- Email: [email protected]
Angiotensin II stimulates PGF2 αrelease in cultured neonatal rat ventricular myocytes via L-type calcium channels
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2001.0291, available online at http://www.idealibrary.com on
Angiotensin II stimulates PGF2a release in cultured neonatal rat ventricular myocytes via L-type calcium channels G. K. Oriji Department of Biology,College of Science and Health,William Paterson University, Wayne,NJ 07470 and Hypertension-Endocrine Branch,National Heart,Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Summary Angiotensin II (Ang II) hasbeen shownto cause Prostaglandin F2a (PGF2a) releaseinneonatalrat ventricular myocytes and smooth muscle cells.In these cells, Ang II has also been shown to regulate growth.We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF2a release.The amount of PGF2a produced was determined by radioimmunoassay. Ang II-induced PGF2a release. Pretreatment of neonatal rat ventricular myocytes with different doses (1078 M,1077 M,1076 M and1075 M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF2a release. Inhibition was first noted at1078 M and was complete at1076 M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (1078 M,1077 M,1076 M and1075 M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF2a release.These results strongly suggest that Ang II-induced PGF2a release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx. & 2001Harcourt Publishers Ltd
INTRODUCTION Angiotensin II (Ang II) causes contraction of vascular smooth muscle in part by increasing the influx of extracellular calcium through calcium channels.1–4 The increase in intracellular levels of calcium may activate phospholipase A2 (PLA2), increase intracellular levels of arachidonate, and enhance the biosynthesis of prostaglandins.5–8 Recently, we showed that Ang II stimulated PGF2a release and hypertrophic growth of neonatal rat ventricular myocytes; this is mediated by protein kinase C (PKC).10 PKC is known to phosphorylate protein at serine or threonine positions.11,12 PKC may phosphorylate PLA2, which may lead to an increase in PG synthesis. Therefore, the aim of this study is to determine the role of extracellular calcium in Ang II-induced PGF2a production. With the observation that PKC activates voltagesensitive L-type calcium channels,13,14 we also used a PKC enzyme assay to study the role of extracellular
Received 17 April 2001 Accepted 12 June 2001 Correspondence to: Dr Gibson K. Oriji, Department of Biology, College of Science and Health,William Paterson University,Wayne, NJ 07470, USA. Tel.: (973) 720 3445; Fax: (973) 720 2338; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
calcium stimulated by PKC signaling pathway in Ang IIinduced PGF2a production.
MATERIALS AND METHODS
Isolation of neonatal rat ventricular myocytes Neonatal rat ventricular myocytes cultures were prepared as per Sen et al., with minor modification.15 Briefly, whole hearts were removed from 1–2-day-old rats. Atria were trimmed away, and ventricles were minced in a balanced salt solution, Hanks buffer salt solution (HBSS), containing NaCl 116 mM, KCl 5.4 mM, NaHPO4 1 mM, MgSO4 1.2 Mm, glucose 5.5 mM, and HEPES 20 mM at pH 7.4. Heart cells were dissociated in HBSS at 378C by a combination of mechanical agitation (Wheaton 356743) and enzymatic digestion with collagenase (5 mg/ml) and elastase (5 mg/ml). After 30 min, the supernatant was discarded and replaced with fresh HBSS. Subsequently, for each of three to four 20 min digestion periods, dissociated cells were collected in 20% serum, concentrated by centrifugation at 500 g for 10 min, resuspended in serum, and kept at 378C. Pooled cells were pelleted by centrifugation, resuspended in HBSS. Myocytes were separated from nonmuscle cardiocytes by centrifugation at 2000 g for 30 min and collected into a 50 ml conical Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
74
Oriji
tube and washed twice in HBSS. Washed cells were counted in a hemacytometer before plating onto gelatin (2%) coated culture dishes (Falcon) at a density of 900 cells/mm in 10 ml of M199 media containing antibiotics (penicillin 100 units/ml), and streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS). The media was replaced 24 h later to remove debris and unattached cells. At one day interval, the media was replaced with M199 containing antibiotics and 10% FBS to feed the cells. The resulting myocardial cell cultures were 495% myocytes, as assessed by immunocytofluorescence with myosin light chain-2 (MLC-2) antisera.16 Primary cell cultures were used for the experiments. Experiments were performed by plating cells in 6-well plates at 161076 cells per well. PGF2a was assayed by radioimmunoassay kit with specific antibody for 6-keto-PGF2a (Kit #515) (Amersham, Arlington Heights, IL 60005). PKC activity was determined in either cytosol or membrane fractions by an enzyme assay (Kit #RPN 77, Amersham, Arlington Heights, IL 60005). Cytosol and membrane fractions were prepared as per Eulalia et al.17
1076 M of diltiazm or conotoxin or thapsigargin in separate experiments before the addition of Ang II (1078 M). The perfusate was collected and PGF2a release was measured (n = 8 for each dose of antagonist).
