Original ArticleHomologous Desensitization of the Endothelin-1 Receptor Mediated Phosphoinositide Response in Cultured Neonatal Rat Cardiomyocytes
Abstract
The goal of the present study was to identify the molecular mechanism underlying desensitization of endothelin-1 receptor-mediated phosphoinositide response in cultured neonatal rat heart cells. Endothelin elicited a concentration-dependent (EC50=2.2 × 10-9 M) increase of inositolphosphate production with a much higher potency then phenylephrine (EC50=1.4 × 10-6 M). Endothelin-1 (10-8 M) evoked phosphoinositide turnover in the presence of 10 mM LiCl, which was greatly attenuated after 30-45 min of continuous stimulation with agonist, apparently resulting in a total absence of further inositolphosphate accumulation. However, when the uncompetitive inositol monophosphatase inhibitor Li+ was only present during the last 30 min of 150 min incubation, the inositolphosphate accumulation was decreased to a steady state of 33% of the initial rate. The loss of responsiveness of cardiomyocytes to endothelin-1 was not brought about by a limiting supply of phospholipase C substrate phosphatidylinositol 4,5-bishosphate. A very rapid resynthesis of this substrate took place as its level remained almost constant during 45 min stimulation with 10-8M endothelin-1 while the accumulation of inositolphosphates was at least 15-fold higher than the initial cellular phosphatidylinositol 4,5-bisphosphate content. After 120 min preincubation of cells with 10-9M endothelin-1 the activation of phospholipase C by a second higher dose (10-8 M) was severely (67%) inhibited at the same time leaving the induction of phosphoinositide turnover by phenylephrine (10-4 M) virtually intact. Preincubation with phenylephrine (3 × 10-6 M) also led to inhibition of the phenylephrine (10-4 M) mediated inositolphosphate response (36% inhibition) while the endothelin-1 (10-8 M) response was not affected. Addition of a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, led to inhibition of the endothelin-1 evoked phosphoinositide turnover but the rate of desensitization was not affected. Inhibition of protein kinase C with staurosporine did not alter the time course of desensitization. In conclusion, the activity of the phosphoinositide cycle in cardiomyocytes is homologously desensitized after stimulation with endothelin-1. The desensitization is not likely to be due to either depletion of phospholipase C substrate or to the activation of protein kinase C by inositol 1,4,5-trisphosphate-mobilized Ca2+ and elevated 1,2-diacylglycerol levels.
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Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and α-B-crystallin
2008, Biochimica et Biophysica Acta - Proteins and ProteomicsWe performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as α-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and α-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.
Down-regulation and recovery of endothelin-1 binding and signaling in rat cardiomyocytes
2001, Biochemical and Biophysical Research CommunicationsDown-regulation and recovery of endothelin (ET) receptors and of ET-dependent phosphoinositide-specific phospholipase C (PI-PLC) signaling was examined in cultured cardiomyocytes from neonatal rats. Three hours treatment with 5 nM ET-1 decreased surface receptors to 30%, and transduction to 19%, of their respective time-zero values. After extensive washing and a 3 h recovery period surface receptors returned to 74% of the time-zero value, with concomitant recovery of signal transduction to 75% of the time-zero value. The recovery of PI-PLC signaling in these cells is in contrast with a previous report, but consistent with recovery of the receptor complement.
Endothelin-1 responsiveness of a 1.4 kb phospholamban promoter fragment in rat cardiomyocytes transfected by the gene gun
2000, Journal of Molecular and Cellular CardiologyThe transcriptional regulation of an isolated rat phospholamban (PL) promoter fragment in rat cardiomyocytes was analyzed by applying a new method to reach substantially higher transfection efficiencies: gene gun biolistics. The gene gun transfection method was optimized for application to primary cultures of rat neonatal cardiomyocytes. Cells, cultured at different densities (0.75–1.50×105cells/cm2) in serum-free medium, were transfected with DNA coated gold particles. A transfection efficiency of up to 10% could be achieved (compared to <1% with other methods) by the gene gun as checked using a RSV- β-Gal construct. Cardiomyocytes were stimulated by endothelin-1 (ET-1) (10−8M) to induce hypertrophy, thereby yielding the characteristic changes in gene expression (upregulation of Atrial Natriuretic Factor (ANF) and downregulation of PL). The basal activity of an ANF promoter fragment (increasing from the lowest to highest density 2.6-fold) and its ET-1 inducibility (only significant upregulation of 2.6-fold, at lowest density) appeared to be dependent on the plating density of the cardiomyocytes. A PL promoter fragment was isolated, sequenced and 1.4 kb was subcloned in a luciferase reporter vector. The basal activity of the PL promoter fragment was not dependent on the plating density. ET-1 did not downregulate the PL promoter, rather a significant upregulation (1.4-fold) was found at the highest plating density. In conclusion, plating density of the cardiomyocytes can influence promoter activity as shown with an ANF promoter fragment. A newly isolated and sequenced rat PL promoter fragment did not direct gene expression as expected on basis of downregulation of the PL gene by ET-1 observed in this model.
