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CARDIOVASCULAR
Oregon Hearing Research Center, Oregon Health and Science University, Portland, Oregon (Z.-G.J., X.-R.S., B.-C.G., H.Z., Y.-Q.Y.); and Department of Otolaryngology, Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, People's Republic of China (H.Z.)
Received October 8, 2006; accepted October 31, 2006.
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Abstract |
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; Busse et al., 2002). The EDHF is an important mechanism regulating blood flow to organs and tissues and is implicated in vascular pathology, such as ischemia, hypertension, atherosclerosis, diabetic and aging vascular malfunction (Faraci and Heistad, 1998), and coronary vascular spasms (Konidala and Gutterman, 2004).
The EDHF seems to be a variable combination of gap junction coupling, endothelial release of K+, NO, epoxyeicosatrienoic acids, and prostanoid in various vascular beds and in different animal species (Busse et al., 2002). We reported recently that ACh induced hyperpolarization and dilation in the cochlear spiral modiolar artery (SMA) via the EDHF (Jiang et al., 2005) and found that ACh-induced EDHF in the SMA was a complex. The induced hyperpolarization originated in the endothelial cell (EC) by activating Ca2+-activated K+ channels (KCas). ACh-induced hyperpolarization in smooth muscle cells (SMCs), however, was mainly (60%) an electrotonic spread of the hyperpolarization from the EC via gap junction coupling. The EC also released K+ into myoendothelial interlayer space via its activated KCa, which in turn activated the inward rectifier potassium channel (Kir) and Na+-K+-ATP pump current in the SMC. Nonetheless, activation of KCa in the EC plays a primary and essential role in the EDHF-mediated vasodilation in the SMA as well as in many other vascular beds.
According to single-channel conductance and pharmacological characteristics, three classes of KCas—big conductance, intermediate conductance, and small conductance (BK, IK, and SK)—are identified in the SMC and/or the EC (Nilius and Droogmans, 2001; Ledoux et al., 2006). Recent work has pointed to IK activation as the main electrogenesis mechanism of ACh-induced hyperpolarization in the EC (Coleman et al., 2001; Eichler et al., 2003; Ledoux et al., 2006). Therefore, it would be interesting to know whether the ACh-induced hyperpolarization in the SMA is sensitive to specific IK blockers or BK blockers.
In this respect, a dihydropyridine, nitrendipine, has been found to be a potent blocker for cloned human IK channel expressed in HEK-293 cells (Jensen et al., 1998). Dihydropyridines (DHPs) are widely used vasodilators for treating hypertension and angina by blocking L-type Ca2+ channels (IL) in vascular SMCs. It is therefore important to know whether nitrendipine and other DHPs effectively suppress the EDHF-mediated hyperpolarization. In addition, it would be interesting to know whether other classes of IL blockers verapamil (a benzothiazepine) and diltiazem (a phenylalkylamine), would affect the ACh-induced hyperpolarization. We report here that the three DHPs—nifedipine, nimodipine, and nitrendipine—share the IK-blocking property and suppress the ACh-induced hyperpolarization in the cochlear artery cells, whereas verapamil and diltiazem had little effect on the IK-mediated ACh-induced hyperpolarization. Preliminary data of this work has been published in a meeting abstract (Jiang et al., 2004).
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Materials and Methods |
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, 2005). Guinea pigs (250–500 g) were anesthetized by intramuscular injection of an anesthetic mixture (1 ml/kg) of 500 mg of ketamine, 20 mg of xylazine, and 10 mg of acepromazine in 8.5 ml of H2O and then killed by exsanguination. Both bullae were rapidly removed and transferred to a Petri dish filled with a physiological solution (Krebs') composed of 125 mM NaCl, 5 mM KCl, 1.6 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 20 mM NaHCO3, and 8.2 mM glucose, and saturated with 95% O2 and 5% CO2 at 35°C, pH 7.4. The SMA was dissected out from the cochlea under a stereomicroscope. The vessels were incubated for 0.5 to 24 h in the Krebs' solution and transferred to a recording bath for intracellular recording. The Oregon Health and Science University Animal Care and Use Committee approved the animal use procedure.
