Antihypertensive Effects of the Putative T-Type Calcium Channel Antagonist Mibefradil Are Mediated by the L-Type Calcium Channel Cav1.2
The role of T-type Ca2+ channels for cardiovascular physiology, in particular blood pressure regulation, is controversial. Selective blockade of T-type Ca2+ channels in resistance arteries has been proposed to explain the effect of the antihypertensive drug mibefradil. In the present study, we used a third generation, time- and tissue-specific conditional knockout model of the L-type Ca2+ channel Cav1.2 (Cav1.2SMAKO mice) to genetically dissect the effects of mibefradil on T- and L-type Ca2+ channels. Myogenic tone and phenylephrine-induced contraction in hindlimb perfusion experiments were sensitive to mibefradil in control mice, whereas the drug showed no effect in Cav1.2-deficient animals. Mean arterial blood pressure in awake, freely moving control mice was reduced by 38±2.5 mm Hg at a dose of 1.25 mg/kg bodyweight mibefradil, but not changed in Cav1.2SMAKO mice. These results demonstrate that the effect of the putative T-type Ca2+ channel-selective blocker mibefradil on blood pressure and small vessel myogenic tone is mediated by the Cav1.2 L-type Ca2+ channel.
Voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and thereby contribute to Ca2+ signaling in numerous cell types. They are classified as either high-voltage activated or low-voltage activated. High-voltage–activated channels include L-, N-, P/Q-, and R-types,1,2 and low-voltage–activated channels are designated as T-type.3 Voltage-gated Ca2+ channels also serve as important drug targets, with most therapeutically useful Ca2+ channel blockers targeting L-type channels. These compounds (eg, isradipine) are widely prescribed as antihypertensive drugs because they block Ca2+ influx via Cav1.2 L-type Ca2+ channels into smooth muscle cells, which constitutes the key determinant of vascular smooth muscle (VSM) tone and blood pressure.4,5 Cav1.2 L-type Ca2+ channels are not the only class of voltage-gated Ca2+ channels in VSM. The presence of T-type Ca2+ channels in vascular smooth muscle has been suggested using electrophysiological and molecular biological techniques.3,6–8
The development and characterization of mibefradil (also termed Ro 40-5967), an antihypertensive drug thought to selectively block T-type Ca2+ channels at the concentrations having a significant effect on blood pressure (&1 to 10 μmol/L), supported the hypothesis that inhibition of VSM T-type Ca2+ channels would produce vasorelaxation.6 In addition, other studies have confirmed the relative selectivity (eg, &10- to 30-fold for T-type over L-type channels) and, thus, the usability of mibefradil as a T-type Ca2+ channel blocking agent for functional studies in the cardiovascular system.9,10 Combined with the antihypertensive and direct vasodilating effects mibefradil on isolated vessels,11–14 a significant body of evidence has been accumulated suggesting the presence and function of T-type Ca2+ channels in regulating vascular contraction and, thus, blood pressure. However, in stark contrast to these findings, a genetic loss-of-function model, a mouse lacking the Cav3.2 T-type Ca2+ channel, surprisingly demonstrated that Ca2+ influx through VSM T-type Ca2+ channels is an essential mediator of normal relaxation, not contraction, of coronary arteries.15
Given these puzzling results on the role of T-type Ca2+ channels for VSM contractility and blood pressure regulation, we hypothesized that the vasorelaxing effect of mibefradil might not be explained by a block of T-type Ca2+ channels but of VSM Cav1.2 L-type Ca2+ channels. To dissect the function of these potential vascular receptors for mibefradil, we used transgenic mice deficient in the VSM L-type Ca2+ channel Cav1.2,4 the major Ca2+ entry pathway in vascular smooth muscle cells.5 In this model, the biophysical and pharmacological properties of the channels that remain expressed, here the Cav3.x T-type Ca2+ channels, can be investigated in isolation.
Materials and Methods
All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Regierung von Oberbayern.