Drugs and chemicals Angiotensin II, diltiazm, conotoxin, thapsigargin, phorbol 12,13-dibutyrate and 4-alpha-phorbol, 12,13-didecanoate were all obtained from Calbiochem Inc., San Diego, CA, USA.
Statistical analysis All data were generated with paired controls. Values are expressed as mean + SEM. Two way analysis of variance was used for comparisons within groups. A value of P50.05 was considered significant.
EXPERIMENTAL PROTOCOL
Protocol 1 This experiment was performed to determine the effects of different doses (0.561079 M, 1079 M, 1078 M, 1077 M and 1076 m) of Ang II on PGF2a release. Neonatal rat ventricular myocytes were stimulated with Ang II for 30 min and the perfusate was collected and PGF2a release was measured (n = 8 for each dose of agonist).
Protocol 2 This experiment was performed to determine the effect of different doses (1078 M, 1077 M, 1076 M and 1075 M) of calcium channel blockers (diltiazm; voltage-sensitive L-type calcium channel blocker or conotoxin; voltagesensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) on PGF2a release. Neonatal rat ventricular myocytes were pretreated for 30 min with different doses (1078 M, 1077 M, 1076 M and 1075 M) of diltiazm or conotoxin or thapsigargin in separate experiments before the addition of Ang II (1078 M). The perfusate was collected and PGF2a release was measured (n = 8 for each dose of antagonist).
Protocol 3 This experiment was performed to determine the effect of 1076 M of different calcium channel blockers (diltiazm or conotoxin or thapsigargin) on PGF2a release. Neonatal rat ventricular myocytes were pretreated for 30 min with Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
Fig. 1 Effects of different doses (0.5 61079 M,1079 M,1078 M, 1077 M and1076 M) of (A) Ang II and (B) Diltiazm+Ang II on PGF2a release in neonatal rat ventricular myocytes. Neonatal rat ventricular myocytes were stimulated with different doses of (A) Ang II for 30 min. Neonatal rat ventricular myocytes were pretreated with different doses (1078 M,1077 M,1076 M and1075 M) of (B) Diltiazm (voltagesensitive L-type calcium channel blocker), for 30 min, in different experiments before the addition of Ang II (1078 M). *P50.021vs control.
& 2001Harcourt Publishers Ltd
Calcium is involved in Ang II-induced PGF2a release 75
RESULTS
Effects of different doses of Ang II and thapsigargin+Ang II or conotoxin+Ang II or thapsigargin+Ang II on PGF2a release in neonatal rat ventricular myocytes
Ang II (1078 M)-induced PGF2a release (Fig. 1). On the other hand, conotoxin or thapsigargin did not cause any significant changes in Ang II (1078 M)-induced PGF2a release (Fig. 2).
Ang II produced significant and dose-dependent (0.5 6 1079 M, 1079 M, 1078 M, 1077 M and 1076 M) increases in PGF2a release (Fig. 1). Pretreatment with different doses (1078 M, 1077 M, 1076 M and 1075 M) of calcium channel blockers (diltiazm; voltage-sensitive L-type calcium channel blocker or conotoxin; voltagesensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced different effects on Ang II-induced PGF2a release. Diltiazm inhibited PGF2a release in a dose-dependent manner. Diltiazm (1076 M) was the minimal dose that completely inhibited
Fig. 2 Effects of different doses of (A) Conotoxin+Ang II and (B) thapsigargin+Ang II on PGF2a release in neonatal rat ventricular myocytes. Neonatal rat ventricular myocytes were pretreated with different doses (1078 M,1077 M,1076 M and1075 M) of (A) Conotoxin (voltage-sensitive N-type calcium channel blocker) and (B) thapsigargin (intracellular calcium channel blocker) for 30 min, in different experiments before the addition of Ang II (1078 M).
& 2001Harcourt Publishers Ltd
Fig. 3 Effects of calcium channel blockers, diltiazm or conotoxin or tharpsigargin, on Ang II-induced PGF2a release. Neonatal rat ventricular myocytes were pretreated with1076 M of each of the calcium channel blockers, (A) diltiazm or (B) conotoxin or (C) tharpsigargin, for 30 min before the addition of Ang II (1078 M) for 60 mins. *P50.011vs antagonist (diltiazm). Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
76
Oriji
Fig. 4 Effects of calcium channel blockers, diltiazm or conotoxin or thapsigargin, on Ang II-induced PKC activation.PKC enzyme assay showed activation, that is, translocation of PKC from cytosolto the membrane fractionin neonatalrat ventricular myocytesthat were treated with1078 Mof Ang II.Translocation wasinhibited by pretreatment with diltiazm (1076 M) (voltage-sensitive L-type calcium channel blocker) and unchanged with conotoxin (1076 M) (voltage-sensitive N-type calcium channel blocker) or thapsigargin (1076 M) (intracellular calcium channel blocker) or 4-aphorbol,12,13-Didecanoate (PDD) (1076 M), aninactive phorbolester (Fig.4).Calcium channelblockersor PDD alone did not affect translocation of PKC (Fig. 4).These results confirm that L-type calcium channel inhibition completely blocked Ang II-induced PGF2a release by activation of PKC. *P50.014 vs antagonist (diltiazm).