Intracellular Recording. A 2- to 5-mm-long segment of the SMA (40–80 µm in diameter) was pinned with minimal stretch to the silicon rubber layer (Sylgard 184; Dow Corning, Midland, MI) in the bottom of the recording bath (0.5-ml volume) and continuously superfused with a 35°C Krebs' solution. The outside connective tissues were cleaned manually under a stereomicroscope (Nikon SMZ-2T; Nikon, Tokyo, Japan). The glass microelectrode was filled with 2 M KCl with a tip resistance of 60 to 200 M
. Intracellular penetration was obtained by advancing the electrode into adventitial surface of the vessel with a micromanipulator (MP-1; Narishige, Tokyo, Japan). Transmembrane potential and current were simultaneously monitored with an NPI preamplifier (SEC10-LX; NPI, Tamm, Germany). The electrical signals were recorded with a computer equipped with pClamp9 software (Molecular Devices, Sunnyvale, CA) using sampling intervals of 0.1, 0.5, or 10 ms. The resting potential (RP) was usually determined 5 min after the initial voltage jump at penetration and checked by the voltage jump at the withdrawal of the electrode. The membrane input resistance was measured by applying 0.2- to 0.5-nA, 0.5- to 2-s current pulses via the recording electrode with the capacitance compensation and bridge-balance well adjusted on the NPI preamplifier (Jiang et al., 2001). The adjustment was achieved by concurrently using an additional data acquisition computer, a monitor displaying fast sweeps (0.5–2 s) of current-voltage signals at a 10-kHz sampling rate to ensure the best bridge-balance during the recording period (Jiang et al., 2005). In addition, five or 10 sweeps were averaged to reduce the baseline noise.
Drug Application and Statistics. Drugs in known concentrations were applied via a bath solution. The solution that passed the recording chamber could be switched, without change in flow rate and temperature, to a solution that contained a drug or a solution of different ionic composition. Drugs used in this study were ACh, charybdotoxin (ChTX), clotrimazole (CLT), (–)-cis-diltiazem (diltiazem), 4-diphenylacetoxy-N-methylpiperidine methiodide (DAMP), iberiotoxin (IbTX), nifedipine, nimodipine, nitrendipine, trifluoperazine (TFP), verapamil (all from Sigma/RBI, Natick, MA), and 18
-glycyrrhetinic acid (18GA; MP Biomedicals, Irvine, CA). Statistical values are expressed as means ± S.E.M.
Immunohistochemistry of IK Channel. Albino guinea pigs (500
600 g) were anesthetized with an overdose of ketamine hydrochloride (100 mg/kg i.m.; Abbott Laboratories, Abbott Park, IL) and xylazine hydrochloride (2 mg/kg i.m.; Phoenix Scientific, Inc., St. Joseph, MO). Cochleae were taken after cardiovascular perfusion with saline followed by 4% paraformaldehyde and then immersed in the same fixative solution for 4 h. The SMA was dissected out from the cochlea, washed in 0.02 M PBS, pH 7.4, and then permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 1 h. After immunoblock in 10% goat serum and 1% bovine serum albumin (BSA) in the PBS for 1 h, the specimens were incubated overnight in a solution containing anti-SK4/IK1 antibody (rabbit polyclonal antibody, SC-32949, 1:100 diluted with 1% BSA-PBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-
smooth muscle actin antibody conjugated with Cy3 (monoclonal, C6198, 1:400 dilution; Sigma-Aldrich). Specimens were washed in 1% PBS for 30 min and incubated in Alexa Fluor 488 anti-rabbit IgG (1:100 diluted with 1% BSA-PBS; Invitrogen, Carlsbad, CA) for 1 h. After wash for 30 min, the vessels were mounted and observed on a Nikon Eclipse TE 300 inverted microscope equipped with a Bio-Rad MRC 1024 confocal laser scanning system (Bio-Rad, Hercules, CA). Negative controls were done by incubating the tissue with 1% BSA-PBS containing no anti-SK4/IK1 primary antibody.
