Anesth Analg 2006;102:799-805
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000195582.22822.e7
ANESTHETIC PHARMACOLOGY
Chromosomal Substitution-Dependent Differences in Cardiovascular Responses to Sodium Pentobarbital
Thomas A. Stekiel, MD,
Stephen J. Contney, MS,
Zeljko J. Bosnjak, PhD,
John P. Kampine, MD, PhD,
Richard J. Roman, PhD, and
William J. Stekiel, PhD
Departments of Anesthesiology and Physiology, Medical College of Wisconsin, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin
Address correspondence and reprint requests to Thomas A. Stekiel, MD, Anesthesia Research, M4280, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. Address e-mail to tstekiel{at}mcw.edu.
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Abstract
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In this study we addressed initial laboratory observations of enhanced cardiovascular sensitivity to sodium pentobarbital (PTB) in normotensive Dahl Salt Sensitive rats (SS) compared to Brown Norway (BN) rats. We also used unique consomic (chromosomal substitution) strains to confirm preliminary observations that such differences were related to chromosome 13. Increasing concentrations of PTB were administered sequentially to SS, BN, and SS strains with BN chromosomal substitutions until the point of cardiovascular collapse. Both spontaneous and controlled ventilation were studied. The effect of large (450 µg/mL) and small (35 µg/mL) concentrations of PTB on in situ transmembrane potential of mesenteric arterial vascular smooth muscle (VSM) cells was also measured in these animals with local sympathetic innervation both intact and eliminated. An analysis of variance was used to identify significant differences among groups. Despite virtually identical plasma clearance of PTB, cardiovascular collapse occurred at approximately 35%–45% smaller cumulative doses of administered PTB in SS and other strains compared with BN and SS.13BN (introgression of BN chromosome 13 into an SS) in both spontaneous and controlled ventilation. In neurally intact preparations, large dose PTB-induced VSM hyperpolarization was 4–5 times greater than the small dose in SS and SS.16BN but not in BN and SS.13BN strains. Denervation eliminated this strain difference. These results suggest that enhanced cardiovascular sensitivity to PTB in SS rats is related to greater hyperpolarization of VSM transmembrane potential in resistance vessels and this effect is associated with chromosome 13.
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Introduction
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In previous studies of the mechanisms by which anesthetics can perturb peripheral vascular regulation, we have observed, incidentally, that the Dahl Salt Sensitive rat strain exhibits noticeably greater analgesic, cardiovascular, and respiratory sensitivity to anesthetics when compared with other rat strains. Pilot studies have indicated that genetically inbred (normotensive) Dahl Salt Sensitive rats (SS/JrHsdMcwi strain, abbreviated as SS) exhibit an enhanced cardiovascular sensitivity to sodium pentobarbital (PTB) and other parenteral anesthetics when compared with both normotensive Brown Norway rats (BN/NhsdMcwi strain, abbreviated as BN) and several single chromosome-substituted (consomic) strains derived from SS and BN parental groups (1). Our preliminary observations also suggest that the substitution of BN chromosome 13 into an SS (SS.13BN consomic strain) eliminates this enhanced sensitivity. The objective of the present study was to further clarify mechanisms underlying the enhanced cardiovascular sensitivity to PTB in normotensive SS compared with control BN rats and to begin to correlate possible mechanisms with individual chromosomes (as a first step toward studies that could eventually map the genetic basis for anesthetic sensitivity). The hypothesis for this study was that PTB produces a greater vascular smooth muscle (VSM) hyperpolarization coupled to a reduction in peripheral vascular tone. Such reduction, in turn, contributes to an enhanced hypotension and resulting circulatory instability in SS compared with other strains. In addition, genetic alterations involving the substitution of chromosome 13 contribute significantly to these phenotypic differences in circulatory control.
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Methods
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The Animal Care and Use Committee at the Medical College of Wisconsin approved all protocols in this study. The terms SS.13BN and SS.16BN (below) refer to consomic rat strains in which a homozygous substitution of BN chromosome 13 or 16 (respectively) was made into an otherwise unchanged SS genetic background. These strains are a part of a larger panel of consomics in which a sequential (one at a time) chromosomal substitution was made in renewable and reliably reproducible animal strains (1). The animals were 9- to 12-wk-old males maintained on a low (0.4%) NaCl diet; SS animals remain normotensive on such a low-salt diet. Mean ± sd weights were 382 ± 40 g, 305 ± 57 g, 369 ± 45 g, and 367 ± 48 g for SS, BN, SS.13BN, and SS.16BN, respectively.
