Phospholipase C-β1 mediates α1-adrenergic receptor-stimulated activation of the sodium–hydrogen exchanger in Chinese hamster lung fibroblasts (CCL39)

The activation of the Na+–H+ exchanger 1 (NHE1) and extracellular-signal regulated kinase (ERK) phospho rylation in Chinese hamster lung fibroblasts (CCL39) was characterized in response to the specific α1-adrenergic agonist, phenylephrine (PE). Addition of 100 µmol PE/L increased the steady-state intracellular pH (pHi) by 0.16 ± 0.03 pH units, as well as increasing the phosphorylation of ERK. The response of NHE1 to PE in CCL39 cells was determined by the use of specific antagonists. Use of 2 specific chemical inhibitors of phosphoinositide-specific phospholipase C (PLC) reduced the ability of PE to activate either the exchanger or ERK. Studies were conducted in PLCβ-deficient cell lines derived from parental CCL39 cells. NHE1 activity in both mutant cell lines was increased in response to phorbal esters or lysophosphatidic acid, whereas the addition of PE only caused a minimal change in either pHi or ERK phosphorylation. These results, combined with reconstitution experiments with exogenously expressed PLCβ1, PLCβ2, or PLCβ3, revealed that stimulation of NHE1 activity by PE in CCL39 cells is a PLCβ1-coupled event. Furthermore, the data indicate that α1-adrenergic signaling of PLCβ is upstream of ERK activation. These data demonstrate that PLCβ1 is primarily involved in the activation of NHE1 in CCL39 fibroblasts.Key words: CCL39, sodium hydrogen exchanger, ERK, α1-adrenergic receptor, phospholipase Cβ.

Thrombin inhibits Bim (Bcl-2-interacting mediator of cell death) expression and prevents serum-withdrawal-induced apoptosis via protease-activated receptor 1

To investigate the role of thrombin in regulating apoptosis, we have used CCl39 cells, a fibroblast cell line in which thrombin-induced cell proliferation has been extensively studied. Withdrawal of serum from CCl39 cells resulted in a rapid apoptotic response that was completely prevented by the inclusion of thrombin. The protective effect of thrombin was reversed by pertussis toxin, suggesting that cell-survival signalling pathways are activated via a Gi or Go heterotrimeric GTPase. Serum-withdrawal-induced death required de novo gene expression and was preceded by the rapid de novo expression of the pro-apoptotic ‘BH3-only’ protein Bim (Bcl-2-interacting mediator of cell death). Thrombin strongly inhibited the up-regulation of both Bim protein and Bim mRNA. The ability of thrombin to repress Bim expression, and to protect cells from apoptosis, was reversed by U0126, a MEK1/2 [MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) 1/2] inhibitor, or LY294002, a phosphoinositide 3′-kinase (PI3K) inhibitor, suggesting that both the Raf→MEK→ERK1/2 and PI3K pathways co-operate to repress Bim and promote cell survival. A PAR1p (protease-activated receptor 1 agonist peptide) was also able to protect cells from serum-withdrawal-induced apoptosis, suggesting that thrombin acts via PAR1 to prevent apoptosis.

Physiological functions of the regulatory domains of the cardiac Na+/Ca2+exchanger NCX1

Physiological functions of the intracellular regulatory domains of the Na+/Ca2+ exchanger NCX1 were studied by examining Ca2+ handling in CCL39 cells expressing a low-affinity Ca2+ regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na+ inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Δ246–672). We found that D447V/D498I was unable to efficiently extrude Ca2+ from the cytoplasm, particularly during a small rise in intracellular Ca2+concentration induced by the physiological agonist α-thrombin or thapsigargin. The same mutant took up Ca2+ much less efficiently than the wild-type NCX1 in Na+-free medium when transfectants were not loaded with Na+, although it appeared to take up Ca2+ normally in transfectants preloaded with Na+. XIP-4YW and, to a lesser extent, Δ246–672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na+-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca2+ handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.

Differential activation of p44mapk (ERK1) by α-thrombin and thrombin-receptor peptide agonist

alpha-Thrombin (thrombin), a potent mitogen for CCL39 hamster lung fibroblasts, stimulates phosphoinositide-specific phospholipase C (PI-PLC) and inhibits adenylate cyclase via cleavage of a specific G-protein-coupled receptor (TH-R), recently cloned from human and hamster cells. This action can be entirely mimicked by the synthetic peptide SFFLRNP, referred to here as TMP (thrombin-mimicking peptide). TMP corresponds to the first seven amino acids of the new N-terminus generated by thrombin cleavage of the hamster TH-R. Although thrombin and TMP apparently generate identical early transmembrane signals, only thrombin is mitogenic on its own. TMP needs to be associated with fibroblast growth factor (FGF), a tyrosine kinase-activating growth factor, to induce cell-cycle re-entry. Here, we have examined the early and late phase of p44 MAP kinase (p44mapk) activation in G0-arrested CCL39 cells after stimulation by thrombin, TMP, FGF or TMP+FGF. We found that: (i) both thrombin and TMP rapidly activate p44mapk in a dose-dependent manner with maximum activation at around 5 min, (ii) after the initial burst of activation, a second and long-lasting wave of activation is observed in response to thrombin (10-100 nM) but not to TMP (up to 300 microM), (iii) FGF alone (25 ng/ml), like thrombin, rapidly and persistently activates p44mapk (20-fold at 5 min and about 3-fold after 2 h), (iv) TMP added together with FGF strongly potentiates the second and sustained phase of p44mapk activation. From these results we propose that: (1) thrombin-induced mitogenesis is mediated only in part by the TH-R recently cloned and (2) activation of p44mapk, in particular the long-lasting phase that correlates with DNA synthesis, is an obligatory event for cell-cycle re-entry.

Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene.

To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human alpha 2-adrenergic receptor (alpha 2-C10) in CCL39 cells and studied the effects of alpha 2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the alpha 2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the alpha 2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, alpha 2-agonists also induced a moderate release of inositol phosphates, indicating that alpha 2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown.Our data show that alpha 2-adrenergic receptors can play a role in the regulation of cell proliferation in an appropriate context; also, the data support the hypothesis that receptors negatively coupled to AC must be taken into account as mediators of growth factor action in fibroblasts, in particular when activated in parallel with receptor tyrosine kinases.