Effects of calcium channel blockers, thapsigargin or conotoxin or thapsigargin, on Ang II-induced PGF2a release in neonatal rat ventricular myocytes Ang II-induced PGF2a release was completely inhibited by pretreatment with calcium channel blocker, diltiazm (1076 M). On the other hand, pretreatment with other calcium channel blockers: conotoxin (1076 M) or thapsigargin (1076 M) did not cause any changes in Ang IIinduced PGF2a release (Fig. 3). These results confirm that the L-type calcium channel is involved in Ang II-induced PGF2a release. PKC enzyme assay showed activation, that is, translocation of PKC from cytosol to the membrane fraction in neonatal rat ventricular myocytes that were treated with 1078 M of Ang II (Fig. 4). Translocation was inhibited by pretreatment with diltiazm and unchanged with conotoxin or thapsigargin or 4-a-phorbol, 12,13-didecanoate (PDD), an inactive phorbol ester (Fig. 4). Calcium channel blockers or PDD alone did not affect translocation of PKC (Fig. 4).
DISCUSSION We showed the following in this study: 1) Ang II stimulated PGF2a release in neonatal rat ventricular myocytes, 2) Ang II-induced PGF2a release is maintained by extracellular calcium via activation of PKC. The inhibition of Ang II-induced PGF2a release by L-type Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
calcium channel blocker and the implication of PKC by enzyme assay suggest that extracellular calcium and PKC mediate these changes. Ang II-induced PGF2a release were inhibited with PKC inhibitor in our previous studies10 and L-type calcium channel blocker in the present study. PKC activates voltage-sensitive L-type calcium channels,13,14 which will enhance calcium influx from the extracellular compartment and therefore maintain the PGF2a release observed in this study. The mechanism by which Ang II causes increased PGF2a release is still unclear, but it appears to involve PKC and L-type calcium channels. Several studies have shown that prostanoids are able to influence the tone of cerebral arteries and arterioles.18–23 Indeed, PG synthesis is altered in various pathological conditions and causes profound modifications in tissue blood flow. It has been shown that the levels of PGF2a in the myocardium were increased by acute6 or chronic24 hemodynamic overload. In addition, PG synthase inhibitors blocked experimentally induced cardiac hypertrophy.25,26 Therefore, it is important to understand the mechanisms involved in PG release. Pretreatment of neonatal rat ventricular myocytes with different doses of diltiazm (voltage-sensitive L-type calcium channel blocker) produced dose-dependent inhibition in Ang II-induced PGF2a release. Inhibition was first noted at 1078 M and was complete at 1076 M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses of calcium channel blockers (thapsigargin; intracellular calcium channel blocker or & 2001Harcourt Publishers Ltd
Calcium is involved in Ang II-induced PGF2a release 77
conotoxin; voltage-sensitive N-type calcium channel blocker) produced no changes on Ang II-induced PGF2a release in neonatal rat ventricular myocytes. In conclusion, Ang II activates PKC, which increases PGF2a production and release via increase in extracellular calcium influx in neonatal rat ventricular myocytes.
ACKNOWLEDGMENTS The author wishes to thank Mrs Mary Oriji for her aid in the preparation of this manuscript. The author also thanks Mrs Margaret Hill and Mr John Tate for their excellent technical assistance.
