L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

Abstract

We investigated the role of intracellular calcium concentration ([Ca2+]i) in endothelin-1 (ET-1) production, the effects of potential vasospastic agents on [Ca2+]i, and the presence of L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells. Primary cultures of endothelial cells isolated from piglet cerebral microvessels were used. Confluent cells were exposed to either the thromboxane receptor agonist U-46619 (1 microM), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 microM) alone or after pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+ channel blocker verapamil (10 microM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 microM) for 15 min. ET-1 production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT), or 3.9 (LPA) fmol/microg protein, respectively. Such elevated ET-1 biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. To investigate the presence of L-type voltage-dependent Ca2+ channels in endothelial cells, the [Ca2+]i signal was determined fluorometrically by using fura 2-AM. Superfusion of confluent endothelial cells with U-46619, 5-HT, or LPA significantly increased [Ca2+]i. Pretreatment of endothelial cells with high K+ (60 mM) or nifedipine (4 microM) diminished increases in [Ca2+]i induced by the vasoactive agents. These results indicate that 1) elevated [Ca2+]i signals are involved in ET-1 biosynthesis induced by specific spasmogenic agents, 2) the increases in [Ca2+]i induced by the vasoactive agents tested involve receptor as well as L-type voltage-dependent Ca2+ channels, and 3) primary cultures of cerebral microvascular endothelial cells express L-type voltage-dependent Ca2+ channels.

Fig. 1

Effects of the Ca²⁺-chelating agent EDTA (0.1 M) on U-46619 (1 μM)-, 5-hydroxytryptamine (5-HT; 0.1 mM)-, or lysophosphatidic acid (LPA; 1 μM)-induced production of endothelin-1 (ET-1) from cerebral microvascular endothelial cells. Confluent endothelial cells, serum deprived for 24 h, were pretreated with EDTA for 15 min before being exposed to U-46619, 5-HT, and LPA and incubated in a 5% CO₂/air incubator at 37°C for 4 h. The medium was collected and assayed for ET-1 at the end of the incubation. Results are presented as means ± SE. *P < 0.05; ANOVA; n = 4.

Fig. 2

Effects of the Ca²⁺ channel blocker verapamil (10 μM) on U-46619 (1 μM)-, 5-HT (0.1 mM)-, and LPA (1 μM)-induced production of ET-1 from cerebral microvascular endothelial cells. Confluent endothelial cells, serum deprived for 24 h, were pretreated with verapamil (10 μM) for 15 min before being exposed to U-46619, 5-HT, or LPA and incubated in a 5% CO₂/air incubator at 37°C for 4 h. The medium was collected and assayed for ET-1 at the end of the incubation. Results are presented as means ± SE. *P < 0.05; ANOVA; n = 4.

Fig. 3

Effects of the receptor-operated Ca²⁺ channel antagonist SKF-96365 HCl (SKF; 10 μM) on U-46619 (1 μM)-, 5-HT (0.1 mM)-, and LPA (1 μM)-induced production of ET-1 from cerebral microvas-cular endothelial cells. Confluent endothelial cells, serum deprived for 24 h, were pretreated with SKF-96365 HCl for 15 min before being exposed to U-46619, 5-HT, or LPA and incubated in a 5% CO₂/air incubator at 37°C for 4 h. The medium was collected and assayed for ET-1 at the end of the incubation. Results are presented as means ± SE. *P < 0.05; ANOVA; n = 4.

Fig. 4

Representative tracings depicting changes in Ca²⁺/fura 2-AM fluorescence induced by thromboxane receptor agonist U-46619 (1 μM; A), 5-HT (0.1 mM; B), or LPA (1 μM; C) in cultured cerebral microvascular endothelial cells. Confluent cell monolayers, serum deprived for 24 h, were preincubated with fura 2-AM for 30 min and superfused with Krebs buffer along with vasoactive agents. Changes in intracellular calcium concentration ([Ca²⁺]_i) were recorded as a ratio (F₃₄₀/F₃₈₀) of fura 2 fluorescence at excitation wavelengths of 340 and 380. The traces are representative of 3 such results.

Fig. 5

Representative tracings depicting changes in Ca²⁺/fura 2-AM fluorescence induced by thromboxane receptor agents U-46619 (1 μM) in the presence and absence of extracellular Ca²⁺ in cultured cerebral microvascular endothelial cells. Confluent cell monolayers, serum deprived for 24 h, were preincubated with fura 2-AM for 30 min and superfused with Krebs buffer along with vasoactive agents. The effects of U-46619 on the regulation of [Ca²⁺]_i were determined in 1.8 mM Ca²⁺ Krebs buffer followed by determination in Ca²⁺-free Krebs. Changes in [Ca²⁺]_i recorded as the ratio (F₃₄₀/F₃₈₀) of fura 2 fluorescence at excitation wavelengths of 340 and 380. The result is a representative of at least 3 such results.

Fig. 6

Effects of membrane depolarization by high K⁺ (60 mM) on the increases in [Ca²⁺]_i induced by 5-HT (0.1 mM; A) or bradykinin (1 μM; B). Responses were recorded before and after the perfusion of endothelial cells with high K⁺. Confluent cell monolayers, serum deprived for 24 h, were pre-incubated with fura 2-AM for 30 min and superfused with Krebs buffer along with vasoactive agents. Changes in [Ca²⁺]_i were recorded as the ratio (F₃₄₀/F₃₈₀) of fura 2 fluorescence at excitation wavelengths of 340 and 380. The traces are representative of 3 such results.

Fig. 7

Effects of nifedipine (4 μM), a voltage-dependent calcium channel blocker on the elevations of [Ca²⁺]_i induced by 5-HT (0.1 mM; A) or bradykinin (1 μM; B). Responses were recorded before and after the perfusion of endothelial cells with nifedipine. Confluent cell monolayers, serum deprived for 24 h, were preincubated with fura 2-AM for 30 min and superfused with Krebs buffer along with vasoactive agents. Changes in [Ca²⁺]_i were recorded as the ratio (F₃₄₀/F₃₈₀) of fura 2 fluorescence at excitation wavelengths of 340 and 380. The tracings are representative of 3 such results.

Fig. 8

Effects of repeated applications of bradykinin (1 μM; A), 5-HT (0.1 mM; B) alone or reapplication after high K⁺ washout on [Ca²⁺]_i-signaling responses from endothelial cells: bradykinin (C) or 5-HT (D). Confluent cell monolayers, serum deprived for 24 h, were preincubated with fura 2-AM for 30 min and superfused with Krebs buffer along with vasoactive agents. Changes in [Ca²⁺]_i were recorded as the ratio (F₃₄₀/F₃₈₀) of fura 2 fluorescence at excitation wavelengths of 340 and 380. The traces are representative of 3 such results.