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Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations

View Article: PubMed Central - PubMed

ABSTRACT

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

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Related in: MedlinePlus

Nifedipine inhibition of VDCC currents obtained in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. VDCC currents were elicited in rat cerebral artery myocytes in response to successive 10 mV depolarizing voltage steps from a holding potential of −80 mV. (A,B) VDCC currents obtained using 10 mM [Ba2+]o as the charge carrier in nominally Ca2+-free extracellular solution in the absence (A) and presence (B) of nifedipine (1 μM). (C) Summary data demonstrating that ~20 % of VDCC currents using 10 mM [Ba2+]o were not inhibited by 1 μM nifedipine. (D,E) VDCC currents obtained using 10 mM [Ba2+]o plus 100 μM [Ca2+]o in the absence (D) and presence (E) of nifedipine (1 μM). (F) Summary data demonstrating that VDCC currents were completely inhibited by 1 μM nifedipine in 10 mM [Ba2+]o plus 100 μM [Ca2+]o.
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Figure 1: Nifedipine inhibition of VDCC currents obtained in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. VDCC currents were elicited in rat cerebral artery myocytes in response to successive 10 mV depolarizing voltage steps from a holding potential of −80 mV. (A,B) VDCC currents obtained using 10 mM [Ba2+]o as the charge carrier in nominally Ca2+-free extracellular solution in the absence (A) and presence (B) of nifedipine (1 μM). (C) Summary data demonstrating that ~20 % of VDCC currents using 10 mM [Ba2+]o were not inhibited by 1 μM nifedipine. (D,E) VDCC currents obtained using 10 mM [Ba2+]o plus 100 μM [Ca2+]o in the absence (D) and presence (E) of nifedipine (1 μM). (F) Summary data demonstrating that VDCC currents were completely inhibited by 1 μM nifedipine in 10 mM [Ba2+]o plus 100 μM [Ca2+]o.

Mentions: Using 10 mM [Ba2+]o as a charge carrier, VDCC currents were elicited in rat cerebral artery myocytes by a series of 10 mV depolarizing steps from a holding potential −80 mV. In nominally Ca2+ free 10 mM Ba2+ bath solution, a maximum VDCC current density of −7.30 ± 0.35 pA/pF (n = 8 cells from four animals) was observed at +10 mV (Figures 1A,C). In the presence of nifedipine (1 μM), VDCC currents were reduced, but not abolished (Figures 1B,C). At +10 mV, the nifedipine-insensitive current density (−1.62 ± 0.48 pA/pF) was ~22% of total VDCC current density measured in these cells. To examine the impact of [Ca2+]o on VDCC currents when 10 mM [Ba2+]o is used as the primary charge carrier, the above voltage-step protocol was repeated using myocytes bathed in a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o (Figures 1D–F). The VDCC current density recorded at +10 mV (−7.28 ± 0.54 pA/pF; n = 11 cells, seven animals) was not significantly different compared to VDCC current density measured in 10 mM [Ba2+]o, alone (Figure 1C). However, the addition of 100 μM [Ca2+]o dramatically influenced the efficacy of nifedipine to inhibit VDCC currents. As illustrated in Figure 1F, inward VDCC currents obtained in the combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o were completely abolished by 1 μM nifedipine. To further investigate the impact of [Ca2+]o on VDCC currents, single depolarizing voltage steps from a holding potential of −80 to +10 mV were used to elicit VDCC currents in the presence of increasing concentrations of nifedipine (10−10–10−6 M) followed by drug washout (Figures 2A–E and Table 2). In 10 mM [Ba2+]o, nifedipine caused a concentration-dependent decrease in VDCC currents with an IC50 of 6.02 ± 0.36 nM (n = 5 cells from five animals) with ~20% of the current insensitive to 1 μM nifedipine. Addition of 100 μM [Ca2+]o to the 10 mM [Ba2+]o bath solution again facilitated the complete inhibition of VDCC currents by 1 μM nifedipine, but did not change VDCC sensitivity to nifedipine (IC50 value 5.12 ± 0.41 nM; n = 4 cell from three animals). Upon washout of 1 μM nifedipine, current density was not significantly different when compared to current density obtained prior to the start of the nifedipine concentration-response curve (Table 2). Current-voltage (I-V) plots shown in Figures 1C,F are consistent with the activation of high-voltage activated VDCCs, i.e., maximum current density was observed at ~+10 mV. To obtain higher resolution with respect to the voltage-dependent activation of VDCC currents, voltage ramp protocols were used that applied a continuous depolarization from a holding potential of −80 to +50 mV over the course of 300 ms (Figures 2D–F). This approach also enabled the study of the same cell using 10 mM [Ba2+]o as the charge carrier in the presence and absence of 100 μM [Ca2+]o (Figures 2D–F). No difference was detected in the membrane potential that evoked maximum VDCC currents in 10 mM [Ba2+]o in the absence or presence of 100 μM [Ca2+]o (9.99 ± 0.08 vs. 10.01 ± 0.08 mV; n = 5 cells from three animals; p > 0.05). Maximum VDCC current density in cells bathed in 10 mM [Ba2+]o was also not significantly different in the absence or presence of 100 μM [Ca2+]o (Figures 2G–I). Voltages for half-maximal inactivation (V0.5inact) were ~−13.9, −32.5, and −19.9 mV in 10 mM [Ba2+]o, 10 mM [Ba2+]o + nifedipine (1 μM) and 10 mM [Ba2+]o + 100 μM [Ca2+]o, respectively (Figures 3A–D and Table 1) Voltages for half-maximal activation (V0.5act) were similar in 10 mM [Ba2+]o ± 100 μM [Ca2+]o, but were shifted modestly (~+8 mV) in 10 mM [Ba2+]o plus 1 μM nifedipine (Figure 3E and Table 1). In addition to 1,4-DHPs (e.g., nifedipine), L-type VDCCs are inhibited by benzothiazepines such as diltiazem. Figure 4 illustrates the concentration dependent inhibition by diltiazem of VDCC currents in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. In contrast to the actions of nifedipine, diltiazem (100 μM) caused complete inhibition of 10 mM [Ba2+]o VDCC currents irrespective of the presence or absence of 100 μM [Ca2+]o. Upon washout of 100 μM diltiazam, current density was not significantly different when compared to a current density obtained prior to the start of the diltiazem concentration-response curve (Table 2).


Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations
Nifedipine inhibition of VDCC currents obtained in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. VDCC currents were elicited in rat cerebral artery myocytes in response to successive 10 mV depolarizing voltage steps from a holding potential of −80 mV. (A,B) VDCC currents obtained using 10 mM [Ba2+]o as the charge carrier in nominally Ca2+-free extracellular solution in the absence (A) and presence (B) of nifedipine (1 μM). (C) Summary data demonstrating that ~20 % of VDCC currents using 10 mM [Ba2+]o were not inhibited by 1 μM nifedipine. (D,E) VDCC currents obtained using 10 mM [Ba2+]o plus 100 μM [Ca2+]o in the absence (D) and presence (E) of nifedipine (1 μM). (F) Summary data demonstrating that VDCC currents were completely inhibited by 1 μM nifedipine in 10 mM [Ba2+]o plus 100 μM [Ca2+]o.
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Figure 1: Nifedipine inhibition of VDCC currents obtained in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. VDCC currents were elicited in rat cerebral artery myocytes in response to successive 10 mV depolarizing voltage steps from a holding potential of −80 mV. (A,B) VDCC currents obtained using 10 mM [Ba2+]o as the charge carrier in nominally Ca2+-free extracellular solution in the absence (A) and presence (B) of nifedipine (1 μM). (C) Summary data demonstrating that ~20 % of VDCC currents using 10 mM [Ba2+]o were not inhibited by 1 μM nifedipine. (D,E) VDCC currents obtained using 10 mM [Ba2+]o plus 100 μM [Ca2+]o in the absence (D) and presence (E) of nifedipine (1 μM). (F) Summary data demonstrating that VDCC currents were completely inhibited by 1 μM nifedipine in 10 mM [Ba2+]o plus 100 μM [Ca2+]o.
Mentions: Using 10 mM [Ba2+]o as a charge carrier, VDCC currents were elicited in rat cerebral artery myocytes by a series of 10 mV depolarizing steps from a holding potential −80 mV. In nominally Ca2+ free 10 mM Ba2+ bath solution, a maximum VDCC current density of −7.30 ± 0.35 pA/pF (n = 8 cells from four animals) was observed at +10 mV (Figures 1A,C). In the presence of nifedipine (1 μM), VDCC currents were reduced, but not abolished (Figures 1B,C). At +10 mV, the nifedipine-insensitive current density (−1.62 ± 0.48 pA/pF) was ~22% of total VDCC current density measured in these cells. To examine the impact of [Ca2+]o on VDCC currents when 10 mM [Ba2+]o is used as the primary charge carrier, the above voltage-step protocol was repeated using myocytes bathed in a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o (Figures 1D–F). The VDCC current density recorded at +10 mV (−7.28 ± 0.54 pA/pF; n = 11 cells, seven animals) was not significantly different compared to VDCC current density measured in 10 mM [Ba2+]o, alone (Figure 1C). However, the addition of 100 μM [Ca2+]o dramatically influenced the efficacy of nifedipine to inhibit VDCC currents. As illustrated in Figure 1F, inward VDCC currents obtained in the combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o were completely abolished by 1 μM nifedipine. To further