Vessel Diameter Measurement. SMA diameter (outside edge to edge) was tracked by a videocamera and computer software as described previously (Jiang et al., 2003). In brief, the SMA segment in the bath was dark field-illuminated by a fiber optic lamp and imaged by a videocamera (Sony XC-13; Sony, Tokyo, Japan) through the trinocular stereomicroscope. The image of the SMA was displayed on a monitor, recorded on a VCR, and digitized by a video capture board (Matrox RainbowRunner Studio) in a Pentium III PC. The digitized video signal was processed online by customer-written edge-detection software. The digital sampling rate for the vessel diameter varied between 2 and 5 Hz. Through a D/A converter board, a voltage signal proportional to the diameter was fed to pClamp9 interface (Digidata 1322A. Molecular Devices) for digital recording (Fig. 6). The digitized images were also saved to disks for further analysis.
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Results |
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, 2005). In brief, the cells sampled usually showed an initial RP either near –40 or –75 mV, called low or high RP, respectively. A low RP cell may quickly shift its RP from the low level to the high level and vise versa (see Fig. 1A in Jiang et al., 2005). Roughly one-half of the recordings were from smooth muscle cells (SMCs), and one-half were from ECs (Jiang et al., 2001). The membrane properties of the SMC and the EC are generally indistinguishable (Jiang et al., 2001). In addition to single-cell labeling with propidium iodide-containing electrode and histological examination (Jiang et al., 2001), it was possible to determine the cell type during the intracellular recording by observing the effects of 30 µM18GA, a gap junction blocker, on the hyperpolarization induced by high 10 mM K+ and/or ACh (Jiang et al., 2005). The SMCs that had a low RP always showed a high K+-induced hyperpolarization (Fig. 1, A and B) that was not sensitive to 18GA, whereas they exhibited an ACh-induced hyperpolarization that was largely blocked by 18GA. Conversely, in the ECs with a low RP, high K+-induced hyperpolarization was blocked by 18GA, but the hyperpolarization response to ACh was not sensitive to 18GA. A portion of the cells in this study was identified for the cell type by one or both methods.
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). The hyperpolarization often had a variable fast transient phase lasting 10 to 30 s followed by a sustained phase lasting to wash-out of ACh (Fig. 2A). The two phases were sometimes separated by a narrow notch (Fig. 5A), or they merged as a single phase (Fig. 1). In many cells, a delayed depolarization was unmasked after the ACh-induced hyperpolarization was pharmacologically suppressed (Fig. 1, A and B). To quantify the ACh-induced hyperpolarization better, we always took the ACh-induced hyperpolarization amplitude at 80 s after the beginning of ACh application, to minimize the influence of the initial transient hyperpolarization and the unmasked depolarization. Unlike the ACh-induced hyperpolarization, the ACh-induced depolarization was insensitive to the KCa blocker 50 nM ChTX (data not shown; Jiang et al., 2005), suggesting that the two responses have different ionic mechanisms. We will report a study on the ACh depolarization separately and thus no further analysis was made in this report regarding the ACh depolarization. To avoid an unwanted membrane resting potential shift from a low RP to high RP (Jiang et al., 2001), which would sabotage the quantitative experiments of the ACh action, we kept 100 (occasionally 200) µMBa2+ present throughout all experiments except when otherwise stated. Because this concentration of Ba2+ eliminated the Kir implication in ACh-induced hyperpolarization in the SMCs (see Introduction; Jiang et al., 2005), it made the ACh-induced hyperpolarization in the SMC almost solely an electrotonic spread of the ACh-induced hyperpolarization of the EC. This largely unified the electrogenesis mechanism of the ACh-induced hyperpolarization in the SMC and the EC, so that the sensitivity of the ACh-induced hyperpolarization to relevant channel blockers became equalized between these two types of cells.
