PAR-1 level. In contrast to the PAR-1 level, the PAR-2 level remained elevated under concomitant stimulation with LPS and PAR-2 agonist. Furthermore, activation of PAR-2, but not of PAR-1, caused production of IL-8 from the epithelial cells. Interestingly, both in the epithelial cell line and in primary epithelial cells, there was a potentiation of the stimulation of the IL-8 synthesis and release by PAR-2 agonist together with LPS. In summary, these results underline the important role of PAR-2 in human lung epithelial cells. Moreover, our study shows an intricate interplay between LPS and PAR agonists in affecting PAR regulation and IL-8 production. protease-activated receptors; lipopolysaccharide; inflammatory mediators

Among the four protease-activated receptors (PARs), PAR-1 plays an important role in normal lung functioning and in the development of lung diseases, including fibrosis. We compared the expression and functional activity of PARs in normal and fibrotic human lung fibroblasts. Both normal and fibrotic cells express PAR-1, -2, and -3, with PAR-2 showing the lowest level. There was no significant difference between normal and fibrotic fibroblasts in expression levels of PAR-1 and PAR-3, whereas a fourfold higher expression level of PAR-2 was observed in fibrotic cells compared with normal cells. [Ca.sup.2+] imaging studies revealed apparently only PAR-1-induced [Ca.sup.2+] signaling in lung fibroblasts. PAR-1 agonists, thrombin and synthetic activating peptide, induced concentration-dependent [Ca.sup.2+] mobilization with E[C.sub.50] values of 5 nM and 1 [micro]M, respectively. The neutrophil protease cathepsin G produced a transient [Ca.sup.2+] response followed by disabling PAR-1, whereas elastase did not affect [Ca.sup.2+] level. PAR-1 activation by thrombin or receptor-activating peptide downregulated expression of all three PARs in lung fibroblasts, with maximal effect at 3-6 h, whereas expression returned toward basal level after 24 h. Furthermore, PAR-1 agonists dose dependently increased PG[E.sub.2] secretion from lung fibroblasts and induction of cyclooxygenase-2 expression. We then found that PG[E.sub.2] downregulated expression of all three PARs. The effect of PG[E.sub.2] was continuously growing with time. Furthermore, PG[E.sub.2] exerts its effect through the EP2 receptor that was confirmed using the selective EP2 agonist butaprost. This novel autocrine feedback mechanism of PG[E.sub.2] in lung fibroblasts seems to be an important regulator in lung physiology and pathology. thrombin; cyclooxygenase; cyclic adenosine 5'-monophosphate; prostanoid

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP [greater than or equal to] UTP > adenosine [greater than or equal to] 2-methylthioadenosine-5'-diphosphate, with [alpha],[beta]-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATP[alpha]S inactive] and PCR analysis indicate involvement of [P2Y.sub.2] and [P2Y.sub.6] receptors. The signal transduction pathway, which, via the [P2Y.sub.2] receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on [Ca.sup.2+]/calmodulin-dependent protein kinase II and nuclear factor-[kappa]B, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1[beta] and interleukin-6, but not tumor ncerosis factor-[alpha]. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells. purinergic receptors; cancer; cytokines; extracellular nucleotides

Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/([beta][gamma]-subunits)-phosphatidylinositol 3-kinase branch, and 2) the [G.sub.q]-PLC-(Ins[P.sub.3] receptor)/[Ca.sup.2+]-PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation. protease-activated receptors; extracellular signal-regulated protein kinase; calcium signaling; epidermal growth factor receptor; transactivation; mitogen-activated protein kinase