REFERENCES 1. Griendling K. K., Rittenhouse S. E., Gimbrone M. A. Jr., Ekstein L. S., Brock T. A., Alexander R. W. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem 1986; 261: 5901–5906. 2. Saito T., Saito O., Ono S., Okada K., Kusano E., Homma S., Fujita N., Asano Y., Ando Y., Akimoto T. Related Articles. Erythropoietin modulates angiotensin II- or noradrenaline-induced Ca(2+) mobilization in cultured rat vascular smooth-muscle cells. Nephrol Dial Transplant 2001; 16(3): 491–499. 3. Yamamoto H., Ushio-Fukai M., Toyofuku K., Nishimura J., Kanaide H., Hirano K. Related Articles. Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery. Br J Pharmacol 2000; 129(3): 425–436. 4. Samain E., Safar M., Renaud J. F., Perret C., Miserey S., Dagher G., Bouillier H. Related Articles. Extracellular signal-regulated kinase pathway is involved in basic fibroblast growth factor effect on angiotensin II-induced Ca(2+) transient in vascular smooth muscle cell from Wistar-Kyoto and spontaneously hypertensive rats. Hypertension 2000; 35(1 Pt 1): 61–67. 5. Peach M. J. Molecular actions of angiotensin. Biochem Pharmacol 1981; 130: 2745–2751. 6. Chazov E. I., Smirnov V. N., Pomoinetsky V. D., Nekrasova A. A., Orlova T. R., Geling N. G. Heart adaptation to acute pressure overload: an involvement of endogenous prostaglandins. Circ Res 1979; 45(2): 205–211. 7. Schramek H. E., Coroneos E., Dunn M. J. Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins. Semin Nephrol 1995; 15(3): 195–204. 8. Escalante B., Cruz B. V. Renal vascular interaction of angiotensin II and prostaglandins in renovascular hypertension. J Cardiovasc Pharmacol 1999; 34(1): 21–27. 9. Harwalkar S., Falck J. R., Dulin N. O., Douglas J. G., Alexander L. D. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proc Natl Acad Sci USA 1998; 95(14): 8098–8102. 10. Oriji G. K. Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2a. Prostaglandins Leukot Essent Fatty Acids 2000; 62(4): 233–237.
& 2001Harcourt Publishers Ltd
11. Taylor C., Marshal I. Calcium and inositol 1,4,5-triphosphate receptors: a complex relationship. Elsevier Science Publishers, (UK) 0376-5067, 1992; 403–407. 12. Song Y. H., Rossi M. W., Korchak H. M., Kilpatrick L. E. Serine phosphorylation of p60 tumor necrosis factor receptor by PKCdelta in TNF-alpha-activated neutrophils. Am J Physiol Cell Physiol 2000; 279(6): C2011–C2018. 13. Smart D., Lambert D. G. Desensitization of the mu-opioid activation of phospholipase C in SH-SY5Y cells: the role of protein kinases C and A and Ca(2+)-activated K+ currents. Br J Pharmacol 1995; 116(6): 2655–2660. 14. Summers B. A., Prabhakar N. R., Overholt J. L. Related Articles. Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway. J Neurophysiol 2000; 84(3): 1636–1644. 15. Sen A., Dunnmon P., Henderson S. A., Gerard R. D., Chien K. R. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem 1988; 263: 19132–19136. 16. Iwaki K., Sukhatme V. P., Shubeita H. E., Chien K. R. Alpha- and beta-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response. J Biol 1990; 265: 13809–13817. 17. Eulalia B., Rapoport R. M., Campbell A. K. Protein Kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats. Eur J Pharm 1992; 227: 343–348. 18. Armstead W. M., Mirro D.W., Leffler C. W., Busija D. W. Permissive role of prostanoids in acetylcholine-induced cerebral constriction. J Pharmacol Exp Ther 1989; 251: 1012–1019. 19. Busija D. W., Leffler C. W. Eicosanoid synthesis elicited by norepinephrine in piglet parietal cortex. Brain Res 1987; 403: 243–248. 20. Busija D. W., Leffier C. S. Dilator effects of amino acid neurotransmitters on piglet pial arterioles. Am J Physiol 1989; 257: HI200–H1203. 21. Busija D. W., Wagerie L. C., Pourcyrous M., Leffler C. W. Acetylcholine dramatically increases prostanoid synthesis in piglet parietal cortex. Brain Res 1988; 439: 122–126. 22. Busija D. W., Leffler C. W. Effects of phorbol esters on pial arteriolar diameter and brain production of prostanoids in piglets. Circ Res 1991; 69: 1253–1258. 23. Pourcyrous M., Leffler C. W., Busija, D. W. Postasphyxial increases in prostaglandins in cerebrospinal fluid in piglets. Pediatr Res 1988; 24: 229–232. 24. Escobar E., Zamorano B., Gazmuri R. Demonstration of prostaglandin E2 and F2a in atrial tissue of patients with heart disease. Am J Cardiol 1983; 52: 424–425. 25. Kentera D., Susic D., Zdravkovic M. Effects of verapamil and aspirin on experimental chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. Respiration 1979; 37(4): 192–196. 26. Palmer R. M., Delday M. I., McMillan D. N., Noble B. S., Bain P., Maltin C. A. Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats. Br J Pharmacol 1990; 101: 835–838.
