CCL39 Cells Research Articles

NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.

Characterizing the Role of the Na+‐H+ Exchanger Isoform 1 (NHE1) in Pulmonary Fibrosis

Pulmonary fibrosis is a chronic, progressive lung disease characterized by stiffening and thickening of the lung tissue leading to reduced respiratory function. When lung tissue becomes damaged, the response is to increase recruitment and proliferation of fibroblasts and increase deposition of extracellular matrix (ECM) proteins such as collagen. This initiates the release of transforming growth factor beta 1 (TGF‐β1), a cytokine known to enhance fibrosis. TGF‐β1 induces myofibroblast differentiation and further stiffening of the ECM by stimulating formation of cytoplasmic microfilament bundles and increasing expression of alpha‐smooth muscle actin (α‐SMA). Signal molecules such as serotonin (5‐HT), and lysophosphatidic acid (LPA) contribute to this by binding to G‐protein coupled receptors that support increased release of TGF‐β1. Fibrosis ultimately results in a state of low blood flow and hypoxia in the lung tissue, a microenvironment associated with upregulation of the sodium‐hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that regulates intracellular pH. Upregulation of NHE1 supports increased cell proliferation and migration, indicating that it may play a role in the behavior of fibroblasts in pulmonary fibrosis. The proliferation, collagen deposition, and myofibroblast differentiation of two fibroblast cell lines will be characterized. CCL39, Chinese hamster lung fibroblasts, and WI‐38, normal human fibroblast cells, will both be studied. CCL39 cells treated with EIPA, a chemical inhibitor of NHE1, show reduced proliferative rates. This suggests that proliferation in these cells is NHE1‐dependent. In addition to proliferation, it is hypothesized that the collagen deposition and myofibroblast differentiation of this cell line will be NHE1‐dependent. Furthermore, it is hypothesized that WI‐38 cells will display similar NHE1‐dependent proliferation, collagen deposition and myofibroblast differentiation as the CCL39 cells. This suggests that CCL39 cells can serve as a predictor of human fibroblast cell behaviors.

Suppressive Effect of Carvedilol on Na+/Ca2+ Exchange Current in Isolated Guinea-Pig Cardiac Ventricular Myocytes

Background and Aims: Carvedilol ((+/-)-1-(carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol), a β-adrenoceptor-blocker, has multi-channel blocking and vasodilator properties. This agent dose-dependently improves left ventricular function and reduces mortality in patients with arrhythmia and chronic heart failure. However, the effect of carvedilol on the cardiac Na⁺/Ca²⁺ exchanger (NCX1) has not been investigated. Methods and Results: We examined the effects of carvedilol and metoprolol, 2 β-blockers, on Na⁺/Ca²⁺ exchange current (I_NCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed I_NCX in a concentration-dependent manner but metoprolol did not. IC₅₀ values for the Ca²⁺ influx (outward) and efflux (inward) components of I_NCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 μmol/l inhibited I_NCX in CCL39 cells expressing wild type NCX1 similar to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 µmol/l abolished ouabain-induced delayed afterdepolarizations. Conclusion: Carvedilol inhibited cardiac NCX in a concentration-dependent manner in isolated cardiac ventricles, but metoprolol did not. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Blocking effect of carvedilol, a beta‐blocker on Na+/Ca2+ exchange current in isolated cardiac ventricular cells of guinea pig

BackgroundCarvedilol has a1, b1, and b2‐adrenergic blocking effects together with the effects such as vasodilating, anti‐ischemic, anti‐oxidant, anti‐apoptotic, anti‐proliferate and anti‐inflammatory activities. Carvedilol inhibited IKr, IKs, ICa‐L and Ito in rabbit cardiac ventricular myocytes. On the other hand, metoprolol, a nonselective b‐blocker also has blocking several channels, including IK1, ICa and Ito in hearts. Murayama et al. (2013) reported that carvedilol and its analogues inhibit delayed after depolarizations (DADs) in Langendorff‐perfused rabbit hearts. It is well known that DADs are caused by the Na+/Ca2+ exchange current (INCX).MethodsWe examined the effects of carvedilol and metoprolol, two b‐blockers, on INCX by using the whole‐cell voltage‐clamp method in isolated guinea pig ventricles and CCL39 fibroblast cells expressing dog cardiac Na+/Ca2+ exchanger (NCX1).ResultsCarvedilol suppressed INCX in a concentration‐dependent manner but metoprolol did not in isolated guinea pig cardiac ventricular myocytes. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 μM and 61.5 μM, respectively. Carvedilol at 100 μM inhibited INCX in CCL39 cells expressing wild type NCX1 similarly to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 μM abolished ouabain‐induced DADs in isolated cardiac ventricular myocytes.ConclusionsCarvedilol suppressed bi‐directional INCX in a concentration‐dependent manner with the IC50 value of approximately 65 μM. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Nicorandil stimulates a Na⁺/Ca²⁺ exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 μM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 μM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 μM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

