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© 1999 International Anesthesia Research Society
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Abstract |
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The aim of this study was to test the hypothesis that halothane interferes with the myogenic response to an increase in intraluminal pressure. Myogenic responsiveness refers to the intrinsic property of vascular smooth muscle to dilate and then constrict in response to an increase in intraluminal pressure, in an attempt to maintain vessel diameter. Vessel segments taken from the rabbit central ear artery were cannulated, pressurized to 60 mm Hg, and perfused with and suspended in Krebs solution. After exposure to extraluminal l-norepinephrine, vessels contracted to an initial diameter (Di) and were subjected to intraluminal pressure increases to 100 mm Hg. Myogenic reactivity was assessed by measurement of the extent of dilatation after the pressure increase from Di to a maximal diameter (Dm) and then the constriction and recovery (against the pressure increase) to a final (Df) diameter. Myogenicity was further assessed by determining the rate of return of the vessel diameter (angle of recovery) and vessel recovery (defined as Dm - Df/Dm - Di) and expressed as a percentage. Myogenicity was determined before and after exposure to halothane in concentrations of between 1–5%. Halothane significantly attenuated the myogenic response at all concentrations studied. The effect of halothane was maximal at a concentration of 5% where there was virtual abolition of the myogenic response with recovery assessed at 6 ± 2.7% (SEM), compared with control (98 ± 2.5%, P < 0.05). The angle of recovery was likewise attenuated. These data suggest that halothane, in a dose-dependent manner, attenuates myogenicity in the isolated rabbit ear artery preparation.
Implications: Blood pressure is controlled partially by the myogenic response. This refers to the capacity of arteries to dilate and then constrict in response to pressure increase. Using arteries from rabbits, we have shown that administration of halothane reduces or abolishes this response. This observation may be a contributing factor to hypotension caused by halothane.
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Introduction |
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The myogenic response of vascular smooth muscle is one of the fundamental control mechanisms involved in cardiovascular homeostasis. The term "myogenicity" refers to the intrinsic property of smooth muscle, notably that of small arteries and arterioles, to dilate and then constrict in response to a rise in intraluminal pressure, in an attempt to maintain vessel diameter in the face of increased distending pressure. This response has been studied for almost 100 years. Although the exact mechanism remains unclear, a more recent study has shown that certain pharmacological agents can attenuate myogenicity (1). The aim of this series of experiments was to investigate the effects of the volatile anesthetic halothane on myogenicity using the model of the pressurized perfused rabbit ear artery.
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Methods |
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The University of Newcastle Animal Care and Ethics Committee granted approval for the study. Nine New Zealand semi–lop-eared rabbits (1.5–2.5 kg) of either sex, and aged between 6–8 mo, were used. The marginal ear vein was cannulated with a 22-gauge catheter and the animal anesthetized with IV pentobarbitone (20–30 mg/kg). Skin and superficial tissues at the base of the ear were carefully dissected clear, and the central ear vein identified, ligated, and then divided to expose the central ear artery lying inferior to it. A proximal segment of this artery, measuring 1–3 cm, was removed, thus avoiding direct contact with the vessel during dissection. Vessels were used within 3 h after storage between 2°–6°C in Krebs solution of the following composition (in mM): NaCl, 118; KCl, 4.69; NaHCO3, 25; CaCl2, 2.52; MgSO4.7H2O, 1.05; dextrose, 5.55; and EDTA, 0.026.
The apparatus used (Fig. 1) was initially developed and described by Speden (2) and Speden and Warren (3), and has undergone several modifications. It is designed to permit measurement of the external diameter of an isolated segment of vascular tissue that is both pressurized and perfused at predetermined levels. This is achieved by the incorporation of an infusion pump, governed by a timing generator (Dick Smith Electronics, Newcastle, Australia), capable of maintaining near-constant intraluminal pressure during changes in vessel diameter.
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Arterial segments were transferred to a 4.2-ml prefilled organ bath, and using a Bausch and Lomb Dissecting Microscope, the proximal end was mounted, tied onto a 19-gauge blunted cannula, flushed out, and filled with Krebs solution before the distal end of the vessel was likewise cannulated. Flushing and filling pressures at this stage were kept below 60 mm Hg. The Krebs solution used as both the intraluminal perfusate and the organ bath exchange solution was drawn from a common reservoir, which was bubbled with a gas mixture (95% O2:5% CO2) at a flow rate of 2 L/min throughout via a CIG Model TM41 anesthetic machine. The intraluminal pressure was then briefly increased to 150 mm Hg to test for leaks, and the distance between the cannulae was adjusted to remove bowing, such that the final length of the artery approximated that in vivo.