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
Angiotensin II stimulates PGF2a release in cultured neonatal rat ventricular myocytes via L-type calcium channels G. K. Oriji Department of Biology,College of Science and Health,William Paterson University, Wayne,NJ 07470 and Hypertension-Endocrine Branch,National Heart,Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Summary Angiotensin II (Ang II) hasbeen shownto cause Prostaglandin F2a (PGF2a) releaseinneonatalrat ventricular myocytes and smooth muscle cells.In these cells, Ang II has also been shown to regulate growth.We used neonatal rat ventricular myocytes to investigate the role of calcium in maintenance of Ang II-induced PGF2a release.The amount of PGF2a produced was determined by radioimmunoassay. Ang II-induced PGF2a release. Pretreatment of neonatal rat ventricular myocytes with different doses (1078 M,1077 M,1076 M and1075 M) of diltiazm (voltage-sensitive L-type calcium channel blocker) produced significant inhibition in Ang II-induced PGF2a release. Inhibition was first noted at1078 M and was complete at1076 M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses (1078 M,1077 M,1076 M and1075 M) of calcium channel blockers (conotoxin; voltage-sensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced no changes in Ang II-induced PGF2a release.These results strongly suggest that Ang II-induced PGF2a release in neonatal rat ventricular myocytes is maintained, at least in part, via increase in extracellular calcium influx. & 2001Harcourt Publishers Ltd
INTRODUCTION Angiotensin II (Ang II) causes contraction of vascular smooth muscle in part by increasing the influx of extracellular calcium through calcium channels.1–4 The increase in intracellular levels of calcium may activate phospholipase A2 (PLA2), increase intracellular levels of arachidonate, and enhance the biosynthesis of prostaglandins.5–8 Recently, we showed that Ang II stimulated PGF2a release and hypertrophic growth of neonatal rat ventricular myocytes; this is mediated by protein kinase C (PKC).10 PKC is known to phosphorylate protein at serine or threonine positions.11,12 PKC may phosphorylate PLA2, which may lead to an increase in PG synthesis. Therefore, the aim of this study is to determine the role of extracellular calcium in Ang II-induced PGF2a production. With the observation that PKC activates voltagesensitive L-type calcium channels,13,14 we also used a PKC enzyme assay to study the role of extracellular
Received 17 April 2001 Accepted 12 June 2001 Correspondence to: Dr Gibson K. Oriji, Department of Biology, College of Science and Health,William Paterson University,Wayne, NJ 07470, USA. Tel.: (973) 720 3445; Fax: (973) 720 2338; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
calcium stimulated by PKC signaling pathway in Ang IIinduced PGF2a production.
MATERIALS AND METHODS
Isolation of neonatal rat ventricular myocytes Neonatal rat ventricular myocytes cultures were prepared as per Sen et al., with minor modification.15 Briefly, whole hearts were removed from 1–2-day-old rats. Atria were trimmed away, and ventricles were minced in a balanced salt solution, Hanks buffer salt solution (HBSS), containing NaCl 116 mM, KCl 5.4 mM, NaHPO4 1 mM, MgSO4 1.2 Mm, glucose 5.5 mM, and HEPES 20 mM at pH 7.4. Heart cells were dissociated in HBSS at 378C by a combination of mechanical agitation (Wheaton 356743) and enzymatic digestion with collagenase (5 mg/ml) and elastase (5 mg/ml). After 30 min, the supernatant was discarded and replaced with fresh HBSS. Subsequently, for each of three to four 20 min digestion periods, dissociated cells were collected in 20% serum, concentrated by centrifugation at 500 g for 10 min, resuspended in serum, and kept at 378C. Pooled cells were pelleted by centrifugation, resuspended in HBSS. Myocytes were separated from nonmuscle cardiocytes by centrifugation at 2000 g for 30 min and collected into a 50 ml conical Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
74
Oriji
tube and washed twice in HBSS. Washed cells were counted in a hemacytometer before plating onto gelatin (2%) coated culture dishes (Falcon) at a density of 900 cells/mm in 10 ml of M199 media containing antibiotics (penicillin 100 units/ml), and streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS). The media was replaced 24 h later to remove debris and unattached cells. At one day interval, the media was replaced with M199 containing antibiotics and 10% FBS to feed the cells. The resulting myocardial cell cultures were 495% myocytes, as assessed by immunocytofluorescence with myosin light chain-2 (MLC-2) antisera.16 Primary cell cultures were used for the experiments. Experiments were performed by plating cells in 6-well plates at 161076 cells per well. PGF2a was assayed by radioimmunoassay kit with specific antibody for 6-keto-PGF2a (Kit #515) (Amersham, Arlington Heights, IL 60005). PKC activity was determined in either cytosol or membrane fractions by an enzyme assay (Kit #RPN 77, Amersham, Arlington Heights, IL 60005). Cytosol and membrane fractions were prepared as per Eulalia et al.17
1076 M of diltiazm or conotoxin or thapsigargin in separate experiments before the addition of Ang II (1078 M). The perfusate was collected and PGF2a release was measured (n = 8 for each dose of antagonist).