Conditional Inactivation of the CaV1.2 Gene in Smooth Muscle Cells
Generation and characterization of the smooth-muscle specific Cav1.2-deficient mice were described previously.4,16 Two different CACNA1C (&Cav1.2) alleles were generated by Cre-mediated recombination in embryonic stem cells (L1 and L2). In L1, exons 14 and 15, which encode the IIS5 and IIS6 transmembrane segments and the pore loop in domain II, were deleted. Additionally, this deletion caused an incorrect splicing from exon 13 to part of an intron upstream of exon 16 and thereby generated a premature stop codon in exon 16 and a loss-of-function allele. L2 contains the “floxed” exons 14 and 15 and encodes a functional CACNA1C gene. To generate Cav1.2SMAKO mice, the Cav1.2L2/L2 mouse (ie, a mouse homozygous for the L2-allele) was crossed with a mouse expressing a tamoxifen-inducible Cre recombinase under control of the SM22 promoter [SM-Cre ERT2(ki)].17 The resulting Cav1.2L2/L2, SM-Cre ERT2(ki)Cre/Cre mice were then mated with Cav1.2+/L1 mice (ie, mice carrying 1 L1-allele and 1 wild-type allele) to obtain the smooth muscle–specific knockout Cav1.2L1/L2, SM-Cre ERT2(ki)+/Cre (ie, Cav1.2SMAKO mice) and control animals (Cav1.2+/L2, SM-Cre ERT2(ki)+/Cre; control). The background mouse strain was C57BL/6. Both lines were viable and showed no gross abnormalities. To induce smooth muscle–specific Cre recombination (conversion of L2 to L1 allele in vivo), adult Cav1.2SMAKO and control mice were treated with freshly prepared tamoxifen solution (Sigma) by IP injection once a day for 5 days at a dosage of 1 mg/d. Tamoxifen was dissolved in miglyol oil (Caelo) at a concentration of 10 mg/mL.
RT-PCR on mRNA of Aortae and Resistance Vessels (Arteria Tibialis)
Aortae and A tibialis were isolated and cleaned of connective tissue. Poly(A) mRNA was isolated from the vessels using Dynabeads Oligo(dT)25 (Dynal Biotech). The following buffers were used: GTC buffer (4 mol/L guanidine thiocyanate, 20 mmol/L Na-acetate [pH 5.4], 0.1 mmol/L dithiothreitol, 0.5% lauroyl sarcosinate [wt/vol], 6.5 μL/mL mercaptoethanol), binding buffer (100 mmol/L Tris-HCl [pH 8.0], 20 mmol/L EDTA, 400 mmol/L LiCl), and washing buffer (10 mmol/L Tris-HCl [pH 8.0], 0.15 mol/L LiCl, 1 mmol/L EDTA). The mRNA was eluted with diethylpyrocarbonate-treated water. Oligo-dT primers and Superscript Reverse Transcriptase II (Life Technologies) were used for cDNA synthesis. Using the following gene-specific, intron-spanning primers, PCR amplification (40 cycles) was performed: Cav3.1 (5′-C A C C A A G T C T G A G T C A G A G C-3′ and 5′-T G A T T T C A T C T C A T G A T G G G G-3′); Cav3.2 (5′-A G A G G A A G A T T T C G A T A A G C T-3′ and 5′-G G C T G C T T C C T G C A C T C T T-3′); Cav3.3 (5′-A A G C T C C C [AC] [AG] G A [AG] G G C C T G G A-3′ and 5′-G T A G T A G G A G C T C C G G G A G C T-3′); and hypoxanthine-guanine phosphoribosyl transferase (5′-G T A A T G A T C A G T C A A C G G G G G A C-3′ and 5′-C C A G C A A G C T T G C A A C C T T A A C C A-3′).
