The Contribution of Protein Kinase C to Cyclopiazonic Acid- Mediated Potentiation of Serotonin-Induced Vascular Calcium Responses

Abstract

We previously showed that serotonin 5-Hydroxytryptamine, 5-HT receptor antagonist methysergide partially inhibited cyclopiazonic acid CPA -potentiated and 5-HT-induced contractions suggesting the contribution of protein kinase C PKC -mediated receptor internalization in attenuated antagonism. In the present study, the effects of CPA and 5-HT receptor antagonist methysergide and ketanserin on 5-HT-induced calcium responses following PKC inhibition were further investigated. For this purpose, intracellular calcium levels were monitored in A7r5 vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. Cells were pre-incubated with specific PKC inhibitor D-sphingosine. In experimental aspects, spontaneous calcium oscillations hindering the monitoring of responses were determined in over confluent cells. 5-HT-induced calcium levels were significantly decreased in D-sphingosine-treated cells compared to control. CPA significantly potentiated 5-HT-induced calcium responses while ketanserin partially but insignificantly inhibited the potentiated responses in the presence of PKC inhibition. In conclusion, increased cell confluency may result in the generation of spontaneous calcium spikes indicating the importance of using optimum cell density for monitoring of intracellular calcium levels. The data suggests that CPA-mediated potentiation and diminished antagonistic effects on 5-HT-induced calcium elevations in vascular smooth muscle cells, are partially mediated by PKC.

Keywords

• Calcium,CPA,D-sphingosine,PKC,Serotonin

Serotonine Bağlı Vasküler Kalsiyum Yanıtlarının Siklopiazonik Asit-Aracılı Potansiyalizasyonuna Protein Kinaz C’nin Katkısı

Abstract

Ö nceki çalışmalar 5-HT reseptör antagonisti metiserjidin, siklopiazonik asitin CPA potansiyalize ettiği serotonine 5-hidroksitriptamin, 5-HT bağlı kontraksiyonları kısmen inhibe ettiği gösterilmiş ve azalan antagonistik etkiye protein kinaz C PKC -aracılı reseptör internalizasyonunun katkıda bulunduğu önerilmiştir. Bu çalışmada, PKC inhibisyonu varlığında CPA ve 5-HT reseptör antagonistleri metiserjid ve ketanserinin 5-HT’ye bağlı vasküler kalsiyum yanıtları üzerindeki etkileri araştırılmıştır. Bu amaçla A7r5 vasküler düz kas hücrelerindeki hücre içi kalsiyum düzeylerindeki değişimler fluoresan indikatör fura-2 ile spektrofluorometri yöntemi ile belirlenmiştir. Hücreler PKC inhibitörü D-sifingozin ile inkübe edilmiştir. Metodolojik açıdan bakıldığında, aşırı yoğun hücrelerde ajanlara alınan yanıtları gizleyecek düzeyde spontan kalsiyum salınımları belirlenmiştir. 5-HT’ye bağlı kalsiyum yanıtları D-sifingozin ile inkübe edilen hücrelerde kontrol hücrelere göre anlamlı düzeyde azalmıştır. PKC inhibisyonu varlığında CPA, 5-HT’ye bağlı kalsiyum yanıtlarını anlamlı düzeyde potansiyalize etmiştir. Bunun yanı sıra ketanserin, potansiyalize yanıtları kısmen ve anlamlı olmayan düzeyde inhibe etmiştir. Sonuç olarak, hücrelerin aşırı yoğunlukta olması ani kalsiyum tepe spike yanıtlarının oluşmasına neden olabilmekte ve hücre içi kalsiyum düzeylerinin izlendiği çalışmalarda hücrelerin optimum yoğunlukta kullanılmasının önemini göstermektedir. Bulgularımız PKC’nin, 5-HT yanıtları üzerindeki CPA-aracılı potansiyalizasyon ve azalmış antagonistik etkiye kısmen aracılık ettiğini göstermiştir