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IK Mediates the ACh-Induced Hyperpolarization. We previously reported that the KCa activation is largely responsible for generation of the ACh-induced hyperpolarization in an EC of the SMA (Jiang et al., 2005), because the hyperpolarization was associated with an increase in input conductance, enhanced by membrane depolarization and blocked by ChTX (by 81%; Fig. 1A) plus apamine (by 10–20%) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, a membrane-permeable Ca2+ chelator, but not by Ba2+. To further identify the role of the IK, we tested the effects on the ACh-induced hyperpolarization of the known IK blockers nitrendipine and clotrimazole (Jensen et al., 1998) and a BK-selective blocker, IbTX (Van Renterghem et al., 1995). Figures 1 and 2 depict the main results. In contrast to a strong blockage by ChTX (Fig. 1A), ACh-induced hyperpolarization was not significantly altered by 100 nM IbTX (Fig. 1C; paired t test, p > 0.05; n = 3), consistent with a notion that the ACh-induced hyperpolarization is generated by an activation of IK, not BK (Ledoux et al., 2006). Similar to ChTX, IbTX alone caused a small (3- to 5-mV) depolarization in the majority of the cells tested (n = 10 and 4).
Nitrendipine (1 µM) suppressed the ACh-induced hyperpolarization by 95.2 ± 18.6% in all the cells tested (n = 7), whereas having no significant effect on the 10 mM K+-induced hyperpolarization (Fig. 1B), suggesting a specific IK blockade without affecting the Kir. Nitrendipine suppression of the ACh-induced hyperpolarization was partially reversible upon wash-out of the drug for longer than 30 min. The inhibition was not significantly different between the identified EC and SMC (Student's t test, p > 0.05; n = 6 and 9).
CLT, an antifungal antibiotic and known IK blocker, concentration-dependently suppressed the ACh-induced hyperpolarization (Fig. 2). A Hill equation fit to the concentration-inhibition relation of four cells revealed an IC50 of 116 nM and a Hill number of 0.82. The inhibition was partially reversible after a 20-min wash with CLT-free solution. Nitrendipine or CLT alone caused little change in the RP.
It is known that IK (or IK1, now termed SK4) belongs to SK protein family and that calmodulins join the SK subunits to form the SK channel polymer complex (Ledoux et al., 2006) where the calmodulin functions as the sensor of intracellular Ca2+. The calmodulin antagonist TFP (10 and 100 µM) caused a concentration-dependent inhibition of ACh-induced hyperpolarization in all cells tested (inhibited 30 ± 5.0 and 92 ± 2.7%, respectively; p < 0.01; n = 4; Fig. 2C). The inhibition was partially reversible after a 10-min wash-out. TFP alone in low concentration (10 µM) caused little change in the RP but induced a 1- to 8-mV depolarization when 100 µM was administrated.
Immunohistochemistry Identification of IK Expression in the SMA Cells. The IK channel protein in the SMA was detected by immunoreaction with anti-SK4/IK1 antibody in two adult guinea pigs tested. The IK immunoreactivity (tagged as green fluorescent color) was localized in all the ECs of the SMA tested (Fig. 3). The fluorescent signal of IK channel protein seemed evenly lined with the surface membrane of the ECs. In contrast, the IK signal was only faintly and unevenly observed in some smooth muscle cells, consistent with the electrophysiological data that the ACh-induced hyperpolarization is generated in the EC.
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15 min during a "wash-in", but the wash-out recovery was notably different. The ACh-induced hyperpolarization often recovered to approximately 60% of the control after a 20-min wash with a drug-free Krebs' solution in the cases of nifedipine and nimodipine, but usually much less (e.g., 20%) recovery was achieved after a 60-min wash-out of nitrendipine, although the fast transient phase recovered faster. Therefore, when estimating the concentration-action relation in a single cell, we consecutively stepped up DHP concentration and used a 15-min wash-in time for each concentration applied. The Hill equation fit to normalized ACh-induced hyperpolarization amplitudes revealed that nimodipine, nitrendipine, and nifedipine had an IC50 value of 3.2, 34, and 455 nM, respectively (Fig. 4).