Isolation of Single Smooth Muscle Cells
The vessel dissection and cell isolation procedures were slightly modified from procedures described previously.4,18 Briefly, control and CaV1.2SMAKO littermates were killed by cervical dislocation. Arteries were cleaned in either ice-cold PBS or physiological salt solution (in mmol/L: 130 NaCl, 5.9 KCl, 1.2 MgCl2, 11 glucose, 10 HEPES; pH 7.4). The arteries were cut into 2 to 4 pieces and equilibrated in PSS or transferred into 0.5 mL Ca-free solution (in mmol/L: 55 NaCl, 80 Na-glutamate, 5.6 KCl, 2 MgCl2, 10 glucose, 10 HEPES; pH 7.4). After a 10-minute equilibration at 37°C, the artery segments were placed into enzyme solution 1 (Ca-free solution containing 1 mg/mL albumin, 0.7 mg/mL papain, and 1 mg/mL dithiothreitol) and digested for 30 minutes. The vessels were then transferred into enzyme solution 2 (containing 1 mg/mL albumin, 0.05 mmol/L CaCl2, 1 mg/mL hyaluronidase, and 1 mg/mL collagenase F/H [70:30]) and digested for another 10 to 12 minutes. The tissue was subsequently washed for 10 minutes in PSS or Ca-free solution 1 containing 1 mg/mL albumin. Single smooth muscle cells were obtained by trituration and stored in the above solution before use at 4°C. Cells were used for 8 to 24 hours.
Membrane potentials and potassium currents were determined in single A tibialis smooth muscle cells from control and CaV1.2SMAKO mice with the perforated patch method. Perforation was induced by the inclusion of 240 μg/mL amphotericin B in the pipette solution. Experiments were performed at 35°C using fire-polished electrodes with resistances of 2.7 to 3.5 MΩ when filled with the following intracellular solutions (in mmol/L): 110 K-aspartate, 30 KCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 10 HEPES, pH 7.2. The extracellular bath solution was PSS plus 2.4 mmol/L CaCl2. Potassium currents were measured in isolated A tibialis cells using the following solutions (in mmol/L): 140 NaCl, 1.8 CaCl2, 1 MgCl2 5.4 KCl, 10 glucose, 10 HEPES, pH 7.4 (bath solution); and 80 K-aspartate, 50 KCl, 12 NaCl, 1 MgCl2, 3 MgATP, 0.1 EGTA, 5 HEPES, pH7.2 (pipette solution). Large-conductance Ca2+-activated K+ (BK) channels were blocked by the addition of 100 nmol/L iberiotoxin in the bath solution supplemented with 0.1% albumin. Additional potassium channels were blocked by exchanging 10 mmol/L NaCl for 10 mmol/L tetraethyl ammonium in the bath solution. Data were collected with an EPC9 amplifier under control of Pulse software (HEKA Electronics) and analyzed with ORIGIN 6.1 (Microcal).
Telemetric Blood Pressure Recordings
Male knockout and control littermate mice (8 to 12 weeks old) were treated with tamoxifen solution (Sigma) by IP injection once a day for 5 days at a dosage of 1 mg/d. Animals were kept on a 12-hour light/dark cycle. Blood pressure signals from the aortic arch were measured in conscious, unrestrained animals with surgically implanted, miniaturized telemetry devices (Datascience Corp). For long-term measurements of mean arterial blood pressure (MAP), mice were implanted with the transmitter, allowed to recover for 2 weeks, and then treated with tamoxifen. Immediately after implantation of the transmitter, mice were returned to their home cages (placed on top of telemetry receivers), where they continued to be monitored daily throughout the study for general condition, body weight, food and water intake, state of surgical wound healing, and any signs of morbidity. MAP, heart rate, and locomotor activity were recorded continuously for 28 days after tamoxifen injection. For short-term measurements of MAP, the transmitters were implanted into mice pretreated with tamoxifen (21 to 28 days before) and the animals were allowed to recover from anesthesia and then were directly measured when fully awake. Vasoactive compounds were applied IP as a bolus (200 μL) in PBS. Sixty-second MAP recordings were obtained every 90 sec for 15 minutes before and until 120 minutes after drug administration. Recordings obtained after a stable drug effect was observed were used for statistical analysis.