Keywords

• CPA,D-sifingozin,kalsiyum,serotonin

References

1. 1. A.J. Ellwood, M.J. Curtis, Involvement of 5-HT(1B/1D) and 5-HT2A receptors in 5-HT-induced contraction of endothelium-denuded rabbit epicardial coronary arteries, Br. J. Pharmacol., 122 (1997) 875-884.
2. 2. L. Rodat-Despoix, V. Aires, T. Ducret, R. Marthan, J.P. Savineau, E. Rousseau, C. Guibert, Signalling pathways involved in the contractile response to 5-HT in the human pulmonary artery, Eur. Respir. J., 34 (2009) 1338-1347.
3. 3. X.J. Yuan, R.T. Bright, A.M. Aldinger, L.J. Rubin, Nitric oxide inhibits serotonin-induced calcium release in pulmonary artery smooth muscle cells, Am. J. Physiol., 272 (1997) 44-50.
4. 4. J.W. Putney, Jr., A model for receptor-regulated calcium entry, Cell Calcium., 7 (1986) 1-12.
5. 5. Z. Moneer, I. Pino, E.J. Taylor, L.M. Broad, Y. Liu, S.C. Tovey, L. Staali, C.W. Taylor, Different phospholipaseC-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells, Biochem. J., 389 (2005) 821-829.
6. 6. V.A. Golovina, M.P. Blaustein, Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum, Science, 275 (1997) 1643- 1648.
7. 7. T. Adachi, Modulation of vascular sarco/endoplasmic reticulum calcium ATPase in cardiovascular pathophysiology, Adv. Pharmacol., 59 (2010) 165-195.
8. 8. L. Lipskaia, F. del Monte, T. Capiod, S. Yacoubi, L. Hadri, M. Hours, R.J. Hajjar, A.M. Lompre, Sarco/ endoplasmic reticulum Ca2+-ATPase gene transfer reduces vascular smooth muscle cell proliferation and neointima formation in the rat, Circ. Res., 97 (2005) 488-495.