investigate the impact of [Ca2+]o on VDCC currents, single depolarizing voltage steps from a holding potential of −80 to +10 mV were used to elicit VDCC currents in the presence of increasing concentrations of nifedipine (10−10–10−6 M) followed by drug washout (Figures 2A–E and Table 2). In 10 mM [Ba2+]o, nifedipine caused a concentration-dependent decrease in VDCC currents with an IC50 of 6.02 ± 0.36 nM (n = 5 cells from five animals) with ~20% of the current insensitive to 1 μM nifedipine. Addition of 100 μM [Ca2+]o to the 10 mM [Ba2+]o bath solution again facilitated the complete inhibition of VDCC currents by 1 μM nifedipine, but did not change VDCC sensitivity to nifedipine (IC50 value 5.12 ± 0.41 nM; n = 4 cell from three animals). Upon washout of 1 μM nifedipine, current density was not significantly different when compared to current density obtained prior to the start of the nifedipine concentration-response curve (Table 2). Current-voltage (I-V) plots shown in Figures 1C,F are consistent with the activation of high-voltage activated VDCCs, i.e., maximum current density was observed at ~+10 mV. To obtain higher resolution with respect to the voltage-dependent activation of VDCC currents, voltage ramp protocols were used that applied a continuous depolarization from a holding potential of −80 to +50 mV over the course of 300 ms (Figures 2D–F). This approach also enabled the study of the same cell using 10 mM [Ba2+]o as the charge carrier in the presence and absence of 100 μM [Ca2+]o (Figures 2D–F). No difference was detected in the membrane potential that evoked maximum VDCC currents in 10 mM [Ba2+]o in the absence or presence of 100 μM [Ca2+]o (9.99 ± 0.08 vs. 10.01 ± 0.08 mV; n = 5 cells from three animals; p > 0.05). Maximum VDCC current density in cells bathed in 10 mM [Ba2+]o was also not significantly different in the absence or presence of 100 μM [Ca2+]o (Figures 2G–I). Voltages for half-maximal inactivation (V0.5inact) were ~−13.9, −32.5, and −19.9 mV in 10 mM [Ba2+]o, 10 mM [Ba2+]o + nifedipine (1 μM) and 10 mM [Ba2+]o + 100 μM [Ca2+]o, respectively (Figures 3A–D and Table 1) Voltages for half-maximal activation (V0.5act) were similar in 10 mM [Ba2+]o ± 100 μM [Ca2+]o, but were shifted modestly (~+8 mV) in 10 mM [Ba2+]o plus 1 μM nifedipine (Figure 3E and Table 1). In addition to 1,4-DHPs (e.g., nifedipine), L-type VDCCs are inhibited by benzothiazepines such as diltiazem. Figure 4 illustrates the concentration dependent inhibition by diltiazem of VDCC currents in 10 mM [Ba2+]o ± 100 μM [Ca2+]o. In contrast to the actions of nifedipine, diltiazem (100 μM) caused complete inhibition of 10 mM [Ba2+]o VDCC currents irrespective of the presence or absence of 100 μM [Ca2+]o. Upon washout of 100 μM diltiazam, current density was not significantly different when compared to a current density obtained prior to the start of the diltiazem concentration-response curve (Table 2).

View Article: PubMed Central - PubMed

ABSTRACT

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

No MeSH data available.


Related in: MedlinePlus