Determination of Cardiovascular Sensitivity to PTB
In this protocol, 20 BN, 21 SS, 20 SS.13BN, and 20 SS.16BN rats were anesthetized with halothane, isoflurane, or sevoflurane for tracheotomy and placement of femoral arterial and venous cannulae (2). An approximately equal number of each strain of animals in the present study received each volatile anesthetic as part of a larger group of animals in which differences in MAC for each animal strain were determined. Results of the latter study have been reported previously (3). After the above described surgical procedures, elimination of the volatile anesthetic occurred within 10–15 min (verified as 0% on an end-tidal anesthetic monitor). At that time, PTB was administered in 2- to 4-mg sequential boluses each separated by a 3-min equilibration period until the point of cardiovascular collapse. This end-point was defined as the time at which CO2 no longer was detectable on the end-tidal medical gas analyzer (i.e., cessation of all effective cardiac pumping). Each strain of animals was divided into two groups, viz. spontaneous and controlled ventilation. Within each strain (group), the animals were simply assigned to spontaneous or controlled ventilation in alternating fashion.
Comparison of Time Course for Elimination of Systemically Administered PTB among SS, BN, SS.13BN and SS.16BN Strains
In this protocol anesthesia was induced and maintained with 1% isoflurane in 6 animals from each of the SS, BN, SS.13BN, and SS.16BN strains. After taking a control blood sample (0 mg/kg PTB), 25 mg/kg of PTB was administered IV to each animal. Subsequent samples were taken at the following time points: 3, 20, 40, 60, 80, 100, and 120 min. Each sample volume was 0.7 mL. Previously described extraction and high-pressure liquid chromatography techniques (4) were used to determine sodium pentobarbital concentrations.
Comparison of VSM Contractile State between SS, BN, SS.13BN 3 and SS.16BN Strains
In this protocol comparative measurements of in situ small mesenteric artery VSM transmembrane potentials (VSM Em) were used as a variable to indirectly assess the contractile state of the VSM in 20 BN, SS, SS.13BN, and SS.16BN animals, respectively. Animal preparation was similar to that described above for the studies assessing cardiovascular sensitivity to PTB, except that 1% inhaled isoflurane anesthesia was maintained throughout the experiment. In addition, a loop of terminal ileum with its attached mesentery was externalized through a midline laparotomy and superfused with temperature-regulated physiological salt solution (PSS). For half of the studies, the vessel preparations were denervated using a 20-min local superfusion with 300 µg/mL of 6-hydroxydopamine. The assignment to either the intact or denervated group was made in alternating fashion for each animal studied in each strain. Single cell VSM Em values were measured in situ as described in detail previously (2). In each of the four animal strains studied (with either intact or denervated mesenteric artery), in situ VSM Em values were measured sequentially before, during and after local superfusion of the vessel preparation with PSS containing either 35 µg/mL or 450 µg/mL PTB. The order in which the PTB doses were administered was randomized.
Additional Cardiovascular Sensitivity Studies
The development of the consomic panel has been a continuing process. During the initial phases of this study, the SS.13BN and the SS.16BN strains were approximately 95% consomic. Thus, in addition to BN chromosome 13 or 16, there were some other segments of BN sequences contained elsewhere within the genomes of these 2 rat strains. This is relevant, as it was assumed that the elimination of the increased cardiovascular sensitivity to PTB in the SS.13BN animals was exclusively related to the substitution of BN chromosome 13. However, genes contained within the other BN chromosomal segments could have been involved as well. Therefore, to validate our results, the cardiovascular collapse studies were repeated in a second series of SS, BN, SS.13BN, and SS.16BN animals (n = 3 for each strain) after these strains were made completely consomic. Three animals from each of 3 other completely consomic strains (SS.8BN, SS.9BN, and SS.20BN) were also included in this study to further establish that the increased cardiovascular anesthetic sensitivity in SS is related to chromosome 13 (and not other chromosomes). In contrast to the initial cardiovascular sensitivity protocol described above (in which both spontaneous and controlled ventilation were studied), only controlled ventilation was used in these additional cardiovascular sensitivity studies.