9. 9. H. Zhang, M. Inazu, B. Weir, M. Buchanan, E. Daniel, Cyclopiazonic acid stimulates Ca2+ influx through non-specific cation channels in endothelial cells, Eur. J. Pharmacol., 251 (1994) 119-125.
10. 10. F. Abe, H. Karaki, M. Endoh, Effects of cyclopiazonic acid and ryanodine on cytosolic calcium and contraction in vascular smooth muscle, Br. J. Pharmacol., 118 (1996) 1711-1716.
11. 11. M. Tosun, R.J. Paul, R.M. Rapoport, Coupling of storeoperated Ca++ entry to contraction in rat aorta, J. Pharmacol. Exp. Ther., 285 (1998) 759-766.
12. 12. K.G. Lamping, W.P. Dole, Acute hypertension selectively potentiates constrictor responses of large coronary arteries to serotonin by altering endothelial function in vivo, Circ. Res., 61 (1987) 904-913.
13. 13. J.A. Lopez, M.L. Armstrong, D.J. Piegors, D.D. Heistad, Effect of early and advanced atherosclerosis on vascular responses to serotonin, thromboxane A2, and ADP, Circulation, 79 (1989) 698-705.
14. 14. K. Suzuki, I. Kusumi, T. Akimoto, Y. Sasaki, T. Koyama, Altered 5-HT-induced calcium response in the presence of staurosporine in blood platelets from bipolar disorder patients, Neuropsychopharmacology, 28 (2003) 1210-1214.
15. 15. C. Selli, Y. Erac, M. Tosun, Cyclopiazonic acid alters serotonin-induced responses in rat thoracic aorta, Vascul. Pharmacol., 61 (2014) 43-48.
16. 16. A. Bhatnagar, D.J. Sheffler, W.K. Kroeze, B. ComptonToth, B.L. Roth, Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors, J. Biol. Chem., 279 (2004) 34614-34623.
17. 17. S. Bhattacharyya, S. Puri, R. Miledi, M.M. Panicker, Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms, Proc. Natl. Acad. Sci., 99 (2002) 14470- 14475.
18. 18. I. Raote, S. Bhattacharyya, M.M. Panicker, Functional selectivity in serotonin receptor 2A (5-ht2a) endocytosis, recycling, and phosphorylation, Mol. Pharmacol., 83 (2013) 42-50.
19. 19. Y. Erac, C. Selli, P. Filik, M. Tosun, Effects of passage number on proliferation and store-operated calcium entry in A7r5 vascular smooth muscle cells, J. Pharmacol. Toxicol. Methods., 70 (2014) 1-5.
20. 20. C. Selli, Y. Erac, B. Kosova, M. Tosun, Posttranscriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca2+ entry in A7r5 vascular smooth muscle cells, Vascul. Pharmacol., 51 (2009) 96-100.
21. 21. C. Selli, Y. Erac, M. Tosun, Simultaneous measurement of cytosolic and mitochondrial calcium levels: Observations in TRPC1-silenced hepatocellular carcinoma cells, J. Pharmacol. Toxicol. Methods., 72 (2015) 29-34.
22. 22. Y.A. Hannun, C.R. Loomis, A.H. Merrill, R.M. Bell, Sphingosine inhibition of protein-kinase-c activity and of phorbol dibutyrate binding invitro and in human-platelets, J. Biol. Chem., 261 (1986) 2604- 2609.
23. 23. W.A. Khan, S.W. Mascarella, A.H. Lewin, C.D. Wyrick, F.I. Carroll, Y.A. Hannun, Use of d-erythro-sphingosine as a pharmacological inhibitor of protein-kinase-c in human platelets, Biochem. J., 278 (1991) 387-392.
24. 24. A. Karibe, J. Watanabe, S. Horiguchi, M. Takeuchi, S. Suzuki, M. Funakoshi, H. Katoh, M. Keitoku, S. Satoh, K. Shirato, Role of cytosolic Ca2+ and protein kinase C in developing myogenic contraction in isolated rat small arteries, Am. J. Physiol. Heart. Circ. Physiol., 272 (1997) 1165-1172.
25. 25. N.W. Seidler, I. Jona, M. Vegh, A. Martonosi, Cyclopiazonic acid is a specific inhibitor of the Ca2+- ATPase of sarcoplasmic reticulum, J. Biol. Chem., 264 (1989) 17816-17823.
26. 26. K.L. Byron, C.W. Taylor, Spontaneous Ca2+ spiking in a vascular smooth muscle cell line is independent of the release of intracellular Ca2+ stores, J. Biol. Chem., 268 (1993) 6945-69452.
27. 27. A.D. Kaye, B.D. Nossaman, I.N. Ibrahim, C.J. Feng, P.J. Kadowitz, Influence of protein-kinase-C inhibitors on vasoconstrictor responses in the pulmonary vascular bed of cat and rat, Am. J. Physiol. Lung. Cell. Mol. Physiol., 268 (1995) 532-538.
28. 28. M. Tosun, Y. Erac, C. Selli, N. Karakaya, Sarcoplasmicendoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta, Am. J. Physiol. Heart. Circ. Physiol., 292 (2007) 1961-1966.
29. 29. H.K. Saini, S.K. Sharma, P. Zahradka, H. Kumamoto, N. Takeda, N.S. Dhalla, Attenuation of the serotonininduced increase in intracellular calcium in rat aortic smooth muscle cells by sarpogrelate, Can. J. Physiol. Pharmacol., 81 (2003) 1056-1063.
30. 30. S.D. Silberstein, Methysergide, Cephalalgia, 18 (1998) 421-35.
31. 31. G. Reiser, F. Donie, F.J. Binmoller, Serotonin regulates cytosolic Ca2+ activity and membrane-potential in a neuronal and in a glial-cell line via 5-ht3 and 5-ht2 receptors by different mechanisms, J. Cell. Sci., 93 (1989) 545-555.
32. 32. T. Matsumoto, T. Kobayashi, K. Ishida, K. Taguchi, K. Kamata, Enhancement of mesenteric artery contraction to 5-HT depends on Rho kinase and Src kinase pathways in the ob/ob mouse model of type 2 diabetes, Br. J. Pharmacol., 160 (2010) 1092-104.