Effects of Non-DHP Ca2+ Channel Blockers on ACh-Induced Hyperpolarization. We previously demonstrated that ACh-induced hyperpolarization in the SMA is a Ca2+-dependent event (Jiang et al., 2005). To test whether the dihydropyridine inhibition of ACh-induced hyperpolarization was related to its blocking action on L-type Ca2+ channels, we observed effects of other classes of Ca2+ channel blockers on the ACh-induced hyperpolarization. Figure 5 depicts the main results. In contrast to nifedipine and other DHPs, the IL blockers 10 µM diltiazem, 100 µMCd2+, or the T-type Ca2+ channel blocker 100 µM Ni2+ caused no significant alteration of the ACh-induced hyperpolarization. Verapamil (10 µM), a blocker for IL, attenuated the ACh-induced hyperpolarization by 21 ± 3.9% (paired t test, p < 0.01; n = 6), which was a significantly weaker inhibition than that of 10 µM nifedipine (Student's t test, p < 0.05).
DHP Blocks ACh-Induced Vasoconstriction. ACh (1–3 µM for 2 min) induced a dilation in six of 10 SMA segments tested (to 58.8 ± 3.7 µm from a control of 56.4 ± 3.5 µmor increased by 4.3 ± 0.27% (paired t test, p < 0.05; n = 6; Fig. 6). The induced dilation was usually slow in its onset and offset, whereas it occasionally showed a fast transient dilation and/or contraction followed by a slow dilation. The quantitative measurement was done only on the peaks of slow-diameter changes that usually occurred approximately 2 min after the ACh application. DAMP (50 nM) completely blocked the ACh-induced dilation 10 min after its application (Fig. 6A). A partial recovery of the dilation was observed after 30-min wash-out, confirming that, like the ACh-induced hyperpolarization (Jiang et al., 2005), the dilation is an M3 receptor-mediated event. The ACh-induced dilation is also near completely suppressed by 50 nM ChTX or 1 µM nitrendipine in all vessels tested (n = 3 and 6, respectively; Fig. 6, B and C). The suppression was partially reversible. Nitrendipine, but not ChTX, alone caused a small dilation (1–3%) in three of five preparations. In the presence of ChTX or nitrendipine, a transient vasoconstriction was unmasked at the beginning of ACh application in most cases, consistent with an intracellular release of Ca2+ in muscle cells that triggered a contraction.
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), IbTX failed to affect the EDHF-attributed hyperpolarization, supporting that the IK, not the BK, mediates the original hyperpolarization in the arteriolar endothelium. Furthermore, our demonstration of clotrimazole inhibition on the ACh-induced hyperpolarization extended the finding that clotrimazole derivatives triarylmethane-34 and triarylmethane-39 both block the IK and ACh-induced current in isolated rat carotid endothelial cells (Eichler et al., 2003). Therefore, IK mediation of the ACh-induced hyperpolarization in the vascular EC is probably across animal species and vascular beds.
IK Mediation of the ACh-Induced Hyperpolarization. Several lines of evidence from this and a previous study (Jiang et al., 2005) supported the notion that IK activation is largely responsible for the ACh-induced hyperpolarization in the endothelial cells. First, a combined application of 100 µM Ba2+ and 25 µM18GRA always almost completely suppresses the ACh-induced hyperpolarization in smooth muscle cells, but it does not affect it in endothelial cells, indicating the endothelial origin of the hyperpolarization (Jiang et al., 2005). Second, the hyperpolarization was associated with an increase in input conductance (decrease in input resistance); the amplitude of hyperpolarization was increased in cells with a less negative RP and decreased in cells with a more negative RP (Jiang et al., 2005), consistent with a K+ channel activation being responsible for the hyperpolarization (Hille, 2001). Third, the calmodulin antagonist trifluoperazine effectively suppressed the ACh-induced hyperpolarization (Fig. 2C), indicating that the responsible channel of the hyperpolarization belongs to the SK family, which requires calmodulin to function, not the BK, which does not require the calmodulin (Ledoux et al., 2006). Fourth, our immunohistochemical data demonstrated that IK proteins are clearly expressed in the EC of the SMA (Fig. 3). Finally and decisively, three classes of known IK channel blockers—ChTX, nitrendipine, and clotrimazole—suppressed the ACh-induced hyperpolarization, whereas BK blocker IbTX and other K+ channel blockers—Ba2+, glipizide, and 4-aminopyridine—had no significant effect.