Anesthetics and Equipment Used
All animals breathed an oxygen concentration of 30% (in an O2-N2 mixture) during each of the experimental protocols to reduce any possibility of hypoxia-induced effects on the measured responses. Ventilation was controlled using a rodent respirator (Model 680; Harvard Apparatus, South Natick, MA) that maintained end-tidal CO2 between 35 and 40 mm Hg. This level corresponded to the level measured in the spontaneously ventilating animals. End-tidal CO2 and anesthetic concentration were measured with a POET 2 infrared pantograph and end-tidal anesthetic monitor (Criticare Systems, Inc. Waukesha, WI). The Beckman Coulter HPLC system used to measure blood concentrations of PTB consisted of a LC-126 SMD pump, a 508 Autosampler, a Gold 168 Fluorescent Detector, and 32 Karat Software (Beckman Coulter, Fullerton, CA). Isoflurane (Abbott Laboratories, North Chicago, IL) was delivered via an Ohio Medical Products vaporizer (Airco Inc., Madison, WI). Halothane (Halocarbon Laboratories, River Edge, NJ) and sevoflurane (Abbott Laboratories) were delivered using a Dräger "Vapor" Model halothane vaporizer and a Dräger Vapor Model 19.1 sevoflurane vaporizer, respectively (Drägerwork Company, Lübeck Germany). PTB was obtained from Sigma Chemical CO (St. Louis, MO).
Data Analysis
The animals studied in each of the protocols were bred as needed and used as they became available. Hence, the order in which the different strains were examined varied randomly based on their availability through natural breeding. A random alternating order was also used for both the "controlled" versus "spontaneous" ventilation studies in the cardiovascular collapse protocol and for the "neurally intact" versus "denervated" studies in the Em protocol.
The variable used for measurement of cardiovascular sensitivity was the cumulative (i.e., total) dose of PTB administered to the point of cardiovascular collapse. The mean cumulative dose (± sd) for each specific rat strain was calculated using results from individual animal preparations. Mean doses for animal strains were compared using an analysis of variance and the Bonferroni/Dunn post hoc test.
The mean ± sd VSM Em value reported for each step in an experimental protocol was calculated from the artery/animal preparations in each strain (SS, BN, SS.13BN, and SS.16BN). An individual VSM Em value for a vessel/animal preparation is the numerical average of at least 5 stable (6–10 s) individual impalements in the VSM medial layer of a vessel. The mean ± sd VSM Em values were compared using an analysis of variance for repeated measures and the "least square means" calculation. The control measurements, which preceded and followed both the large and the small concentrations of PTB, were combined to produce a single "pooled control." Also, the membrane potential changes (
VSM Em) between control Em and Em during large or small PTB doses) were compared using a standard analysis of variance and the Bonferroni/Dunn post hoc test.
For the protocols used to determine clearance of PTB from the circulation, the mean ± sd concentration in blood was measured at each selected sample time for each animal strain (SS, BN, SS.13BN, and SS.16BN). An analysis of variance was used to compare differences among means of the animal strains. Differences among mean values were considered significant at P 
0.05.
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Results
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All animals tolerated larger concentrations of PTB when ventilation was controlled compared with spontaneous ventilation. However, under both modes of ventilation, the administered cumulative dose required to produce cardiovascular collapse was significantly smaller in SS and SS.16BN animals compared with BN or SS.13BN animals (Fig. 1a). Regardless of the mode of ventilation, there was no difference in the dose required for cardiovascular collapse between SS and SS.16BN or between BN and SS.13BN. When repeated in the animals that had been verified as "completely consomic," results were identical. In addition, the doses required for cardiovascular collapse in the SS.8BN, SS.9BN and SS.20BN strains were identical to those in the SS and SS.16BN animals (Fig. 1b).
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Figure 1. A, Greater sensitivity of salt sensitive (SS) and SS.16BN versus Brown Norway (BN) and SS.13BN rats to IV administration of pentobarbital. Data shown are means ± sd of plasma pentobarbital concentrations that produced cardiovascular collapse under conditions of spontaneous and controlled ventilation. *significant difference versus BN and SS.13BN, n = 11 for spontaneous ventilation SS and 10 for all others. B, Illustration of similarity of cardiovascular sensitivity to IV pentobarbital in respective partial or completely consomic animal strains. Columns labeled BN, SS, SS.13BN, and SS.16BN are measurements illustrated in Figure 1A; n = 10 for each. Repeat BN, SS, SS.13BN, and SS.16BN columns represent repeated measurements made in additional animals that were verified to be completely consomic; n = 3 for each. Additional cardiovascular sensitivities (measured in SS.8BN, SS.9BN, and SS.20BN, n = 3 each) are virtually identical to SS and SS.16BN and are significantly different from BN and SS.13BN. Each column represents mean ± sd plasma pentobarbital concentration that produced cardiovascular collapse under conditions of controlled ventilation. *significant difference versus BN, repeat BN, SS.13BN, and repeat SS.13BN.