After death, the infrarenal aorta was prepared, and a catheter (1 mm; Hugo Sachs, Germany) was introduced, advanced to the iliac arteries, and tied with a 6/0 prolene stitch (Ethicon). The inferior caval vein was slit open longitudinally to prevent venous congestion. A roller pump was used to perfuse the hindlimb constantly with filtered Krebs–Henseleit (KH) solution. A pressure transducer and a compliance chamber were connected to a side port of the perfusion system. The flow rate was gradually increased to achieve a perfusion pressure of 100 mm Hg. At this perfusion pressure, a considerable amount of spontaneous myogenic tone is present, which made preconstriction unnecessary. When a substance produced a pronounced change in resistance, flow rate was adjusted. When a stable pressure plateau was reached, phenylephrine (PE) was applied in increasing concentrations as a bolus (200 μL) in KH solution, in the presence or absence of inhibitors (mibefradil, Ni2+) in the perfusate. For inhibitor studies, agonist-induced responses were recorded in the absence and then in the presence of the inhibitor to facilitate paired comparison of the results. Vasodilator responses were measured as changes in perfusion pressure.
Data are given as mean values±SEM. Statistical significances were evaluated by either ANOVA followed by Dunnet’s ad hoc tests for unpaired comparisons or by paired Student’s t test.
Results and Discussion
To investigate how the effects of mibefradil on blood pressure are mediated without the need to rely on potentially nonselective L-type Ca2+ channel blockers, we used a third generation, time- and tissue-specific conditional knockout model with an inactivation of the CACNA1C (&Cav1.2) gene. These animals (Cav1.2SMAKO mice) express the tamoxifen-dependent CreERT2 recombinase under the control of the endogenous smooth muscle–specific SM22 gene promoter.4 Treatment of these premutant mice with tamoxifen activates the CreERT2 recombinase and results in effective ablation of Cav1.2 selectively in smooth muscle in vivo (see Materials and Methods for details).
T-Type Ca2+ Channels Are Present in Murine Aorta and Resistance Vessels
As a prerequisite for the functional experiments, we first wanted to characterize the expression of the 3 T-type Ca2+ channel subunits in murine aortae and resistance vessels (A tibialis). RNA was extracted from both vessel types of 3 mice and analyzed along with RNA obtained from total brain, which served as a positive control. Amplification products for all three T-type Ca2+ channels Cav3.1 to Cav3.3 were detected in brain by RT-PCR amplification for 40 cycles (Figure 1A). Sequencing corroborated the correct identity of the amplification products. We observed significant expression of Cav3.2 mRNA in both aortae and tibialis vessels (Figure 1B and 1C). Cav3.3 transcripts were weakly detectable in aortae (Figure 1D) and more strongly expressed in A tibialis (Figure 1B). No amplification products were observed for Cav3.1 mRNA in both vessel types.
No Downregulation of T-type Ca2+ Channel Expression in Cav1.2SMAKO Mice
Knocking out the CACNA1C gene, the major Ca2+ influx pathway in smooth muscle cells,5 might influence other ion channels and transporters playing a role in the regulation of resting membrane potential, intracellular Ca2+ homeostasis, and tone. Therefore, reduced expression of T-type Ca2+ channel subunits in Cav1.2SMAKO animals had to be ruled out before using this model to assign the vascular effects of mibefradil to L- and T-type Ca2+ channels, respectively. We compared the mRNA expression of Cav3.x transcripts in aortae of control and Cav1.2SMAKO mice by semiquantitative RT-PCR using hypoxanthine-guanine phosphoribosyl transferase as an internal control. The experiments revealed no differences in the expression of the Cav3.2 and Cav3.3 isoforms between control and Cav1.2-deficient mice (Figure 1C and 1D; n=3 for each genotype and isoform). Cav3.1 was not detectable in either control or Cav1.2SMAKO mice. These results indicate that the deletion of Cav1.2 in smooth muscle cells did not modulate the expression of Cav3.x T-type Ca2+ channels.