The intraluminal perfusion pressure was recorded using a precalibrated pressure transducer (Statham, P23 Db) mounted between the artery segment and the variable outflow resistor. A cover slip was placed across the bath well to remove the meniscus of the bath solution. The outflow resistor was adjusted to maintain a pressure of 60 mm Hg, and intraluminal flow set at 1 mL/min.
Generally, the distance between the cannulae tips was between 8–12 mm and the diameter of the pressurized perfused vessel segments was in the range of 1150–1400 µm. Vessels were allowed to equilibrate under these conditions for at least 30 min. The organ bath fluid was replaced by means of Watson-Marlow 101 U variable speed pump capable of flow rates of between 3 and 4 mL/min.
Temperature was maintained within the limits of 32°–33°C by the use of circulating water and monitored by a digital thermometer (Analog Devices 2036-T-4112) using a thermocouple mounted within the bath.
The artery was illuminated from below with an Olympus LSE light source directed through a red filter and the external diameter of the vessel calculated from the width of the shadow cast by the tissue on a video angiometer, which consisted of a linear array of 1024 photodiodes (Reticon RL1024G) mounted at right angles to the long axis of the vessel. This photodiode array was scanned every 10 ms and the resulting signal processed through circuitry designed by Speden and Warren (4), similar to that described by Sakaguchi et al (5). The signal produced was monitored on an oscilloscope and recorded to the nearest 5 µm on a chart recorder.
After equilibration, vessels were activated by constriction from their resting diameter by the infusion of l-norepinephrine solution added to the extraluminal bath. The final l-norepinephrine bath concentration was in the range of 4.5–6.75 x 10-7 M, and was adjusted to achieve a constricted diameter equivalent to approximately half that of the resting diameter.
Pressure increases were then produced using the Timing Generator coupled to the infusion pump. Pressure increases were made from 60 to 100 mm Hg over 500 ms and held for 120 s. This pressure range was selected because it has been shown (6) that average systolic/diastolic pressures in conscious rabbits are approximately 94/66; therefore, pressures in this range simulate those found in vivo.
Between five and eight repetitions of intraluminal pressure increases were performed on each constricted vessel to determine myogenic characteristics under control conditions. Halothane (Fluothane, ICI Pharmaceuticals, Victoria, Australia), in concentrations of 1%–5%, was then added to the perfusing gas mixture. After 10 min of continuous perfusion at the chosen concentration, the protocol of pressure increases was repeated. Halothane was then added to the perfusing gas mixture via an Ohmeda Fluotec Mark 3 Vaporizer set at 1%–5%. The vaporizer was precalibrated against a Spacelabs Model 90518 Agent monitor at a fresh gas flow of 2 L/min to determine the delivered halothane concentrations. The calibration curve showed excellent linear correlation between vaporizer settings and delivered volatile anesthetic concentrations across the range 1–5% (r2 = 0.989; P < 0.001). A 20-min wash-out period was incorporated into the experimental protocol where vessels were exposed to more than one concentration of halothane, and all experiments were completed within 3 h. At the conclusion of the experimental protocols, vessels were retested to ensure myogenic responsiveness remained intact.
To assess the degree of myogenicity, several variables were determined for each response. Initial diameter (Di) represents the baseline diameter after the addition of vasoconstrictor.
Maximal diameter (Dm) represents the Dm attained after application of the pressure jumps. Maximal-Initial difference (MI) is the amount of initial distention that the vessel undergoes as a result of the pressure increase (equal to Dm - Di). In vessels subjected to pressure jumps, after the initial distention, vessels constrict against the pressure increase to a new final diameter (Df), which is usually, although not always, slightly more than Di.
An examination of the typical myogenic response in Fig. 2 shows that after Dm has been reached, the rate of return of the vessel toward Df is relatively rapid. The angle of this initial rate of return can be measured and is referred to as the angle of recovery, measured in degrees. Percent recovery is another variable used to describe myogenic behavior. It is calculated as: 
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Statistical analysis was undertaken using repeated-measures analysis of variance followed by Dunnett’s test (7). Throughout the text, data are shown as mean ± SEM, and differences at P < 0.05 were considered significant.
