Mechanism of Differential Cardiovascular Response to Propofol in Dahl Salt-Sensitive, Brown Norway, and Chromosome 13-Substituted Consomic Rat Strains: Role of Large Conductance Ca and Voltage-Activated Potassium Channels

Cardiovascular sensitivity to general anesthetics is highly variable among individuals in both human and animal models, but little is known about the genetic determinants of drug response to anesthetics. Recently, we reported that propofol (2,6-diisopropylphenol) causes circulatory instability in Dahl salt-sensitive SS/JRHsdMcwi (SS) rats but not in Brown Norway BN/ NHsdMcwi (BN) rats and that these effects are related to genes on chromosome 13. Based on the hypothesis that propofol does target mesenteric circulation, we investigated propofol modulation of mesenteric arterial smooth muscle cells (MASMC) in SS and BN rats. The role of chromosome 13 was tested using SS-13/Mcwi and BN-13/Mcwi consomic strains with chromosome 13 substitution. Propofol (5 M) produced a greater in situ hyperpolarization of MASMC membrane potential in SS than BN rats, and this effect was abrogated by iberiotoxin, a voltage-activated potassium (BK) channel blocker. In inside-out patches, the BK channel number, Po, and apparent Ca sensitivity, and propofol sensitivity all were significantly greater in MASMC of SS rats. The density of wholecell BK current was increased by propofol more in SS than BN myocytes. Immunolabeling confirmed higher expression of BK subunit in MASMC of SS rats. Furthermore, the hyperpolarization produced by propofol, the BK channel properties, and propofol sensitivity were modified in MASMC of SS-13/Mcwi and BN-13/Mcwi strains toward the values observed in the background SS and BN strains. We conclude that differential function and expression of BK channels, resulting from genetic variation within chromosome 13, contribute to the enhanced propofol sensitivity in SS and BN-13/Mcwi versus BN and SS-13/Mcwi strains. The sensitivity to general anesthetics is highly variable among strains of rats and individual patients. This is thought to be due in part to genetic differences, but little is known about the pharmacogenetics of the response to anesthetics or the mechanisms involved. We recently identified a major strain difference in the cardiovascular sensitivity to anesthetics whereby Dahl salt-sensitive SS/JRHsdMcwi (SS) rats exhibited a much higher cardiovascular sensitivity to general anesthetic agents than control salt-resistant Brown Norway BN/NHsdMcwi (BN) rats. This was evidenced by the cardiovascular collapse that occurred at lower concentrations of infused pentobarbital (Stekiel et al., 2004, 2006) or propoThis work was supported in part by the National Institutes of Health General Medicine Institute [Grant GM 068725] (to T.A.S.); the National Institutes of Health National Heart, Lung and Blood Institute [Grant HL036279] (to R.J.R.); and the National Institutes of Health National Heart, Lung and Blood Institute [Grant HL082798] (SCOR grant to A. W. Cowley, support for the chromosome 13-substituted consomic rats). Part of this work was previously presented: Stadnicka A, Contney SJ, Bosnjak ZJ, Stekiel WJ, Stekiel TA (2006) Propofol effects on maxiKCa channels in mesenteric artery myocytes of Dahl SS, BN and SS13BN rats. Anesthesiology A1612 and Abstracts2View on CD-ROM; 59th Annual Meeting of the American Society of Anesthesiologists; 2006 October 14–18; Chicago, IL; American Society of Anesthesiologists, Park Ridge, IL. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.154104. ABBREVIATIONS: SS, salt sensitive; BN, Brown Norway; BK, large conductance calcium and voltage-activated potassium channels; RGD, Rat Genome Database; Em, membrane potential; NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2one; IbTX, iberiotoxin; MASMC, mesenteric arterial smooth muscle cell(s); Vh, holding potential(s); PBS, phosphate-buffered saline; IBK, whole-cell BK outward current; Ang II, angiotensin II. 0022-3565/09/3303-727–735 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 330, No. 3 U.S. Government work not protected by U.S. copyright 154104/3506457 JPET 330:727–735, 2009 Printed in U.S.A. 727 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from fol (Stekiel et al., 2007) in SS compared with BN. In follow-up studies, using a panel of consomic strains in which individual chromosomes from the BN rat are introgressed one at time into the homogenous genetic background of the SS rat, and vice versa (Cowley at al., 2001; Roman et al., 2002; Kunert et al., 2006), we determined that introgression of BN chromosome 13 into the genetic background of