Our data suggest that the small-conductance Ca2+-activated K+ channel plays a minor (5%) role in generation of the ACh-induced hyperpolarization in the preparation used. The incomplete (81%) inhibition by 50 nM ChTX might simply be due to its less-than-sufficient concentration to reach a full block of IK, because the IC50 of the toxin was estimated to be 30 nM for the IK in brain microvascular endothelial cells (Van Renterghem et al., 1995) and for the cloned human IK in HEK-293 cells (Jensen et al., 1998). The strong suppression by clotrimazole was probably not due to its inhibitory action on cytochrome P450 or voltage-gated K+ channels (Yuan et al., 1995; Eichler et al., 2003), since the ACh-induced hyperpolarization was not affected by 17-octadecynoic acid, a known cytochrome P450 inhibitor (Jiang et al., 2005). In addition, the estimated IC50 (116 nM) of clotrimazole is very close to that determined from HEK-293 cells that expressed human IK channels (153 nM; Jensen et al., 1998), and it is 2-fold of that determined from the IK expressed in glioblastoma GL-15 cell lines (63 nM; Fioretti et al., 2004). Taken together, we have for the first time a quantitative estimation that IK activation is responsible for approximately 95% of the hyperpolarization induced by ACh in the endothelial cells.
IL Is Not Involved in DHP Inhibition on ACh-Induced Hyperpolarization. The ACh-induced Hyperpolarization is a Ca2+-dependent event, and the DHPs are well established L-type Ca2+ channel blockers. Our data demonstrated that the inhibition of ACh-induced hyperpolarization by the DHPs was not related to DHP antagonism on the IL channels. First, the known IL blockers diltiazem and Cd2+ showed no effect on the ACh-induced hyperpolarization. Verapamil caused 20% inhibition on the ACh-induced hyperpolarization; this could be related to its side action on other channels, rather than to its Ca2+ antagonism, since its inhibition on voltage-gated K+ channels and Na+ channels has been well documented (Lin et al., 1995; Yokoo et al., 1998). It is noteworthy that these data are consistent with a previous report that diltiazem and verapamil showed little inhibition on human IK channels expressed in HEK-293 cells (Jensen et al., 1998). Second, the potency of individual DHP in inhibiting ACh-induced hyperpolarization seems unrelated to its potency of IL inhibition. For example, the IC50 of nifedipine, nitrendipine, and nimodipine for IL was reported to be 36, 108, and 5.3 nM, respectively (Hermsmeyer et al., 1988; McCarthy and TanPiengco, 1992; Hirakawa et al., 1994), whereas it was 455, 34, and 3.2 nM for the IK inhibition (Fig. 4). The presently estimated IC50 of nitrendipine on the IK was very close to that determined on the cloned human IK expressed in HEK-293 cells (27 nM; Jensen et al., 1998). Our data are also consistent with a current belief that vascular endothelial cells do not express the voltage-gated Ca2+ channel, IL (Nilius and Droogmans, 2001; Ledoux et al., 2006).