Effect of ether glycerol lipids on interleukin-1β release and experimental autoimmune encephalomyelitis

We have assessed the effect of two ether glycerol lipids, 77-6 ((2S, 3R)-4-(Tetradecyloxy)-2-amino-1,3-butanediol) and 56-5 ((S)-2-Amino-3-O-hexadecyl-1-propanol), which are substrates for sphingosine kinases, on inflammatory responses. Treatment of differentiated U937 macrophage-like cells with 77-6 but not 56-5 enhanced IL-1β release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1β release was reduced by pretreatment of cells with the caspase-1 inhibitor, Ac-YVAD-CHO, thereby indicating a role for the inflammasome. The enhancement of LPS-stimulated IL-1β release in response to sphingosine, but not 77-6, was reduced by pretreatment of cells with the cathepsin B inhibitor, CA074Me, indicating a role for lysosomal destabilization in the effect of sphingosine. Administration of 56-5 to mice increased disease progression in an experimental autoimmune encephalomyelitis model and this was associated with a considerable increase in the infiltration of CD4+ T-cells, CD11b+ monocytes and F4/80+ macrophages in the spinal cord. 56-5 and 77-6 were without effect on the degradation of myc-tagged sphingosine 1-phosphate 1 receptor in CCL39 cells. Therefore, the effect of 56-5 on EAE disease progression is likely to be independent of the inflammasome or the sphingosine 1-phosphate 1 receptor. However, 56-5 is chemically similar to platelet activating factor and the exacerbation of EAE disease progression might be linked to platelet activating factor receptor signaling.

Retracted: Mice lacking the Raf‐1 kinase inhibitor protein exhibit exaggerated hypoxia‐induced pulmonary hypertension

Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP-/-) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. RKIP-/- mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP-/- mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP-/- mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.

Sodium hydrogen exchanger and phospholipase D are required for α 1-adrenergic receptor stimulation of metalloproteinase-9 and cellular invasion in CCL39 fibroblasts

Matrix metalloproteinase 9 (MMP-9) plays a critical role in digesting the extracellular matrix and has a vital function in tumor metastasis and invasion; this protease activity is significantly increased in non-small cell lung cancers. The sodium hydrogen exchanger isoform 1 (NHE1) functions as a focal point for signal coordination and cytoskeletal reorganization. NHE1 is thought to play a central role in establishing signaling components at the leading edge of a migrating cell. Therefore, we studied the relationship between NHE1 and MMP-9 activity in Chinese hamster lung fibroblasts (CCL39) stimulated with phenylephrine (PE). We show that PE increases MMP-9 gelatinolytic activity in CCL39 cells. The inhibition of phospholipase D (PLD) signaling abrogated PE-induced MMP-9 activity. The role of PLD as an essential signaling intermediate was confirmed when the addition of permeable phosphatidic acid increased MMP-9 activity in the same cells. PE-induced invasion was increased 1.9-fold over controls and the PE response was lost when 1-butanol was used to block PLD signaling. Cells pre-treated with the NHE1 inhibitor, 5-( N-ethyl- N-isopropyl) amiloride (EIPA) prior to PE addition resulted in a notable decrease in MMP-9 activation and cell invasion as compared to untreated PE-stimulated cells. CCL39 NHE1 null cells demonstrated no increase in MMP-9 protease activity or cell invasion in response to PE treatment. Reconstitution of NHE1 expression recovered the PE-induced activation of protease activity and cell invasion. MMP-9 processing was altered in cells expressing a proton transport defective NHE1 but retained the ability to respond to PE. Conversely, cells expressing an ezrin, radixin, moesin (ERM)-binding deficient NHE1 had a lower MMP-9 activity and the protease did not respond to PE addition. Parallel studies on NCI-H358 non-small cell lung cancer (NSCL) cells showed that PE stimulated both MMP-9 activity and cell invasion in an NHE1 dependent manner. This work describes for the first time a PE-induced relationship between NHE1 and MMP-9 and a new potential mechanism by which NHE1 could promote tumor formation and metastasis.

