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Calcium as first messenger through calcium receptor (CaR) in vascular smooth muscle cells
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ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
h2AR coupling to Gi might be due to the spatial restriction of h1AR in t-tubules and h2AR in caveolae. We examine whether this spatial constraint of h1-AR in t-tubules influences its Gi coupling here. Rat ventricular myocytes were treated with 1.5 M formamide for 15 min at room temperature to disrupt the t-tubules. T-tubule structure was visualised with di-8-ANEPPS, and contraction amplitude was measured at 37 -C, 1 mM Ca, 0.5 Hz using the IonOptix system. Formamide-treated rat cardiac myocytes exhibited a significant reduction in t-tubules, with 81.5 T 3.5% of cells fully detubulated. Basal amplitude of contraction was reduced from 8.4 T 1.9% to 4.1 T 0.7% shortening (P < 0.05) after detubulation. Formamide-treated cells demonstrated a decreased response to isoprenaline (10 nM P < 0.05). The maximum amplitude of contraction was reduced from 12.1 T 2.5% to 10.6 T 2.6% (n = 6) in the presence of the Gistimulating agent carbachol for normal rat cardiac myocytes P = NS, and from 9.05 T 1.12% to 5.46 T 0.58% (n = 7) for formamide-treated cells (P < 0.02). The percentage reduction by carbachol was significantly enhanced in formamide-treated cells, P < 0.05. Loss of t-tubules has therefore enhanced the h1AR coupling to Gi. This would support the hypothesis that the initial lack of h1 AR coupling to Gi was due to the partial constraint of h1AR in t-tubules. doi:10.1016/j.yjmcc.2006.03.025
011. Use of the mitochondrial Ca2+-transport inhibitors Ru360 and clonazepam to investigate cell Ca2+-signalling in adult cardiomyocytes: A cautionary tale Christopher J. Bell, Guy A. Rutter, Elinor J. Griffiths. Department of Biochemistry, Bristol Heart Institute, University of Bristol, UK Previously, we used mitochondrially targeted aequorin (mAq) to show that mitochondrial Ca2+ ([Ca2+]m) transients occur on a beat-to-beat basis in adult cardiomyocytes. The aim of the current work was to determine whether [Ca2+]m regulates cytosolic Ca2+-signalling by using inhibitors of the mitochondrial Ca2+-transport pathways, namely Ru360, which inhibits the Ca2+-uniporter, and clonazepam, which inhibits the mitochondrial Na/Ca exchanger (mNCX). Adult rat ventricular myocytes were cultured in the presence of adenovirus containing aequorin targeted to either mitochondria or cytosol. [Ca2+] was measured in electrically stimulated cells by collecting aequorin light output using a photon counting camera, from a field of typically 10 –50 cells. However, in our experiments Ru360 did not appear to enter myocytes, in that it had no effect on [Ca2+]m. Clonazepam inhibited mitochondrial Ca2+-efflux, as indicated by a sustained, non-oscillatory, increase in [Ca2+]m, but also produced a small decrease in systolic cytosolic, [Ca2+] ([Ca2+]c). This was not due to an effect on the SR store of Ca2+, since the caffeine-releasable [Ca2+] was identical in absence or presence of clonazepam: 2.3 AM T 0.14 (n = 6) vs.