SS (consomic SS13/Mcwi strain) normalized the propofol sensitivity of SS rats. Conversely, propofol sensitivity of BN rats was elevated to the level typical for SS rats in the consomic BN-13/Mcwi strain in which SS chromosome 13 was introgressed into the genetic background of BN rats (T. A. Stekiel, S. J. Contney, A. Stadnicka, C. Moreno, and R. J. Roman, unpublished data). The major characteristic of the anesthetic-induced cardiovascular collapse is an irreversible drop in the arterial blood pressure, suggesting a significant contribution from the compromised peripheral vascular capacitance and resistance. To this end, general anesthetics such as isoflurane and pentobarbital were reported to cause vasodilation of peripheral resistance vessels, specifically the small mesenteric arteries and veins, in part by hyperpolarizing the membrane potential of vascular smooth muscle cells (Yamazaki et al., 1998, 2002; Kokita et al., 1999; Stekiel et al., 2001; Nagakawa et al., 2003). This effect was attenuated by blockers of the large conductance Ca and voltage-activated potassium (BK) channels (Kokita et al., 1999; Nagakawa et al., 2003). BK channels are the class/superfamily of potassium channels found in the plasma membrane of the vascular smooth muscle cells that are activated by membrane depolarization and/or by local elevation of cytosolic Ca released via ryanodine-sensitive channels on the smooth muscle sarcoplasmic reticulum. Structurally, the arterial smooth muscle BK channel is composed of four pore-forming subunits and four accessory 1 subunits, which function to enhance the channel Ca affinity and voltage sensitivity (Toro et al., 1998). The plasma membrane BK channels exist in complexes with voltage-gated calcium channels, ryanodine receptors, protein kinases, and phosphatases and other signaling proteins (Ghatta et al., 2006; Lu et al., 2006). BK channels contribute to regulation of vascular tone by participating in both vasodilation and vasoconstriction (Nelson et al., 1995; Brenner et al., 2000; Alioua et al., 2002; Toro et al., 2004). The intravenous anesthetic propofol is now commonly used for induction and maintenance of general anesthesia in both outpatient/ambulatory and inpatient surgery because of its highly desirable clinical profile: characteristic rapid onset and emergence and relatively few persistent side effects (White, 2008). However, as is also the case with other general anesthetics, there is great variability in sensitivity to propofol that may lead to circulatory instability among patients and strains of rats (Stekiel et al., 2007). The present study addressed the mechanisms of differential cardiovascular sensitivity to propofol. We hypothesized that circulatory instability induced by propofol is in part due to its effects on mesenteric circulation, in particular, the small mesenteric arteries, which are important contributors to systemic vascular resistance, and involves hyperpolarization of vascular smooth muscle cells via activation of BK channels. We investigated whether variability in sensitivity to propofol could be attributed to genetic differences in the properties, function, and expression of the BK channel. The availability of reciprocal, consomic strains with chromosome 13 substitution (SS-13/Mcwi and BN-13/Mcwi) allowed us to directly test whether chromosome 13 plays a role in differential cardiovascular sensitivity to anesthetics, as a first step to identifying the genes involved in this effect. Materials and Methods Animals. Experiments were performed on four strains of rats. The two parental strains were the Dahl salt-sensitive (SS/JrHsdMcwi, abbreviated as SS, RGD ID 61499) and the Brown Norway (BN/ NHsdMcwi, abbreviated as BN, RDG ID 61498). The two consomic strains with a single homozygous chromosome 13 substitution were SS-13/Mcwi (RDG ID 629523) and BN-13/Mcwi (RDG ID 2303972) strains. In the SS-13/Mcwi strain, chromosome 13 of the BN rat was introgressed onto the homogenous genetic background of the SS rat. In the BN-13/Mcwi strain, the reciprocal substitution was created by introgression of chromosome 13 of the SS rat into the genetic background of the BN rat. Consomic strains were obtained from PhysGen (the Program for Genomic Applications at the Medical College of Wisconsin, Milwaukee, WI) and were derived as described previously (Cowley et al., 2004). From birth, all rats were fed a low-salt diet (0.4% NaCl) to minimize the development of hypertension and associated end-organ damage in SS rats. Age-matched male rats (250–400 g; 8–12 weeks old) were used for experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. In