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In vessels with intact innervation, both the small (35 µg/mL) and large (450 µg/mL) concentrations of PTB in PSS superfusate hyperpolarized the small mesenteric artery VSM (Fig. 2a). The small concentration produced a similar hyperpolarization in all four animal strains. However, the large concentration produced a fourfold to fivefold significantly greater hyperpolarization in SS and SS.16BN compared to BN or SS.13BN strains (Fig. 2b).
In locally denervated vessel preparations, both the small and large concentrations of PTB hyperpolarized the arterial VSM in all four strains (as was observed in the neurally intact vessel preparations) (Fig. 3a). However, in the denervated preparations, the hyperpolarization responses (Em) to large PTB concentrations were significantly more than those to the small PTB concentrations in all four strains (rather than just in SS and SS.16BN). Also, in the denervated preparations both PTB concentrations produced a greater hyperpolarization in SS and SS.16BN compared with BN or SS.13BN (Fig. 3b).
No significant difference was observed among the four animal strains (SS, BN, SS.13BN and SS.16BN) for the peak blood PTB concentration after its bolus injection and for the concentration at each 20-min time point over the subsequent 2-h period required for its elimination (Fig. 4).
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Figure 4. Lack of difference among salt sensitive (SS), Brown Norway (BN), SS.13BN, and SS.16BN in the rate of elimination of pentobarbital from blood after a single 25 mg/kg IV pentobarbital bolus injection; n = 6 for each strain.
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Discussion
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Significant differences in susceptibility to the same concentration of anesthetic at a particular effector site may be the result of differential genetic expression leading to variation in the response of certain regulatory mechanisms to drugs (5). In the present study, the observed smaller blood concentration of PTB required to produce cardiovascular collapse in the SS relative to the BN animals clearly demonstrates that this type of difference exists between the SS and the BN parental strains. In addition, the results in the consomic strains suggest this differential genetic expression is somehow associated with chromosome 13 (but not chromosome 16) in these animals. The smaller blood concentration of PTB required to produce cardiovascular collapse in the SS compared with the BN rats under conditions of spontaneous versus controlled ventilation also suggests that a strain difference exists in the effect of PTB on mechanisms regulating ventilation. This effect on ventilation also appears to be related to chromosome 13 because the critical cumulative concentration of PTB required for cardiovascular collapse in spontaneously breathing animals was similar for the SS and SS.16BN but smaller than for the SS.13BN and BN strains.
Clearly, the use of PTB (as an anesthetic) and its stepwise titration to the point of cardiovascular collapse in the present study differs substantially from choice of anesthetic and its dosing in the clinical setting. However, the rationale for using this approach was based on the incidental observation (during previous other studies in our laboratory) that SS animals become over-dosed at a smaller concentration of IV (or intraperitoneally) administered PTB than other strains. The results of the present study clearly confirm these observations. In addition, in this study the effects of PTB on hemodynamic variables, such as heart rate and arterial blood pressure, showed considerable variability and no consistent pattern of response to cumulative step increases in PTB concentration among the strains tested. In contrast, the end-point of cardiovascular collapse was distinct, unambiguous, repeatable, and clearly occurred at a lower cumulative concentration in SS and SS.16BN rats compared with BN and SS.13BN.
Sequential doses of PTB accumulate and elimination is determined by metabolism of the drug (6). Therefore, the observation of significant strain differences in cardiovascular collapse in response to PTB raises a major question, namely: are such differences merely attributable to different pharmacokinetic clearances rather than to differences in anesthetic actions on cellular mechanisms affecting cardiovascular control? The lack of a difference in the initial peak blood concentration (after the 25 mg/kg bolus) or at any of the subsequent measurement intervals strongly suggests no significant difference in pharmacokinetic clearance among any of the strains tested. This supports our conclusion that the chromosome 13-related differences in PTB-induced cardiovascular collapse result from a difference in PTB's effect on cellular mechanisms regulating peripheral circulation. Moreover, in the Em studies a greater VSM hyperpolarization was observed for the SS and SS.16BN rats compared with the BN and SS.13BN animals. Furthermore, for these measurements VSM was exposed to the same concentration of PTB in all four groups, thus eliminating any causal effect attributable to pharmacokinetic differences.