2.44 AM T 0.17 (n = 5), respectively. We also attempted to inhibit mitochondrial Ca2+ uptake by dissipating the mitochondrial membrane potential using FCCP and oligomycin. This abolished mitochondrial Ca2+ transients, but also depressed systolic [Ca2+]c. Thus, our aim of elucidating whether or not [Ca2+]m plays a role in regulating [Ca2+]c cannot currently be achieved since none of the inhibitors gave unambigous results in cultured adult rat cardiomyocytes.This work was funded by the British Heart Foundation. doi:10.1016/j.yjmcc.2006.03.026
012. Calcium as first messenger through calcium receptor (CaR) in vascular smooth muscle cells S. Smajilovic a,*, J. Lerche-Hansen a, E.H.T. Christoffersen a, K. Kastberg a, E. Lewin a, S.P. Sheikh a, E.F. Terwilliger b, M.E. Brown c, S. Haunso a, J. Tfelt-Hansen a. a Copenhagen University Hospital, Rigshospitalet, Denmark. b Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, USA. c Brigham and Woman’s Hospital and Harvard Medical School, Boston, USA. *[email protected]. The function of the vascular smooth muscle cell (VSMC) as a contractile cell is dependent on the calcium ion. Calcium is a multifunctional regulator of diverse cellular functions. Calcium ions act by changes in intracellular calcium levels through the action of calcium channels, exchangers and pumps. However, it has been demonstrated that calcium may also act outside the cell as a first messenger through a seven transmembrane receptor, the calcium receptor (CaR). Noncovalent binding of calcium to the CaR activates intracellular signal pathways, including G proteins and extracellularregulated kinases (ERK1/2). In the literature, it is debated whether the CaR is expressed in VSMCs. Here, we report the expression of the CaR messenger RNA and protein in cultured rat aortic VSMCs. Moreover, elevated calcium stimulated DNA and protein synthesis, and induced an increase in the cell number. These effects of calcium were attenuated by the pre-treatment with NPS 2390, a negative allosteric modulator of the CaR, suggesting an involvement of the CaR in calcium-induced cell growth of the VSMCs. Stimulation with calcium induced phosphorylation of ERK1/ 2, but surprisingly did not cause inositol phosphate accumulation. Furthermore, inhibition of MAPK kinase 1 (MEK1) abolished calcium-induced DNA synthesis. In conclusion, we show that aortic VSMCs express a functional CaR and that elevated calcium induces cell proliferation, the effect might be mediated by the CaR and MEK1/ERK pathway. These findings might be of importance in connection to atherosclerosis, which is an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium. doi:10.1016/j.yjmcc.2006.03.027
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920 – 1015
h2AR coupling to Gi might be due to the spatial restriction of h1AR in t-tubules and h2AR in caveolae. We examine whether this spatial constraint of h1-AR in t-tubules influences its Gi coupling here. Rat ventricular myocytes were treated with 1.5 M formamide for 15 min at room temperature to disrupt the t-tubules. T-tubule structure was visualised with di-8-ANEPPS, and contraction amplitude was measured at 37 -C, 1 mM Ca, 0.5 Hz using the IonOptix system. Formamide-treated rat cardiac myocytes exhibited a significant reduction in t-tubules, with 81.5 T 3.5% of cells fully detubulated. Basal amplitude of contraction was reduced from 8.4 T 1.9% to 4.1 T 0.7% shortening (P < 0.05) after detubulation. Formamide-treated cells demonstrated a decreased response to isoprenaline (10 nM P < 0.05). The maximum amplitude of contraction was reduced from 12.1 T 2.5% to 10.6 T 2.6% (n = 6) in the presence of the Gistimulating agent carbachol for normal rat cardiac myocytes P = NS, and from 9.05 T 1.12% to 5.46 T 0.58% (n = 7) for formamide-treated cells (P < 0.02). The percentage reduction by carbachol was significantly enhanced in formamide-treated cells, P < 0.05. Loss of t-tubules has therefore enhanced the h1AR coupling to Gi. This would support the hypothesis that the initial lack of h1 AR coupling to Gi was due to the partial constraint of h1AR in t-tubules. doi:10.1016/j.yjmcc.2006.03.025
