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Pharmacological evidences for the stimulation of calcium-sensing receptors by nifedipine in gingival fibroblasts.

Hattori T, Ara T, Fujinami Y - J Pharmacol Pharmacother (2011)

Bottom Line: This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR.Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation.These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Pharmacology, Matsumoto Dental University, Shiojiri 399-0781, Japan.

ABSTRACT

Objective: To investigate pharmacologically whether CaSRs are involved in the Ca(2+) antagonist-induced [Ca(2+)]i elevation in gingival fibroblasts.

Materials and methods: Gin-1 cells, normal human gingival fibroblasts, were used as the material. The [Ca(2+)] i was measured with fura-2/AM, a Ca(2+)-sensitive fluorescent dye.

Results: At first, we confirmed the existence of CaSRs in these cells by showing that [Ca(2+)] i was elevated by high concentrations of extracellular Ca(2+) and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP(3)) receptors, NSCCs, and, importantly, by the CaSR antagonist, NPS2390. Furthermore, the action of gentamicin was potentiated by activators of PLC and protein kinase C (PKC). This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca(2+) channel blocker) stimulates CaSRs to elevate [Ca(2+)] i via a similar mechanism. Nifedipine Ca(2+) responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP(3) receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation.

Conclusion: These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

No MeSH data available.


Related in: MedlinePlus

Inhibition of the nifedipine-induced [Ca2+]i elevation by calphostin C and TMB-8. Cells were incubated with calphostin C (2 µM) or TMB-8 (500 prior to the addition of nifedipine (10 µM). Upper panel: representative trace showing the effect of calphostin C on the nifedipine-induced [Ca2+]i elevation. Lower panel: Ca2+responses to nifedipine in the absence (white bars) or presence (hatched bars) of inhibitors. Data are mean ± SEM, N = 30 (calphostin C) or 42 (TMB- 8). ****P < 0.001 compare to corresponding pretreated values
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Figure 0007: Inhibition of the nifedipine-induced [Ca2+]i elevation by calphostin C and TMB-8. Cells were incubated with calphostin C (2 µM) or TMB-8 (500 prior to the addition of nifedipine (10 µM). Upper panel: representative trace showing the effect of calphostin C on the nifedipine-induced [Ca2+]i elevation. Lower panel: Ca2+responses to nifedipine in the absence (white bars) or presence (hatched bars) of inhibitors. Data are mean ± SEM, N = 30 (calphostin C) or 42 (TMB- 8). ****P < 0.001 compare to corresponding pretreated values

Mentions: Nifedipine (10 µM) also stimulated CaSRs, and U73122 (10 µM), xestospongin C (2 µM), and SKF-96365 (10 µM) significantly inhibited the [Ca2+]i elevation induced by 10 µM of nifedipine [Figure 6]. These effects were qualitatively the same as those for the gentamicin-induced [Ca2+]i elevation. A similar effect was observed with calphostin C (a PKC inhibitor)[17] and TMB-8 (a Ca2+ store release inhibitor).[18] Calphostin C (2 µM) and TMB-8 (500 µM) showed a significant inhibition of the nifedipine-induced [Ca2+]i elevation [Figure 7]. The cell culture which was incubated for 10 min with NPS2390 between two pulses of 10 mM nifedipine separated by 12 min [Figure 8, upper panel] also inhibited the nifedipine-induced [Ca2+]i elevation, in a concentration-dependent manner (concentration range: 1–10 µM; Figure 8, lower panel). It is important to note that, in a similar experiment where cells were perfused for 10 min with the vehicle instead of NPS2390, the sequential administration of two pulses of nifedipine (10 µM) separated by 12 min caused changes in the [Ca2+]i of 61.78 ± 10.99 nM and 58.71 ± 11.04 nM, respectively (N = 34, data not shown). There was no significant difference between the first and second responses, demonstrating that the effect of NPS2390 was not an artifact of, for example, the store depletion.