Resting Membrane Potential Is Not Changed Cav1.2SMAKO Vascular Smooth Muscle Cells
Given that the expression of T-type Ca2+ channels was unchanged in Cav1.2SMAKO animals, we next determined resting membrane potential and potassium currents in A tibialis smooth muscle cells from control and knockout mice. In principle, a reduction in the expression or function of potassium channels in Cav1.2SMAKO mice could result in membrane depolarization inducing complete inactivation of T-type Ca2+ channels. In line with this view, others have already questioned the significance of T-type Ca2+ channels for blood pressure regulation even at the physiological membrane potentials prevailing in vascular smooth muscle.3 The resting membrane potential of Cav1.2-deficient smooth muscle cells was not significantly different from that of control cells (−40±1.5 mV for control mice, n=8; −39±7.2 mV for Cav1.2SMAKO mice, n=5). Similar values have been reported for A tibialis cells by others.18 We next determined potassium currents in the absence and presence of iberiotoxin and TEA. Potassium current in control and Cav1.2SMAKO cells were 52±9.8 and 49.4±12.5 pA/pF, respectively. Addition of iberiotoxin decreased the current to 18.2±3 and 16.9±2.3 pA/pF in control and Cav1.2SMAKO cells, respectively. The currents were further reduced by 10 mmol/L TEA to 9.5±2.9 and 5.8±0.7 pA/pF in control and Cav1.2SMAKO cells, respectively. These values were determined at +40 mV in 6 to 13 individual cells. These data demonstrate that, in Cav1.2SMAKO animals, no gross changes in smooth muscle membrane potential or potassium channel density are likely to influence the analysis of mibefradil effects on vascular tone.
Perfused Hindlimb Experiments
Pressure-induced vasoconstriction or myogenic tone of small arteries and arterioles is a major component in the control of vascular resistance and, thus, blood pressure.5 Therefore, we first analyzed the effect of mibefradil on peripheral resistance in control and Cav1.2SMAKO mice resistance vessels (arterioles and capillaries) using the perfused hindlimb system. Flow rate was gradually increased to achieve a perfusion pressure of approximately 100 mm Hg; at this perfusion pressure, a considerable amount of spontaneous myogenic tone is present.19 Resistance at 100 mm Hg perfusion pressure was reduced to &60% in Cav1.2SMAKO compared with control hindlimbs (Figure 2A), indicating the loss of autoregulatory myogenic tone in animals deficient for Cav1.2.4 Mibefradil treatment of control preparations using a concentration in the perfusate supposed to be specific for T-type Ca2+ channels6,14 and equivalent to the therapeutic plasma level in vivo8 (10 μmol/L) reduced peripheral vessel resistance in the hindlimbs to Cav1.2SMAKO levels (control mice: 13.2±0.6 mm Hg/mL per minute; control mice+mibefradil: 8.1±0.3 mm Hg/mL per minute; Cav1.2SMAKO mice: 6.7±0.1 mm Hg/mL per minute; P<0.01). However, mibefradil had no effect on peripheral vessel resistance in Cav1.2SMAKO mice (Cav1.2SMAKO+mibefradil: 6.8±0.2 mm Hg/mL per minute). This result clearly supported the concept that the effect of mibefradil on pressure-induced myogenic response can be explained solely by its action on Cav1.2 Ca2+ channels without the need to postulate other Ca2+ influx pathways. In addition, when Ni2+ (0.1 mmol/L) was added to the perfusate to block T-type Ca2+ channels, peripheral vessel resistance was not affected (control mice+Ni2+: 13.3±1.2 mm Hg/mL per minute). Given the relatively high IC50 of Ni2+ block of Cav1.2 L-type Ca2+ channels20 (&0.8 mmol/L) and low IC50 of block of Cav3.2 T-type Ca2+ channels21 (&10 μmol/L), this result may suggest minimal contribution of T-type Ca2+ channels to peripheral resistance in hindlimb vessels.
Arterial blood pressure is not only determined by autoregulatory myogenic mechanisms but also by an interplay of vasoconstrictors (most importantly angiotensin II and norepinephrine) and vasodilators.5 Therefore, we tested the effect of mibefradil on contractile responses to α-adrenergic stimulation. Perfusion pressure changes to bolus application of 200 μL of 1 mmol/L PE were blunted by mibefradil treatment in control mice (contractile response in control mice: 52±4%; in control mice+mibefradil: 30±3%; P<0.01) (Figure 2B), indicating that mibefradil inhibited the PE-induced resistance increase. Again, mibefradil had no further effect in Cav1.2SMAKO mice (Cav1.2SMAKO mice without mibefradil: 31.5±2%; Cav1.2SMAKO mice with mibefradil: 34±2%) (Figure 2C). These results make it unlikely that block of T-type Ca2+ channels plays a role in the action of mibefradil on the α-adrenergic vasoconstrictor response in the microvasculature.