The Em results in the present studies indicate that there is a greater propensity for VSM hyperpolarization (and consequent reduction of VSM tone) in response to PTB in the SS and the SS.16BN compared with the BN and the SS.13BN animals. These results clearly correlate with results from the cardiovascular collapse experiments. The results also suggest that the greater hyperpolarization in mesenteric resistance vessels produced by PTB in SS and SS.16BN strains is indicative of an earlier loss of VSM tone that may contribute significantly to the observed enhanced cardiovascular sensitivity (reflected by the earlier cardiovascular collapse in these strains compared with BN and SS.13BN). The smaller PTB-induced hyperpolarization observed in the BN and the SS.13BN compared with the SS and SS.16BN strains agrees with the observation of a reversal of the enhanced cardiovascular sensitivity to PTB in SS strains after introgression of BN chromosome 13 into the SS strains. Such a peripheral mechanism would not exclude the possibility that other factors could be involved in this strain difference as well. These factors could include altered mechanisms regulating ventilation (as discussed above in relation to results from the spontaneous versus controlled ventilation groups) as well as altered mechanisms regulating myocardial function (which remains to be studied). However, arterial blood pressure is a function of two major components, namely cardiac output and peripheral vascular resistance, and the observed strain differences in PTB's effect on the latter (in the current results) are clear. It is also recognized that the VSM Em values are only indirect measurements of force generation in any particular vessel. However, there is extensive support for tight electromechanical coupling of VSM Em and Ca2+-dependent force generation in situ as well as in vitro in small mesenteric capacitance and resistance vessels (7,8).
Collectively, these data suggest that the SS strain consistently demonstrates greater sensitivity to PTB when compared with BN and SS.13BN strains. This sensitivity appears to encompass widely different responses (e.g., cardiovascular and ventilatory). Differential alteration of potassium channel function is a plausible explanation for these consistently observed strain differences. Potassium channels are essential participants in the regulation of cellular functions in a diverse variety of tissues including VSM and central nervous system cells (9,10). Functions of both of these cell types are perturbed by anesthetics (11–13). Such a mechanism could also account for the differential hyperpolarization that was observed in the denervation protocols. In these preparations, the SS and SS.16BN strains were still proportionally more sensitive to PTB than the BN and the SS.13BN. The greater hyperpolarization observed in the denervated preparations compared with the intact preparations suggests that the effect of PTB on intrinsic (possibly potassium channel-related) mechanisms are separate from (and possibly compensated by) sympathetic tone to the VSM. Clearly, verification of the involvement of potassium channels (as suggested) will require comparison of specific potassium channel activities (e.g., from patch clamp measurements among SS, BN, and SS.13BN strains and other consomics).
The repeat studies of differential cardiovascular sensitivity to PTB (in SS, BN, SS.13BN, and SS.16BN strains) were small in number (n = 3). However, they were intended only as screening studies to confirm the association of this response specifically with chromosome 13 because the SS.13BN and SS.16BN animals in the first group were almost, but not completely (approximately 95%), consomic. The fact that all of the consomics in this repeat group were verified to be 100% consomic eliminated any possibility that genetic variability in the SS background could account for the observed difference (unrelated to chromosome 13). Likewise, the screening studies in the other consomics tested (SS.8BN, SS.9BN, and SS.20BN strains) strongly suggest that chromosomes 8, 9, and 20 are not associated with mechanisms involved in this response. The results in these animals were almost identical to those in the SS and the SS.16BN strains and were significantly different from the responses in the BN and the SS.13BN. To determine conclusively whether any of the other chromosomes are involved, it will be necessary to study all of the remaining strains in the consomic panel. However, it is clear that chromosome 13 is involved. We recognize that there are approximately 1500 genes on any one chromosome and localizing a response to an entire chromosome is not specific. However, it is a first step that lends itself to more detailed mapping strategies such as the development (and study) of congenic (partial chromosomal substitution) substrains and gene expression studies (to characterize differences in response to anesthetic drugs) in the future.
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Footnotes
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Supported, in part, by Grant No. GM068725-01A3 from the National Institutes of Health, Bethesda, MD.
Accepted for publication October 6, 2005.
Presented, in part, at the Annual Meetings of the American Society of Anesthesiologists in New Orleans, Louisiana, October 16, 2001 and in Orlando, Florida, October 15, 2002.
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Abstract
Introduction
(450 µg/mL – 0 µg/mL) 
= 0 µg/mL, denervated vessel is significantly more polarized than innervated vessel. 
= both the (35 µg/mL – 0 µg/mL) 