Pharmacological evidences for the stimulation of calcium-sensing receptors by nifedipine in gingival fibroblasts.

Hattori T, Ara T, Fujinami Y - J Pharmacol Pharmacother (2011)

Inhibition of the nifedipine-induced [Ca2+]i elevation by calphostin C and TMB-8. Cells were incubated with calphostin C (2 µM) or TMB-8 (500 prior to the addition of nifedipine (10 µM). Upper panel: representative trace showing the effect of calphostin C on the nifedipine-induced [Ca2+]i elevation. Lower panel: Ca2+responses to nifedipine in the absence (white bars) or presence (hatched bars) of inhibitors. Data are mean ± SEM, N = 30 (calphostin C) or 42 (TMB- 8). ****P < 0.001 compare to corresponding pretreated values
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117567&req=5

Figure 0007: Inhibition of the nifedipine-induced [Ca2+]i elevation by calphostin C and TMB-8. Cells were incubated with calphostin C (2 µM) or TMB-8 (500 prior to the addition of nifedipine (10 µM). Upper panel: representative trace showing the effect of calphostin C on the nifedipine-induced [Ca2+]i elevation. Lower panel: Ca2+responses to nifedipine in the absence (white bars) or presence (hatched bars) of inhibitors. Data are mean ± SEM, N = 30 (calphostin C) or 42 (TMB- 8). ****P < 0.001 compare to corresponding pretreated values
Mentions: Nifedipine (10 µM) also stimulated CaSRs, and U73122 (10 µM), xestospongin C (2 µM), and SKF-96365 (10 µM) significantly inhibited the [Ca2+]i elevation induced by 10 µM of nifedipine [Figure 6]. These effects were qualitatively the same as those for the gentamicin-induced [Ca2+]i elevation. A similar effect was observed with calphostin C (a PKC inhibitor)[17] and TMB-8 (a Ca2+ store release inhibitor).[18] Calphostin C (2 µM) and TMB-8 (500 µM) showed a significant inhibition of the nifedipine-induced [Ca2+]i elevation [Figure 7]. The cell culture which was incubated for 10 min with NPS2390 between two pulses of 10 mM nifedipine separated by 12 min [Figure 8, upper panel] also inhibited the nifedipine-induced [Ca2+]i elevation, in a concentration-dependent manner (concentration range: 1–10 µM; Figure 8, lower panel). It is important to note that, in a similar experiment where cells were perfused for 10 min with the vehicle instead of NPS2390, the sequential administration of two pulses of nifedipine (10 µM) separated by 12 min caused changes in the [Ca2+]i of 61.78 ± 10.99 nM and 58.71 ± 11.04 nM, respectively (N = 34, data not shown). There was no significant difference between the first and second responses, demonstrating that the effect of NPS2390 was not an artifact of, for example, the store depletion.

Bottom Line: This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR.Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation.These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Pharmacology, Matsumoto Dental University, Shiojiri 399-0781, Japan.

ABSTRACT

Objective: To investigate pharmacologically whether CaSRs are involved in the Ca(2+) antagonist-induced [Ca(2+)]i elevation in gingival fibroblasts.

Materials and methods: Gin-1 cells, normal human gingival fibroblasts, were used as the material. The [Ca(2+)] i was measured with fura-2/AM, a Ca(2+)-sensitive fluorescent dye.

Results: At first, we confirmed the existence of CaSRs in these cells by showing that [Ca(2+)] i was elevated by high concentrations of extracellular Ca(2+) and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP(3)) receptors, NSCCs, and, importantly, by the CaSR antagonist, NPS2390. Furthermore, the action of gentamicin was potentiated by activators of PLC and protein kinase C (PKC). This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca(2+) channel blocker) stimulates CaSRs to elevate [Ca(2+)] i via a similar mechanism. Nifedipine Ca(2+) responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP(3) receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation.

Conclusion: These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

No MeSH data available.


Related in: MedlinePlus