NHE1 is critical in urokinase plasminogen activator‐mediated invasion and migration processes in lung cells

Cancer cell motility is a complex series of events, which includes the coordination of proteases and cytoskeletal proteins at the leading edge of the tumor. Matrix metalloproteinases (MMP) play a critical role in digesting the extracellular matrix (ECM). Degradation of the extracellular matrix by MMP in migrating cells provides a vital function for tumor metastasis. The sodium hydrogen exchanger (NHE), functions as a coordinator of motility by acidification of the ECM and as a docking site for cytoskeletal proteins. CCL39 cells were used as a model to dissect the role on NHE in UPA signaling. In these cells, 10 nM UPA gave rise to a two fold increase in pH, activated MMP9 and doubled the number of cells invading through matrigel. EIPA, a NHE1 inhibitor blocked the UPA‐mediated activation of MMP in CCL39 and H1299 cells. UPA activation of MMP9 and CCL39 cells expressing mutant NHE was also determined. UPA stimulated tumor formation in both H1299 and H358 cancer cells well over controls. EIPA inhibition of cell proliferation and growth in a soft‐agar assay was investigated in both human lung cancer cell lines. We show that urokinase plasminogen activator (UPA) stimulates MMP activity in an NHE dependent fashion and that UPA activation of tumor formation and invasion required NHE. NIH 1 R15 HL074924‐01A1

The role of the sodium hydrogen exchanger in stress fiber formation in human lung cancer stimulated with urokinase plasminogen activator, lysophosphatic acid, and phenylephrine

One of cancers hallmarks is the ability of cells to spread from the tumor of origin to other areas of the body, a process called metastastis. The Sodium‐Hydrogen Exchanger (NHE) plays a role in pH regulation and cytoskeletal attachment to the plasma membrane. In studies using CCL39 lung fibroblasts, phenylephrine (PE) induced stress fibers, and caused cells to become tumor like. In wound assay studies, PE stimulated stress fiber formation in cells immediately adjacent to the wound area and in migrating cells. These data indicate that stress fiber formation has a direct involvement in cell migration. In this study, we relate data from CCL39 cells to stress fiber formation in human lung cancer. We studied stress fiber formation in H358, H460, and H1299 cells. Our data shows that in H358 cells both 50μM PE and 10nM urokinase‐type plasminogen activator (uPA) significantly increase stress fiber formation. In H460 cells, 3μM lysophosphatidic acid (LPA) and UPA significantly increase stress fiber formation, and in H1299 all three hormones significantly increase stress fiber formation. Our data also shows that using ethylisopropylamiloride to inhibit NHE decreases the level of stress fiber formation in H460 and H1229 cells. These studies indicate that NHE plays a vital role in stress fiber formation and cellular migration, metastasis, in human lung cancer cells.

Regulation of the Cardiac Na+/Ca2+ Exchanger by Calcineurin and Protein Kinase C

Na+/Ca2+ exchanger (NCX) activity is markedly inhibited in hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment. This inhibition is reversed partially and independently by acute inhibition of calcineurin and protein kinase C (PKC) activities. Similar NCX inhibition occurs in CCL39 cells expressing cloned wild-type NCX1, when they are infected with adenoviral vectors carrying activated calcineurin A and then treated acutely with phorbol myristoyl acetate or protein phosphatase-1 inhibitors. The data obtained with these cells suggest that calcineurin activity, PKCalpha-mediated NCX1 phosphorylation, and the central loop of NCX1 (possibly its beta1 repeat) are required for the observed NCX inhibition. We observe partial inhibition of NCX activity independent of NCX1 phosphorylation when CCL39 cells are infected with activated calcineurin A but not further treated with phorbol myristoyl acetate or phosphatase inhibitors. Calcineurin thus appears to downregulate NCX activity via two independent mechanisms, one involving NCX1 phosphorylation and the other not involving NCX1 phosphorylation. These data indicate the existence of a novel regulatory mechanism for NCX1 involving calcineurin and PKC, which may be important in cardiac pathology.