Drugs and chemicals Angiotensin II, diltiazm, conotoxin, thapsigargin, phorbol 12,13-dibutyrate and 4-alpha-phorbol, 12,13-didecanoate were all obtained from Calbiochem Inc., San Diego, CA, USA.
Statistical analysis All data were generated with paired controls. Values are expressed as mean + SEM. Two way analysis of variance was used for comparisons within groups. A value of P50.05 was considered significant.
EXPERIMENTAL PROTOCOL
Protocol 1 This experiment was performed to determine the effects of different doses (0.561079 M, 1079 M, 1078 M, 1077 M and 1076 m) of Ang II on PGF2a release. Neonatal rat ventricular myocytes were stimulated with Ang II for 30 min and the perfusate was collected and PGF2a release was measured (n = 8 for each dose of agonist).
Protocol 2 This experiment was performed to determine the effect of different doses (1078 M, 1077 M, 1076 M and 1075 M) of calcium channel blockers (diltiazm; voltage-sensitive L-type calcium channel blocker or conotoxin; voltagesensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) on PGF2a release. Neonatal rat ventricular myocytes were pretreated for 30 min with different doses (1078 M, 1077 M, 1076 M and 1075 M) of diltiazm or conotoxin or thapsigargin in separate experiments before the addition of Ang II (1078 M). The perfusate was collected and PGF2a release was measured (n = 8 for each dose of antagonist).
Protocol 3 This experiment was performed to determine the effect of 1076 M of different calcium channel blockers (diltiazm or conotoxin or thapsigargin) on PGF2a release. Neonatal rat ventricular myocytes were pretreated for 30 min with Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
Fig. 1 Effects of different doses (0.5 61079 M,1079 M,1078 M, 1077 M and1076 M) of (A) Ang II and (B) Diltiazm+Ang II on PGF2a release in neonatal rat ventricular myocytes. Neonatal rat ventricular myocytes were stimulated with different doses of (A) Ang II for 30 min. Neonatal rat ventricular myocytes were pretreated with different doses (1078 M,1077 M,1076 M and1075 M) of (B) Diltiazm (voltagesensitive L-type calcium channel blocker), for 30 min, in different experiments before the addition of Ang II (1078 M). *P50.021vs control.
& 2001Harcourt Publishers Ltd
Calcium is involved in Ang II-induced PGF2a release 75
RESULTS
Effects of different doses of Ang II and thapsigargin+Ang II or conotoxin+Ang II or thapsigargin+Ang II on PGF2a release in neonatal rat ventricular myocytes
Ang II (1078 M)-induced PGF2a release (Fig. 1). On the other hand, conotoxin or thapsigargin did not cause any significant changes in Ang II (1078 M)-induced PGF2a release (Fig. 2).
Ang II produced significant and dose-dependent (0.5 6 1079 M, 1079 M, 1078 M, 1077 M and 1076 M) increases in PGF2a release (Fig. 1). Pretreatment with different doses (1078 M, 1077 M, 1076 M and 1075 M) of calcium channel blockers (diltiazm; voltage-sensitive L-type calcium channel blocker or conotoxin; voltagesensitive N-type calcium channel blocker or thapsigargin; intracellular calcium channel blocker) produced different effects on Ang II-induced PGF2a release. Diltiazm inhibited PGF2a release in a dose-dependent manner. Diltiazm (1076 M) was the minimal dose that completely inhibited
Fig. 2 Effects of different doses of (A) Conotoxin+Ang II and (B) thapsigargin+Ang II on PGF2a release in neonatal rat ventricular myocytes. Neonatal rat ventricular myocytes were pretreated with different doses (1078 M,1077 M,1076 M and1075 M) of (A) Conotoxin (voltage-sensitive N-type calcium channel blocker) and (B) thapsigargin (intracellular calcium channel blocker) for 30 min, in different experiments before the addition of Ang II (1078 M).
& 2001Harcourt Publishers Ltd
Fig. 3 Effects of calcium channel blockers, diltiazm or conotoxin or tharpsigargin, on Ang II-induced PGF2a release. Neonatal rat ventricular myocytes were pretreated with1076 M of each of the calcium channel blockers, (A) diltiazm or (B) conotoxin or (C) tharpsigargin, for 30 min before the addition of Ang II (1078 M) for 60 mins. *P50.011vs antagonist (diltiazm). Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
76
Oriji
Fig. 4 Effects of calcium channel blockers, diltiazm or conotoxin or thapsigargin, on Ang II-induced PKC activation.PKC enzyme assay showed activation, that is, translocation of PKC from cytosolto the membrane fractionin neonatalrat ventricular myocytesthat were treated with1078 Mof Ang II.Translocation wasinhibited by pretreatment with diltiazm (1076 M) (voltage-sensitive L-type calcium channel blocker) and unchanged with conotoxin (1076 M) (voltage-sensitive N-type calcium channel blocker) or thapsigargin (1076 M) (intracellular calcium channel blocker) or 4-aphorbol,12,13-Didecanoate (PDD) (1076 M), aninactive phorbolester (Fig.4).Calcium channelblockersor PDD alone did not affect translocation of PKC (Fig. 4).These results confirm that L-type calcium channel inhibition completely blocked Ang II-induced PGF2a release by activation of PKC. *P50.014 vs antagonist (diltiazm).