Cross-talk between receptor-mediated phospholipase C-β and D via protein kinase C as intracellular signal possibly leading to hypertrophy in serum-free cultured cardiomyocytes
1997, Journal of Molecular and Cellular CardiologyPhospholipase C-β(PLC-β) signalling via protein kinase C (PKC) has been recognized as a major route by which stimuli such asα1-adrenergic agonists, endothelin-1 (ET-1) and angiotensin II (Ang II) induce hypertrophy of myocytes. The goal of this study was to evaluate the role of phospholipase D (PLD) in contributing to the formation of the PKC activator 1,2-diacylglycerol (1,2-DAG) and to study the mechanism(s) of PLD activation by agonists. Stimulation of serum-free cultured neonatal rat cardiomyocytes with ET-1 (10−8m), phenylephrine (PHE, 10−5m) or Ang II (10−7m) resulted in a rapid (0–10 min) activation of PLC-βto an extent (ET-1>PHE>Ang II) that correlated with the magnitude of stimulation of protein synthesis ([3H]leucine incorporation into protein) measured after 24 h. Phorbol 12-myristate 13-acetate (PMA, 10−6m) and ET-1 were equipotent in stimulating protein synthesis. ET-1 and PMA, but not PHE and Ang II stimulated [3H]choline formation from labelled PtdCho after a lag-phase of about 10 min. That this [3H]choline formation was due to the action of PLD was confirmed by measurement of phosphatidylgroup-transfer from cellular [14C]palmitoyl-phosphatidylcholine to exogenous ethanol. ET-1 and PHE, to much lesser extent, produced a rapid (0–5 min) translocation of PKC-εimmunoreactivity from the cytosol to the membrane fraction, whereas no intracellular redistribution of PKC-α, -δand -ξimmunoreactivities was observed. PMA caused translocation of PKC-α, PKC-εas well as PKC-δ. Cellular redistribution of PKC activity measured by [32P]-incorporation into histone III-S was not observed with ET-1 and PHE, but only with PMA stimulation. Down-regulation of PKC isozymes by 24 h pretreatment of cells with PMA or blockade of PKC by chelerythrine (10−4m) inhibited ET-1 and PMA stimulated [3H]choline production. Staurosporine (10−6m) had, however, no effect. In conclusion, the results indicate that in serum-free cultured cardiomyocytes, ET-1 initially activates PLC-βand after a lag-phase PLD, whereas PHE and Ang II activate only PLC-β. PLC-βstimulated by ET-1, may cross-talk with PLD via translocation of PKC-ε. These signals are possibly linked to the hypertrophic response.
Angiotensin II-mediated growth and antigrowth effects in cultured neonatal rat cardiac myocytes and fibroblasts
1997, Journal of Molecular and Cellular CardiologyAngiotensin II (Ang II) stimulates cardiovascular growth and remodeling via AT1receptors. Recent experiments have shown that Ang II may also exert antiproliferative effects via AT2receptors. We studied the effects of Ang II on protein and DNA content and synthesis rate in unstimulated and endothelin-1 (ET-1)-stimulated neonatal rat cardiomyocytes and fibroblasts, isolated from 1–3-day-old Wistar strain pups. Total protein and total DNA, as well as [3H]leucine and [3H]thymidine incorporation were measured following incubation with either vehicle, Ang II, ET-1 or Ang II+ET-1, both in the presence or absence of the AT1receptor blocker losartan or the AT2receptor blocker PD123319. In myocytes, ET-1 increased total protein (+38% relative to control) as well as [3H]leucine (+66%) and [3H]thymidine (+77%) incorporation. Ang II did not affect any of these parameters, nor did it influence the ET-1-induced responses. However, in the presence of PD123319 Ang II stimulated [3H]leucine (+24%) and [3H]thymidine (+30%) incorporation. In fibroblasts, ET-1 and Ang II did not significantly affect total DNA and [3H]thymidine incorporation. Ang II tended to increase total protein in these cells, an effect which was significant only in the presence of PD123319 (+17%). Ang II stimulated [3H]leucine incorporation (+24%) in fibroblasts. This effect was absent with losartan and enhanced in the presence of PD123319. These data demonstrate that AT1receptor-mediated proliferative effects of Ang II in neonatal cardiac cells may become apparent only when its AT2receptor-mediated antigrowth effects are blocked. The net growth effect of Ang II therefore depends on the cellular AT1/AT2receptor ratio. Ang II does not appear to interfere with ET-1-induced effects.
Regulation of phospholipases C and D in rat ventricular myocytes: Stimulation by endothelin-1, bradykinin and phenylephrine
1997, Journal of Molecular and Cellular CardiologyThe physiological activator of protein kinase C (PKC), diacylglycerol, is formed by hydrolysis of phosphoinositides (PI) by phospholipase C (PLC) or phosphatidylcholine by phospholipase D (PLD). We have measured activation of these phospholipases by endothelin-1 (ET-1), bradykinin (BK), or phenylephrine (PE) in ventricular myocytes cultured from neonatal rat. The stimulation of PI hydrolysis after 10 min by 0.1 μmET-1 (about 12-fold) was much greater than for BK or PE (each about four-fold), and did not correlate with translocation of nPKCδor nPKCε(Clerk A, Bogoyevitch MA, Andersson MB, Sugden PH, 1994.J Biol Chem269: 32848–32857; Clerk A, Gillespie-Brown J, Fuller SJ, Sugden PH, 1996.Biochem J317: 109–118). However, ET-1 and BK stimulated a similar rapid increase in [3H]InsP3formation (<30 s), which was much greater than that seen with PE. This early phase correlated with PKC translocation. Acute or chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment with Ro-31-8220 showed that the stimulation of PI hydrolysis by PE, but not ET-1 or BK, was inhibited by activation of PKC. Furthermore, ET-1 and BK heterologously desensitized the stimulation of PI hydrolysis by PE. ET-1 or BK homologously uncoupled their own receptors from [3H]InsP3formation, but there was no evidence of heterologous desensitization with these two agonists. Anomalously, chronic exposure to TPA increased the stimulation of PI hydrolysis by BK, but this probably resulted from an increase in BK receptor density. PLD was also rapidly activated by TPA, ET-1, BK or PE. Experiments with Ro-31-8220 showed that the stimulation of PLD by ET-1 and BK was mediated through activation of PKC. We discuss the characteristics of the activation of PI hydrolysis and PLD by ET-1, BK, and PE with respect to the translocation of PKC.