011. Use of the mitochondrial Ca2+-transport inhibitors Ru360 and clonazepam to investigate cell Ca2+-signalling in adult cardiomyocytes: A cautionary tale Christopher J. Bell, Guy A. Rutter, Elinor J. Griffiths. Department of Biochemistry, Bristol Heart Institute, University of Bristol, UK Previously, we used mitochondrially targeted aequorin (mAq) to show that mitochondrial Ca2+ ([Ca2+]m) transients occur on a beat-to-beat basis in adult cardiomyocytes. The aim of the current work was to determine whether [Ca2+]m regulates cytosolic Ca2+-signalling by using inhibitors of the mitochondrial Ca2+-transport pathways, namely Ru360, which inhibits the Ca2+-uniporter, and clonazepam, which inhibits the mitochondrial Na/Ca exchanger (mNCX). Adult rat ventricular myocytes were cultured in the presence of adenovirus containing aequorin targeted to either mitochondria or cytosol. [Ca2+] was measured in electrically stimulated cells by collecting aequorin light output using a photon counting camera, from a field of typically 10 –50 cells. However, in our experiments Ru360 did not appear to enter myocytes, in that it had no effect on [Ca2+]m. Clonazepam inhibited mitochondrial Ca2+-efflux, as indicated by a sustained, non-oscillatory, increase in [Ca2+]m, but also produced a small decrease in systolic cytosolic, [Ca2+] ([Ca2+]c). This was not due to an effect on the SR store of Ca2+, since the caffeine-releasable [Ca2+] was identical in absence or presence of clonazepam: 2.3 AM T 0.14 (n = 6) vs.
2.44 AM T 0.17 (n = 5), respectively. We also attempted to inhibit mitochondrial Ca2+ uptake by dissipating the mitochondrial membrane potential using FCCP and oligomycin. This abolished mitochondrial Ca2+ transients, but also depressed systolic [Ca2+]c. Thus, our aim of elucidating whether or not [Ca2+]m plays a role in regulating [Ca2+]c cannot currently be achieved since none of the inhibitors gave unambigous results in cultured adult rat cardiomyocytes.This work was funded by the British Heart Foundation. doi:10.1016/j.yjmcc.2006.03.026
012. Calcium as first messenger through calcium receptor (CaR) in vascular smooth muscle cells S. Smajilovic a,*, J. Lerche-Hansen a, E.H.T. Christoffersen a, K. Kastberg a, E. Lewin a, S.P. Sheikh a, E.F. Terwilliger b, M.E. Brown c, S. Haunso a, J. Tfelt-Hansen a. a Copenhagen University Hospital, Rigshospitalet, Denmark. b Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, USA. c Brigham and Woman’s Hospital and Harvard Medical School, Boston, USA. *[email protected]. The function of the vascular smooth muscle cell (VSMC) as a contractile cell is dependent on the calcium ion. Calcium is a multifunctional regulator of diverse cellular functions. Calcium ions act by changes in intracellular calcium levels through the action of calcium channels, exchangers and pumps. However, it has been demonstrated that calcium may also act outside the cell as a first messenger through a seven transmembrane receptor, the calcium receptor (CaR). Noncovalent binding of calcium to the CaR activates intracellular signal pathways, including G proteins and extracellularregulated kinases (ERK1/2). In the literature, it is debated whether the CaR is expressed in VSMCs. Here, we report the expression of the CaR messenger RNA and protein in cultured rat aortic VSMCs. Moreover, elevated calcium stimulated DNA and protein synthesis, and induced an increase in the cell number. These effects of calcium were attenuated by the pre-treatment with NPS 2390, a negative allosteric modulator of the CaR, suggesting an involvement of the CaR in calcium-induced cell growth of the VSMCs. Stimulation with calcium induced phosphorylation of ERK1/ 2, but surprisingly did not cause inositol phosphate accumulation. Furthermore, inhibition of MAPK kinase 1 (MEK1) abolished calcium-induced DNA synthesis. In conclusion, we show that aortic VSMCs express a functional CaR and that elevated calcium induces cell proliferation, the effect might be mediated by the CaR and MEK1/ERK pathway. These findings might be of importance in connection to atherosclerosis, which is an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium. doi:10.1016/j.yjmcc.2006.03.027