Effects of calcium channel blockers, thapsigargin or conotoxin or thapsigargin, on Ang II-induced PGF2a release in neonatal rat ventricular myocytes Ang II-induced PGF2a release was completely inhibited by pretreatment with calcium channel blocker, diltiazm (1076 M). On the other hand, pretreatment with other calcium channel blockers: conotoxin (1076 M) or thapsigargin (1076 M) did not cause any changes in Ang IIinduced PGF2a release (Fig. 3). These results confirm that the L-type calcium channel is involved in Ang II-induced PGF2a release. PKC enzyme assay showed activation, that is, translocation of PKC from cytosol to the membrane fraction in neonatal rat ventricular myocytes that were treated with 1078 M of Ang II (Fig. 4). Translocation was inhibited by pretreatment with diltiazm and unchanged with conotoxin or thapsigargin or 4-a-phorbol, 12,13-didecanoate (PDD), an inactive phorbol ester (Fig. 4). Calcium channel blockers or PDD alone did not affect translocation of PKC (Fig. 4).
DISCUSSION We showed the following in this study: 1) Ang II stimulated PGF2a release in neonatal rat ventricular myocytes, 2) Ang II-induced PGF2a release is maintained by extracellular calcium via activation of PKC. The inhibition of Ang II-induced PGF2a release by L-type Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77
calcium channel blocker and the implication of PKC by enzyme assay suggest that extracellular calcium and PKC mediate these changes. Ang II-induced PGF2a release were inhibited with PKC inhibitor in our previous studies10 and L-type calcium channel blocker in the present study. PKC activates voltage-sensitive L-type calcium channels,13,14 which will enhance calcium influx from the extracellular compartment and therefore maintain the PGF2a release observed in this study. The mechanism by which Ang II causes increased PGF2a release is still unclear, but it appears to involve PKC and L-type calcium channels. Several studies have shown that prostanoids are able to influence the tone of cerebral arteries and arterioles.18–23 Indeed, PG synthesis is altered in various pathological conditions and causes profound modifications in tissue blood flow. It has been shown that the levels of PGF2a in the myocardium were increased by acute6 or chronic24 hemodynamic overload. In addition, PG synthase inhibitors blocked experimentally induced cardiac hypertrophy.25,26 Therefore, it is important to understand the mechanisms involved in PG release. Pretreatment of neonatal rat ventricular myocytes with different doses of diltiazm (voltage-sensitive L-type calcium channel blocker) produced dose-dependent inhibition in Ang II-induced PGF2a release. Inhibition was first noted at 1078 M and was complete at 1076 M. Conversely, pretreatment of neonatal rat ventricular myocytes with different doses of calcium channel blockers (thapsigargin; intracellular calcium channel blocker or & 2001Harcourt Publishers Ltd
Calcium is involved in Ang II-induced PGF2a release 77
conotoxin; voltage-sensitive N-type calcium channel blocker) produced no changes on Ang II-induced PGF2a release in neonatal rat ventricular myocytes. In conclusion, Ang II activates PKC, which increases PGF2a production and release via increase in extracellular calcium influx in neonatal rat ventricular myocytes.
ACKNOWLEDGMENTS The author wishes to thank Mrs Mary Oriji for her aid in the preparation of this manuscript. The author also thanks Mrs Margaret Hill and Mr John Tate for their excellent technical assistance.