Telemetric Blood Pressure Measurements
Given that the vasodilating effect of mibefradil in skeletal muscle resistance arteries was evidently mediated by the Cav1.2 channel, we next examined the in vivo effects of mibefradil on blood pressure in awake, freely moving mice. We followed the changes in MAP in control and Cav1.2SMAKO mice by telemetry (Figure 3A). In general, as shown previously,4 inactivation of the Cav1.2 gene resulted in a decrease of MAP form 118±4 to 85±3 mm Hg. The MAP was decreased in control mice by 38±2.5 mm Hg after IP injection of increasing doses of mibefradil (1,25 μg/kg body weight mibefradil to 1.25 mg/kg body weight mibefradil; Figure 3A and 3B). Although this drop in MAP indicates the efficiency of mibefradil as an antihypertensive agent, mibefradil had no effect on blood pressure in mice lacking the L-type Ca2+ channel Cav1.2 (Figure 3A and 3C), again indicating that the antihypertensive effect is solely mediated by the Cav1.2 channel. Heart rate increased in Cav1.2SMAKO and control mice after mibefradil injection to a similar extent (control: 456±32 to 512±14 bpm; Cav1.2SMAKO: 432±56 to 534±18 bpm).
Although the properties of cloned Cav3.x T-type Ca2+ channels are, in principle, consistent with possible roles for these channels in Ca2+ homeostasis of smooth muscle cells, their function in vascular tissue is unclear. The antihypertensive actions of mibefradil, a compound thought to selectively block T-type Ca2+ channels at therapeutic concentrations (&1 to 10 μmol/L), suggested the hypothesis that inhibition of VSM T-type Ca2+ channels would produce vasorelaxation.6 Because of the existence of multiple receptors for this drug (for a review, see Perez-Reyes22), it is difficult to single out the correct in vivo mechanism for the antihypertensive effects of mibefradil.
Here, we have investigated whether mibefradil achieves its beneficial vascular effects by a block of L- or T-type Ca2+ channels. We used a spatiotemporally controlled Cre/lox mouse model specifically lacking 1 of the putative receptors of mibefradil, the Cav1.2 L-type Ca2+ channel, in the vessels. In this model, the pharmacological properties of the channels that remain expressed, here the Cav3.x T-type Ca2+ channels, can be investigated free from interference by the Cav1.2 channel.
Our major findings were as follows: (1) mibefradil lowers MAP and attenuates peripheral resistance in mice; (2) although in vitro studies have reported a 10- to 30 fold selectivity of mibefradil for T-type voltage-gated channels,6–9 its effect on MAP and peripheral resistance are mediated by the Cav1.2 L-type Ca2+ channel; and (3) additionally, control experiments demonstrated that T-type Ca2+ channel mRNA expression and SMC membrane potentials are not changed in Cav1.2SMAKO animals.
These results do not rule out a modest contribution of T-type Ca2+ channels in the regulation of vascular tone, although they suggest that the contribution of T-type channels to the contraction of resistance vessels is minimal. This notion is in excellent agreement with the recent finding that deletion of the Cav3.2 T-type channels impaired coronary relaxation, not contraction, in mice.15 Given the controversial literature on the role of T-type Ca2+ channel as a target for cardiovascular pathology,6,14,15,22–25 studies on transgenic mice lacking T-type Ca2+ channels without the need to rely on the unspecific blocker mibefradil might help to further clarify the functional role of these channels.
This work was supported by the Volkswagenstiftung, Deutsche Forschungsgemeinschaft (SFB 391), and Fond der Chemischen Industrie. We are indebted to Susanne Paparisto for excellent technical assistance.
Original received August 10, 2004; first resubmission received November 16, 2004; second resubmission received October 20, 2005; accepted November 10, 2005.
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