It is interesting to note that the initial fast transient ACh-induced hyperpolarization was more resistant to all IK blockers used in this study than the delayed sustained hyperpolarization (Figs. 1, 2, 3). The transient hyperpolarization was rarely completely blocked by a high concentration of IK blockers, and its recovery during wash-out was long before the recovery of the sustained ACh-induced hyperpolarization. The cause of the apparent different sensitivity of the two phases to IK blockers remains to be found. It is noteworthy that ACh-induced increase in cytosolic calcium in the ECs often shows two phases, a fast transient phase followed by a slower sustained phase, very much corresponding to the two phases of hyperpolarization observed in the SMA and other arterioles (Nilius and Droogmans, 2001). There is evidence that the fast-phase elevation is mainly due to Ca2+ release from internal Ca2+ storage via the coupling between the M3 receptor and the inositol-1,4,5-triphosphate-sensitive mechanism (Fukao et al., 1997; Nilius and Droogmans, 2001) and that the second sustained phase relies on influx from the extracellular space, possibly via a store-operated Ca2+ channel, TRP channels, and other nonspecific cation channels (Nilius and Droogmans, 2001). We speculate that the different sources of Ca2+ may activate two distinct populations of IK channels that have different binding dynamics and/or accessibilities for the blockers.
Significance of DHP Blockade on IK-Mediated EDHF. A recent report verified that IK deficiency causes impaired EDHF and hypertension in a mouse model (Si et al., 2006). We found that 1 µM nitrendipine near completely suppressed the ACh-induced dilation (Fig. 6). Although it is possible that IL inhibition may also play a role, nitrendipine has a lower IC50 value for IK inhibition than that for IL inhibition; it is likely that its blocking action on IK, thus the hyperpolarization, may be primarily responsible for the inhibition of ACh-induced dilation.
In summary, the present study demonstrated that ACh-induced hyperpolarization in the SMA is mainly due to an activation of IK channels in the endothelial cells; dihydropyridines suppressed the ACh-induced hyperpolarization by blocking the IK with a potency order of nimodipine > nitrendipine > nifedipine. IK activation is a primary and essential step in EDHF-mediated hyperpolarization and vasodilation, and activation of the EDHF mechanism by ACh and other vasoactive agents may play an important role in keeping blood flow to vital organs in several clinical conditions. Therefore, clinicians should note that an administration of dihydropyridines might compromise the EDHF-mediated vasodilation in these patients.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ACh, acetylcholine; EDHF, endothelium-derived hyperpolarization factor; SMC, smooth muscle cell; EC, endothelial cell; KCa, calcium-activated potassium channel; SMA, spiral modiolar artery; Kir, inward rectifier potassium channel; BK, big-conductance calcium-activated K+ channel; IK, intermediate-conductance Ca2+-activated K+ channel; SK, small-conductance Ca2+-activated K+ channel; HEK, human embryonic kidney; DHP, dihydropyridine; IL, L-type calcium current or channel; RP, resting potential; ChTX, charybdotoxin; CLT, clotrimazole; DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; IbTX, iberiotoxin; TFP, trifluoperazine; 18GA, 18-glycyrrhetinic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SMC, smooth muscle cell; Nif, nifedipine.
Address correspondence to: Dr. Zhi-Gen Jiang, Oregon Hearing Research Center, NRC04, Oregon Health and Science University, Portland, OR 97239. E-mail: jiangz{at}ohsu.edu
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References |
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Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, and Weston AH (2002) EDHF: bringing the concepts together. Trends Pharmacol Sci 23: 374–380.[CrossRef][Medline]
Coleman HA, Tare M, and Parkington HC (2001) K+ currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels. J Physiol (Lond) 531: 359–373.
Eichler I, Wibawa J, Grgic I, Knorr A, Brakemeier S, Pries AR, Hoyer J, and Kohler R (2003) Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K+-channels suppresses EDHF-mediated vasodilation. Br J Pharmacol 138: 594–601.[CrossRef][Medline]
Faraci FM and Heistad DD (1998) Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev 78: 53–97.
Fioretti B, Castigli E, Calzuola I, Harper AA, Franciolini F, and Catacuzzeno L (2004) NPPB block of the intermediate-conductance Ca2+-activated K+ channel. Eur J Pharmacol 497: 1–6.[CrossRef][Medline]
Fukao M, Hattori Y, Kanno M, Sakuma I, and Kitabatake A (1997) Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br J Pharmacol 120: 1328–1334.[CrossRef][Medline]
Hermsmeyer K, Sturek M, and Rusch NJ (1988) Nitrendipine inhibition of calcium current in rat vascular muscle cells. J Cardiovasc Pharmacol 12: S100–S103.