Situ Measurement of Mesenteric Arterial Smooth Muscle Membrane Potential. Rats were anesthetized with 2.5% isoflurane (Forane; Baxter, Deerfield, IL) in 30% O2, 70% N2 carrier administered via an Ohio Medical Products vaporizer (Airco Inc., Madison, WI). The end tidal CO2 (35–40 mm Hg) and isoflurane concentrations were monitored with POET2 infrared capnograph and end tidal agent monitor (Criticare Systems, Inc., Waukesha, WI). Surgical preparation included tracheotomy and cannulation of femoral artery and femoral vein. A midline laparotomy was performed, and a loop of terminal ileum was externalized, placed into a temperature-controlled recording chamber, and superfused with physiologic salt solution containing 119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4, 1.6 mM CaCl2, 24 mM NaHCO3, 1.18 mM NaHPO4, and 0.026 mM EDTA, pH 7.4. Physiologic salt solution was maintained at 37°C and was continuously gassed with 90% N2, 5% O2, 5% CO2. A segment of small mesenteric artery (200 m o.d.) was cleared of perivascular connective tissue and fat without disturbing luminal flow and was stabilized with 50m-o.d. stainless steel pins anchored in the silastic base of the recording chamber. Inhaled isoflurane was adjusted to 1% to maintain anesthesia, and animals were allowed to breathe spontaneously. Membrane potential (Em) was measured in situ from the vessel adventitial side with a borosilicate glass microelectrode (FHC Inc., Bowdoinham, ME) filled with 3 M KCl (tip diameter, 0.1 m; impedance, 40–60 M ). Microelectrodes were inserted manually using a hydraulic micromanipulator (Trent Wells Inc., Coulterville, CA). Data were recorded using a Grass RPS7C polygraph (Astro-Med/Grass Inc., West Warwick, RI) and Superscope II, version 1.44 digital data acquisition system (GW Instruments, Somerville, MA). Recordings were made before, during, and after vessel superfusion with 5 M propofol (International Union of Pure and Applied Chemistry name, 2,6-diisopropylphenol; SigmaAldrich, St. Louis, MO). At least five Em recordings, each of 6 s or greater duration, were averaged to obtain each successive data point under each experimental condition. A similar experimental protocol was followed when investigating the effects of propofol-induced hyperpolarization of Em in the presence of the BK channel blocker iberiotoxin (IbTX; 100 nM) or the BK opener NS1619 (5 M). Isolation of Mesenteric Arterial Myocytes. Secondand thirdorder (200–300m-o.d.) arteries were dissected from the mesentery of 2.5% isoflurane-anesthetized, age-matched SS, BN, and consomic 728 Stadnicka et al. at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from SS-13/Mcwi and BN-13/Mcwi rats. Mesenteric arterial smooth muscle cells (MASMC) were isolated by the procedure described in Kubo et al. (1997). In brief, after removing perivascular fat, small mesenteric arteries were excised and placed for 15 min into an ice-cold buffer composed of 137 mM NaCl, 5.6 mM KCl, 0.42 mM Na2HPO4, 0.44 mM NaH2PO4, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, and 10 mM glucose, pH 7.3 adjusted with NaOH. The tissue was transferred to the digestion buffer supplemented with 0.1 mM CaCl2 and containing 1.5 mg/ml papain, 1.0 mg/ml dithioerythritol, and 0.5 mg/ml bovine serum albumin for 30-min incubation at 35°C. This was followed by 20-min incubation at 35°C in the digestion buffer containing 1.5 mg/ml collagenase type F, 1.0 mg/ml hyaluronidase type I-S, and 0.5 mg/ml bovine serum albumin (all from SigmaAldrich). At the end of the incubation, the tissue was washed in enzyme-free buffer and triturated with a large-bore transfer pipette to release cells. Dispersed cells were kept at 4°C and were used for patch-clamp experiments within 5 h after isolation. Patch-Clamp Experiments. BK channel currents were monitored in the excised inside-out patch and the whole-cell configurations of the patch-clamp technique (Hamill et al., 1981), using an EPC7 patch-clamp amplifier (ALA Scientific Instruments, Westbury, NY), Digidata 1322A interface (Molecular Devices, Sunnyvale, CA), and an IBM computer running pClamp9 software (Molecular Devices). Borosilicate glass (no filament) pipettes (Garner, Clairmount, CA) had resistances of 6 to 9 M and 3 to 5 M for single-channel and whole-cell recordings, respectively. The pipette/extracellular solution for single-channel recordings consisted of 145 mM KCl, 0.5 mM MgCl2, 0.5 mM CaCl2, and 10 mM HEPES, pH 7.4. The bath/intracellular solution at pH 7.3 consisted of 145 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, 2.0 mM EGTA, and CaCl2 added at concentrations yielding 0.1, 1.0, or 10 M free