Sodium Hydrogen Exchanger 1 is required for Matrix Metalloproteinase 9 activity by Phenylephrine

Matrix metalloproteinases (MMP) are a group of enzymes that play a critical role in digesting the extracellular matrix. Degradation of the extracellular matrix by MMP in migrating cells provides a vital function for tumor metastasis and angiogenesis. The link between extracellular MMP activity and the sodium hydrogen exchanger (NHE) has been suggested but not yet identified. We studied the relationship between NHE and MMP activity in CCL39 fibroblasts containing NHE1, PS120 cells (NHE1 null derived from CCL39 cells) and PS127 cells (PS120 cells expressing NHE1). Initial studies with CCL39 cells found resting cells had moderate MMP9 activity. This activity increased 2.5 fold after 12 hour phenylephrine (PE) stimulation. Western blot analysis of culture media identified MMP9. We found MMP9 activity to be dependent upon the expression and activation of NHE1. In both CCL39 and PS127 cells, MMP9 was activated in the presence of 100 μM PE. In PS120 cells no MMP was activated in the presence of PE. Incubation of cells with amiloride prior to PE addition also resulted in a notable decrease in MMP9 activation compared to control cells. Incubation of cells with 0.5% butanol prior to PE stimulation decreased MMP9 activity similar to the control level, while expression of either dominant negative phospholipase D1 or 2 caused a decrease in MMP9 activity less than untransfected cells. This work, for the first time, describes an agonist-induced relationship between NHE1 and MMP and a new potential role for NHE1 in tumor formation. This work was supported by a grant from the NIH, Award number 1 R15 HL074924-01A1.

Heat shock protein 27 downregulates the transferrin receptor 1-mediated iron uptake

It has been reported that over-expression of human heat shock protein 27 (hsp27) in murine cells decreased the intracellular iron level [Arrigo, A. P., Virot, S., Chaufour, S., Firdaus, W., Kretz-Remy, C., & Diaz-Latoud, C. (2005). Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxidants & Redox Signalling, 7, 412–422]. However, the mechanism involved is unknown. In this study, the regulation of transferrin receptor 1 (TfR1)-mediated iron uptake by human hsp27 was investigated in CCL39 cells by overexpression of human hsp27 and its dominant-negative mutant (hsp27-3G). The results showed that overexpression of hsp27 diminished intracellular labile iron pool, increased the binding activity of iron regulatory protein (IRP) to iron responsive element (IRE) and the cell surface-expressed TfR1s. However, the increased surface-expressed TfR1s resulted in decrease rather than increase of iron uptake. Further study revealed that overexpression of hsp27 decelerated transferrin endocytosis and recycling, while overexpressed hsp27-3G had a reversal effect. Moreover, flowcytometric analysis showed an enhanced actin polymerization in the cells overexpressing hsp27. In particular, fluorescence imaging of cytoskeleton displayed highly stabilized microfilaments and preferential localization of hsp27 in cortical area of the actin cytoskeleton. In contrast, disruption of actin cytoskeleton by cytochalasin B resulted in acceleration of the endocytosis and recycling of Tf, as well as increase of iron uptake. Meanwhile, the possible involvement of ferroportin 1 in down-regulation of intracellular iron level by overexpression of hsp27 was checked. However, the outcome was negative. Our findings indicate that hsp27 down-regulates TfR1-mediated iron uptake via stabilization of the cortical actin cytoskeleton rather than the classical IRP/IRE mode. The study may also imply that hsp27 protects cells from oxidative stress by reducing cellular iron uptake.

The conditional kinase ΔMEKK1:ER* selectively activates the JNK pathway and protects against serum withdrawal-induced cell death

The conditional protein kinase ΔMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of ΔMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of Bim EL and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of Bim EL but caused only a partial reversion of the cyto-protective effect of ΔMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing ΔMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IκB kinase. Activation of ΔMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death.