Hille B (2001) Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA.
Hirakawa Y, Kuga T, Kanaide H, and Takeshita A (1994) Actions of a new Ca2+ channel antagonist, CD832, on two types of Ca2+ channels in smooth muscle. Eur J Pharmacol 252: 267–274.[CrossRef][Medline]
Jensen BS, Strobaek D, Christophersen P, Jorgensen TD, Hansen C, Silahtaroglu A, Olesen SP, and Ahring PK (1998) Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel. Am J Physiol 275: C848–C856.
Jiang ZG, Nuttall AL, Zhao H, Dai CF, Guan BC, Si JQ, and Yang YQ (2005) Electrical coupling and release of K+ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery. J Physiol (Lond) 564: 475–487.
Jiang Z-G, Qiu J-H, Ren T-Y, and Nuttall AL (1999) Membrane properties and the excitatory junction potentials in smooth muscle cells of cochlea spiral modiolar artery in guinea pigs. Hear Res 138: 171–180.[CrossRef][Medline]
Jiang Z-G, Shi XR, Zhao H, Si JQ, and Nuttall AL (2003) Basal nitric oxide production contributes to membrane potential and vasotone regulation of guinea pig in vitro spiral modiolar artery. Hear Res 189: 92–100.[CrossRef]
Jiang Z-G, Si J-Q, Lasarev MR, and Nuttall AL (2001) Two resting potential levels regulated by inward rectifying potassium channels in guinea pig cochlea spiral modiolar artery. J Physiol (Lond) 537: 829–842.
Jiang ZG, Zhao H, and Yang YQ (2004) Distinct channels responsible for acetylcholine-induced hyperpolarization and depolarization in guinea pig in vitro spiral modiolar artery. Assoc Res Otolaryngol Midwint Res Meet Abstr 27: 996.
Konidala S and Gutterman DD (2004) Coronary vasospasm and the regulation of coronary blood flow. Prog Cardiovasc Dis 46: 349–373.[CrossRef][Medline]
Ledoux J, Werner ME, Brayden JE, and Nelson MT (2006) Calcium-activated potassium channels and the regulation of vascular tone. Physiology (Bethesda) 21: 69–78.
Lin X, Hume RI, and Nuttall AL (1995) Dihydropyridines and verapamil inhibit voltage-dependent K+ current in isolated outer hair cells of the guinea pig. Hear Res 88: 36–46.[CrossRef][Medline]
McCarthy RT and TanPiengco PE (1992) Multiple types of high-threshold calcium channels in rabbit sensory neurons: high-affinity block of neuronal L-type by nimodipine. J Neurosci 12: 2225–2234.[Abstract]
Nilius B and Droogmans G (2001) Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415–1459.
Si H, Heyken WT, Wolfle SE, Tysiac M, Schubert R, Grgic I, Vilianovich L, Giebing G, Maier T, Gross V, et al. (2006) Impaired endothelium-derived hyperpolarizing factor-mediated dilations and increased blood pressure in mice deficient of the intermediate-conductance Ca2+-activated K+ channel. Circ Res 99: 537–544.
Van Renterghem C, Vigne P, and Frelin C (1995) A charybdotoxin-sensitive, Ca(2+)-activated K+ channel with inward rectifying properties in brain microvascular endothelial cells: properties and activation by endothelins. J Neurochem 65: 1274–1281.[Medline]
Yokoo H, Shiraishi S, Kobayashi H, Yanagita T, Yamamoto R, and Wada A (1998) Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells. Naunyn-Schmiedeberg's Arch Pharmacol 357: 526–531.[CrossRef][Medline]
Yuan XJ, Tod ML, Rubin LJ, and Blaustein MP (1995) Inhibition of cytochrome P-450 reduces voltage-gated K+ currents in pulmonary arterial myocytes. Am J Physiol 268: C259–C270.
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