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Results |
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In total, 11 vessels were used in this study, and a pooled summary of the results is shown in Fig. 3a–f.
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After l-norepinephrine activation, control vessels constricted to a diameter of 842 (±12.3) µm (Fig. 3a), and in response to the 40 mm Hg pressure increase, dilated by almost 100 µm to 945 (±12.7) µm (Fig. 3b). However, return to the initial diameter was rapid, with a steep angle of return of 10.5 (±0.84) degrees (Fig. 3e), and a recovery of 98 (±2.5%) (Fig. 3f). After the addition of halothane, initial vessel diameters remained essentially unchanged from control (Fig. 3a). In response to pressure increases, vessels tended to dilate more than control, but this reached statistical significance only at the two highest concentrations of 4 and 5% (Fig. 3b). During the recovery phase, halothane markedly attenuated the myogenic response at all concentrations, and indeed at the higher concentration of 5% the response was virtually abolished, with vessels showing an initial angle of recovery of 73 (±4.3) degrees (control: 10.5 ± 0.84 degrees), representing a diminished rate of muscle contraction (Fig. 3e). The principal measurement of myogenicity, vessel recovery (Fig. 3f), was attenuated in a concentration-dependent manner, with recovery diminishing from 98 (±2.5%) in control vessels, to just 6 (±2.7%) at the 5% halothane concentration. The reduction in recovery was statistically significant (P < 0.05) at all concentrations.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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These experiments demonstrate that halothane has little effect on resting tone in the isolated rabbit ear artery, but increases compliance and attenuates the myogenic response in a dose-related manner. Since the myogenic response is a fundamental, homeostatic mechanism for controlling blood flow to organ beds (8), it follows that if this process were disrupted by an increasing concentration of halothane, the perfusion of the rabbit ear vascular bed would become progressively pressure-dependent.
There have been few investigations of the effects of drugs, other than calcium channel antagonists, on vascular myogenicity, although using the methodology described in this study, it has previously been demonstrated that the IV induction anesthetic propofol is a powerful attenuator of myogenicity (1). A study by Park et al. (9) examined the effects of the volatile anesthetics halothane and isoflurane on myogenicity using the rat coronary artery. In these experiments, external diameter measurements of cannulated epicardial vessels (range 75–120 µm) were made while being subjected to pressure increments of 10 mm Hg from 10 to 120 mm Hg. The protocol was then repeated after exposure to isoflurane (1–3%) and halothane (1–2%). It was determined that isoflurane had no effect on coronary myogenicity. In contrast, in the presence of halothane, the vessel behaved passively to pressure increases, without evidence of myogenic constriction. The latter response was concentration independent, and the authors therefore suggested the inhibition was noncompetitive.
In contrast to those findings, we found that the myogenic response of the rabbit ear artery preparation was sensitive to halothane at all concentrations, and that attenuation of the response in the measured variables occurred in a concentration-dependent manner.
Comparison of these data with those of Park et al. (9) highlight the difficulties of research in this area. It is now apparent that myogenic responses can differ between animal species (10) and among vessel types within the same species (11–13). An added complexity is that myogenicity is only one mechanism controlling organ blood flow in vivo. For example, in the unanesthetized rabbit, the ear is an important thermoregulatory organ and control of ear blood flow depends on a complex integration of local and reflex neural factors (14,15). It would not be surprising therefore that there may be differences between myogenic mechanisms in this thermoregulatory vascular bed and other perfusion areas such as the coronary circulation. Such factors must be considered when comparing the effects of anesthetics on vascular beds with differing physiological functions.
Despite intensive research, the mechanisms underlying myogenicity have not been fully determined, although there are currently at least four broad hypotheses (10). Of these, McCarron et al. (8) suggested two dominant proposals. The first is that stretch increases cytosolic calcium concentration by depolarization, and thereby increases opening of voltage-dependent calcium channels. Recent experiments by this group support this theory. The myogenic responses in rat posterior cerebral arteries with external diameters of approximately 120 µm were examined. It was ascertained that the vasoconstriction observed in these vessels was dependent on calcium entry through voltage-sensitive calcium channels and could be abolished by dihydropyridine antagonists. The second alternative hypothesis is that myogenic contraction relies upon stretch-induced activation of protein kinase C (PKC), resulting in an increased calcium sensitivity, without the requirement for an increase in intracellular calcium. Experimental evidence supporting a role for the voltage-gated calcium channel in the response have also been reported by Bulow (16) using small femoral artery branches of the rat and Takenada et al. (17) on afferent renal vessels of the same animal.