intracellular Ca , as calculated with WinMAXC32 software (C. Patton, Stanford University, Pacific Grove, CA). Single-channel currents were filtered at 500 Hz (eight-pole Bessel filter) and recorded at a 5s sampling interval in a gap-free mode at the membrane potential of 40 mV. The unitary current amplitude, conductance, and Po were determined from all-points histograms using pClamp9 software (Molecular Devices) and Origin7 (OriginLab Corp., Northampton, MA). The number of BK channels per patch, unitary current amplitude, single-channel conductance, and Po were determined from the amplitude histograms. The mean Po was calculated by the binomial distribution [1 (Pc) ], where n is the number of active channels in the patch and Pc is the channel closed probability derived from the Gaussian fits to the amplitude histograms. In single-channel analysis, the detection threshold for opening and closing was set using a half-amplitude criterion. The number of BK channels per patch was estimated under conditions of maximal channel opening at the membrane potential of 80 mV and in the presence of 10 M free intracellular Ca (Nelson et al., 1995; Meera et al., 1996). The extracellular solution for whole-cell outward K current recordings was composed of 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4. The pipette solution for whole-cell recordings at the holding potential (Vh) of 60 mV contained 130 mM K-aspartate, 30 mM KCl, 10 mM NaCl, 1 mM MgCl2, 0.5 mM EGTA, and 10 mM HEPES, pH 7.3. The pipette solution for recordings at Vh of 35 mV was composed of 150 mM KCl, 5 mM NaCl, 1.0 mM MgCl2, 10 mM HEPES, 2 mM EGTA, and CaCl2 that was added from 1 M stock in the amounts (calculated with WinMAXC32; see above) yielding 0.1 and 1 M free intracellular Ca . The pH was 7.2. Whole-cell K currents were evoked by 300-ms voltage steps from Vh of 60 or 35 mV to potentials ranging from 60 to 80 or 60 mV in 20-mV increments. Recordings were filtered at 1 kHz, digitized, and stored for offline analysis. Identity of single BK channels was confirmed by blockade with 100 nM penitrem A (tremorin A, C37H44CINO6) and 1 M paxilline (C27H33NO4). Whole-cell currents were blocked with 1 M paxilline or 300 nM IbTX. All experiments were performed at room temperature (20– 24°C). The 100 mM stock of propofol in dimethyl sulfoxide was diluted in recording buffer. Dimethyl sulfoxide alone had no effect on BK channel activity. All standard chemicals and iberiotoxin, paxilline, penitrem A, and NS1619 were purchased from Sigma-Aldrich. Immunohistochemistry. Small mesenteric arteries were obtained from isoflurane-anesthetized parental SS and BN rats for immunolabeling of BK channel proteins. Freshly dissected vessels were washed in ice-cold 0.1 M phosphate-buffered saline (PBS) and slit open longitudinally. The vessels were fixed for 10 min in suspension in 1% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, and rinsed with PBS. Thereafter, the vessels were incubated for 1 h at 37°C with the rabbit polyclonal primary antibodies for BK channel subunit (anti-KCa 1.1/KCNMA1; Alomone Labs, Jerusalem, Israel; 1:50 dilution in PBS) and 1 subunit (anti-slob1/KCNMB1; Alomone Labs; 1:100 dilution in PBS). Antibodies preincubated with the control antigens were used as negative controls. After incubation with primary antibodies, the vessels were washed three times for 5 min each with PBS, incubated 30 min at 37°C with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen/ Molecular Probes, Carlsbad, CA) at a 1:1000 dilution in PBS, and washed in PBS. Nuclear staining was accomplished with 3-min exposure to 1 M TO-PRO-3 (Invitrogen/Molecular Probes) at room temperature. After a final rinse in PBS, the vessels were mounted on positively charged Colorfrost Plus microscope slides (Thermo Fisher Scientific, Waltham, MA). Immunofluorescence was measured in 10 randomly selected visual fields from each vessel. Images were captured at 400 magnification, under fluorescence confocal laser-scanning microscope (Nikon, Tokyo, Japan) using a krypton/argon laser imaging system. Fluorescence excitation and emission were set at 488 and 520 nm, respectively, for Alexa Fluor 488 and at 633 and 661 nm, respectively, for TO-PRO-3. Data were analyzed with MetaMorph software (Universal Imaging Corporation). Results are expressed as averaged fluorescence intensity. Statistical Analysis. Mean values S.E.M. are presented. Statistical analysis was performed using the analysis of variance with a post hoc Bonferroni/Dunn analysis, and n indicates the number of rats used in each experimental group. Differences were accepted as significant at p 0.05.