Phospholipase C-β1mediates 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) phosphorylation in Chinese hamster lung fibroblasts (CCL39) was characterized in response to the specific alpha1-adrenergic agonist, phenylephrine (PE). Addition of 100 micromol 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 PLCbeta-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 PLCbeta1, PLCbeta2, or PLCbeta3, revealed that stimulation of NHE1 activity by PE in CCL39 cells is a PLCbeta1-coupled event. Furthermore, the data indicate that alpha1-adrenergic signaling of PLCbeta is upstream of ERK activation. These data demonstrate that PLCbeta1 is primarily involved in the activation of NHE1 in CCL39 fibroblasts.

Calcineurin Inhibits Na+/Ca2+ Exchange in Phenylephrine-treated Hypertrophic Cardiomyocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX1) is the predominant mechanism for the extrusion of Ca(2+) from beating cardiomyocytes. The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased hearts remains unclear. In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C terminus of calcineurin Abeta, containing the autoinhibitory domain, binds to the beta1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca(2+) regulatory site. The association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with that in the normal control. In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na(+)(i)-dependent (45)Ca(2+) uptake or the rate of Na(+)(o)-dependent (45)Ca(2+) efflux. Depressed NCX activity was partially and independently reversed by the acute inhibition of calcineurin and protein kinase C activities with little effect on myocyte hypertrophic phenotypes. Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the beta1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate. Our data suggest that NCX1 is a novel regulatory target for calcineurin and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and protein kinase C are chronically activated.

Fish somatostatin sst3 receptor: comparison of radioligand and GTPγS binding, adenylate cyclase and phospholipase C activities reveals different agonist‐dependent pharmacological signatures

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.

Different cation sensitivities and binding site domains of Na+–Ca2+–K+ and Na+–Ca2+ exchangers

We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li(+) stimulated reverse-mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K(+). Thus, both exchangers exhibited differential sensitivities to not only K(+) but also many other external cations. We attempted to locate the putative binding sites within the alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.

α-Thrombin Rapidly Induces Tyrosine Phosphorylation of a Novel, 74–78-kDa Stress Response Protein(s) in Lung Fibroblast Cells

We demonstrated previously that exposure of CCL39 lung fibroblasts to alpha-thrombin rapidly inhibits interleukin 6-induced tyrosine phosphorylation of signal transducers and activators of transcription 3 (Stat3). While studying the cross-talk between alpha-thrombin and interleukin 6, we observed that the phospho-specific (tyrosine) anti-Stat3 antibody specifically cross-reacted with a 74-78-kDa protein(s) in alpha-thrombin-treated cells. In this study, we demonstrate that in alpha-thrombin-treated CCL39 cells, the 74-78-kDa protein(s) rapidly undergoes tyrosine phosphorylation. The phosphorylation by alpha-thrombin was detected as early as 5 min and reached a maximum at 15 min; however, low levels were present at 2 h. alpha-Thrombin receptor agonist peptide (SFLLRN) induced its tyrosine phosphorylation, suggesting that alpha-thrombin mediates the effects via protease-activated receptor type 1. Anti-Stat3 antibodies specific to different regions of Stat3 failed to recognize the 74-78-kDa protein(s), suggesting that it is unrelated to Stat3. Cell fractionation experiments showed that it is localized to the cytoplasm. Mass spectrometric analysis of the immunoprecipitated protein showed that the 74-78-kDa protein(s) is related to glucose-regulated protein 75 (GRP-75), a member of the heat shock/stress-response protein family. Consistent with these data, we observed tyrosine phosphorylation of GRP-75 in alpha-thrombin-treated cells. Exposure of cells to pervanadate, a stress-inducing agent, stimulated its tyrosine phosphorylation; however, cytokines and growth factors were ineffective. This is the first report of tyrosine phosphorylation of GRP-75-related stress protein(s) by alpha-thrombin and suggests that this pathway may contribute to the ability of alpha-thrombin to prevent apoptosis in cells exposed to stress or in the injured tissue.

Two G protein-coupled receptors activate Na +/H + exchanger isoform 1 in Chinese hamster lung fibroblasts through an ERK-dependent pathway

The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific α 1-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 μM phenylephrine and 10 μM LPA. α 1-Adrenergic stimulation also led to a rise in steady-state pH i of 0.16±0.02 pH units, and incubation with LPA induced a 0.43±0.06 pH unit increase in pH i. Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the α 1-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.