However, other ion channels have also been shown to be implicated. Wesselman et al. (18) used cannulated rat mesenteric artery segments and measured both diameter and membrane potential. They found that myogenic responsiveness and pressure-induced depolarization were inhibited by charybdotoxin, used to block K+-Ca2+ channels, suggesting that these too might be involved in mediating the response.
In support of the alternative hypothesis, Liu et al. (19) examined myogenicity in exteriorized first-order rat cremaster arterioles, and while confirming that myogenicity was attenuated by blockade of voltage-dependent calcium channels, also determined a role for PKC. Activators of PKC, such as indolactam, increased vascular tone and induced myogenicity, while the PKC inhibitors staurosporine and calphostin significantly attenuated the response.
From the current experiments, it is not possible to determine the mechanism for the observed myogenic attenuation by halothane, although it has been reported (20,21) that halothane does affect the activity of PKC, at least in skeletal muscle preparations. The underlying mechanisms require further investigation.
In summary, we have demonstrated that, in the isolated perfused central ear artery of the rabbit, halothane—in concentrations used in clinical practice—has a consistent effect on variables of myogenicity, which is most marked at the higher concentrations of 4%–5%, where the response undergoes significant attenuation. These data may help explain the decrease in blood pressure associated with halothane use in vivo.
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Acknowledgments |
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This work was supported by a research grant from the Australian and New Zealand College of Anaesthetists.
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References |
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MacPherson RD, Rasiah RL, McLeod LJ. Propofol attenuates the myogenic response of vascular smooth muscle. Anesth Analg 1993;76:822–9.
[Abstract/Free Full Text] - Speden RN. Active reactions of the rabbit ear artery to distension. (Lond) 1984;351:631–43.
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Speden RN, Warren DM. Myogenic adaptation of rabbit ear arteries to pulsatile internal pressures. J Physiol (Lond) 1987;391:313–23.
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Speden RN, Warren DM. The interaction between noradrenaline activation and distension activation of the rabbit ear artery. J Physiol (Lond) 1986;375:283–302.
[Abstract/Free Full Text] - Sakaguchi M, Ohhashi T, Azuma T. A photoelectric diameter gauge utilizing the image sensor. Pflugers Arch 1979;378:263–8.[Web of Science][Medline]
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McCarron JG, Crichton CA, Langton PD, et al. Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries. J Physiol (Lond) 1997;498 (Pt 2):371–9.
[Abstract/Free Full Text] - Park KW, Dai HB, Lowenstein E, Sellke FW. Steady-state myogenic response of rat coronary microvessels is preserved by isoflurane but not by halothane. Anesth Analg 1996;82:969–74.[Abstract]
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Sun D, Messina EJ, Kaley G, Koller A. Characteristics and origin of myogenic response in isolated mesenteric arterioles. Am J Physiol 1992;263 (5 Pt 2):H1486–91.
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- White SW, Traugott FM, Quail AW. Central nervous system 5-hydroxytryptamine and noradrenaline specificity of ear vascular and ventilation reflexes in thermoregulating rabbits. J Auton Nerv Syst 1985;12:131–44.[Web of Science][Medline]
- Bulow A. Differentiated effects of vasodilators on myogenic reactivity during partial inhibition of myogenic tone in pressurized skeletal muscle small arteries of the rat. Acta Physiol Scand 1996;157:419–26.[Web of Science][Medline]
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Wesselman JP, Schubert R, VanBavel ED, et al. KCa-channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries. Am J Physiol 1997;272 (5 Pt 2):H2241–9.
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Liu J, Hill MA, Meininger GA. Mechanisms of myogenic enhancement by norepinephrine. Am J Physiol 1994;266 (2 Pt 2):H440–6.
[Abstract/Free Full Text] - Tsuchiya M, Okimasu E, Ueda W, et al. Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils. FEBS Lett 1988;242:101–5.[Web of Science][Medline]
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Firestone S, Firestone LL, Ferguson C, Blanck D. Staurosporine, a protein kinase C inhibitor, decreases the general anesthetic requirement in Rana pipiens tadpoles. Analg 1993;77:1026.
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