REFERENCES 1. Griendling K. K., Rittenhouse S. E., Gimbrone M. A. Jr., Ekstein L. S., Brock T. A., Alexander R. W. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells. J Biol Chem 1986; 261: 5901–5906. 2. Saito T., Saito O., Ono S., Okada K., Kusano E., Homma S., Fujita N., Asano Y., Ando Y., Akimoto T. Related Articles. Erythropoietin modulates angiotensin II- or noradrenaline-induced Ca(2+) mobilization in cultured rat vascular smooth-muscle cells. Nephrol Dial Transplant 2001; 16(3): 491–499. 3. Yamamoto H., Ushio-Fukai M., Toyofuku K., Nishimura J., Kanaide H., Hirano K. Related Articles. Changes in the cytosolic Ca2+ concentration and Ca(2+)-sensitivity of the contractile apparatus during angiotensin II-induced desensitization in the rabbit femoral artery. Br J Pharmacol 2000; 129(3): 425–436. 4. Samain E., Safar M., Renaud J. F., Perret C., Miserey S., Dagher G., Bouillier H. Related Articles. Extracellular signal-regulated kinase pathway is involved in basic fibroblast growth factor effect on angiotensin II-induced Ca(2+) transient in vascular smooth muscle cell from Wistar-Kyoto and spontaneously hypertensive rats. Hypertension 2000; 35(1 Pt 1): 61–67. 5. Peach M. J. Molecular actions of angiotensin. Biochem Pharmacol 1981; 130: 2745–2751. 6. Chazov E. I., Smirnov V. N., Pomoinetsky V. D., Nekrasova A. A., Orlova T. R., Geling N. G. Heart adaptation to acute pressure overload: an involvement of endogenous prostaglandins. Circ Res 1979; 45(2): 205–211. 7. Schramek H. E., Coroneos E., Dunn M. J. Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins. Semin Nephrol 1995; 15(3): 195–204. 8. Escalante B., Cruz B. V. Renal vascular interaction of angiotensin II and prostaglandins in renovascular hypertension. J Cardiovasc Pharmacol 1999; 34(1): 21–27. 9. Harwalkar S., Falck J. R., Dulin N. O., Douglas J. G., Alexander L. D. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proc Natl Acad Sci USA 1998; 95(14): 8098–8102. 10. Oriji G. K. Angiotensin II stimulates hypertrophic growth of cultured neonatal rat ventricular myocytes: roles of PKC and PGF2a. Prostaglandins Leukot Essent Fatty Acids 2000; 62(4): 233–237.
& 2001Harcourt Publishers Ltd
11. Taylor C., Marshal I. Calcium and inositol 1,4,5-triphosphate receptors: a complex relationship. Elsevier Science Publishers, (UK) 0376-5067, 1992; 403–407. 12. Song Y. H., Rossi M. W., Korchak H. M., Kilpatrick L. E. Serine phosphorylation of p60 tumor necrosis factor receptor by PKCdelta in TNF-alpha-activated neutrophils. Am J Physiol Cell Physiol 2000; 279(6): C2011–C2018. 13. Smart D., Lambert D. G. Desensitization of the mu-opioid activation of phospholipase C in SH-SY5Y cells: the role of protein kinases C and A and Ca(2+)-activated K+ currents. Br J Pharmacol 1995; 116(6): 2655–2660. 14. Summers B. A., Prabhakar N. R., Overholt J. L. Related Articles. Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway. J Neurophysiol 2000; 84(3): 1636–1644. 15. Sen A., Dunnmon P., Henderson S. A., Gerard R. D., Chien K. R. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem 1988; 263: 19132–19136. 16. Iwaki K., Sukhatme V. P., Shubeita H. E., Chien K. R. Alpha- and beta-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response. J Biol 1990; 265: 13809–13817. 17. Eulalia B., Rapoport R. M., Campbell A. K. Protein Kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats. Eur J Pharm 1992; 227: 343–348. 18. Armstead W. M., Mirro D.W., Leffler C. W., Busija D. W. Permissive role of prostanoids in acetylcholine-induced cerebral constriction. J Pharmacol Exp Ther 1989; 251: 1012–1019. 19. Busija D. W., Leffler C. W. Eicosanoid synthesis elicited by norepinephrine in piglet parietal cortex. Brain Res 1987; 403: 243–248. 20. Busija D. W., Leffier C. S. Dilator effects of amino acid neurotransmitters on piglet pial arterioles. Am J Physiol 1989; 257: HI200–H1203. 21. Busija D. W., Wagerie L. C., Pourcyrous M., Leffler C. W. Acetylcholine dramatically increases prostanoid synthesis in piglet parietal cortex. Brain Res 1988; 439: 122–126. 22. Busija D. W., Leffler C. W. Effects of phorbol esters on pial arteriolar diameter and brain production of prostanoids in piglets. Circ Res 1991; 69: 1253–1258. 23. Pourcyrous M., Leffler C. W., Busija, D. W. Postasphyxial increases in prostaglandins in cerebrospinal fluid in piglets. Pediatr Res 1988; 24: 229–232. 24. Escobar E., Zamorano B., Gazmuri R. Demonstration of prostaglandin E2 and F2a in atrial tissue of patients with heart disease. Am J Cardiol 1983; 52: 424–425. 25. Kentera D., Susic D., Zdravkovic M. Effects of verapamil and aspirin on experimental chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. Respiration 1979; 37(4): 192–196. 26. Palmer R. M., Delday M. I., McMillan D. N., Noble B. S., Bain P., Maltin C. A. Effects of the cyclo-oxygenase inhibitor, fenbufen, on clenbuterol-induced hypertrophy of cardiac and skeletal muscle of rats. Br J Pharmacol 1990; 101: 835–838.
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(2), 73^77