Endothelium-independent Vasoconstricting and Vasodilating Actions of Halothane on Rat Mesenteric Resistance Blood Vessels 

The vessel isolation and cannulation methods were similar to those previously described. [33,35] Mesenteric tissue was removed from halothane-anesthetized Long Evans rats (200–250 g) and pinned to the sylgard coating in the base of a water-jacketed dissection dish. Proximal arteries were followed distally, and small distal branches (50–200 micro meter OD) were carefully dissected from surrounding tissue and removed (Figure 1(A)). The vessels were transferred to a specially constructed vessel bath in which the ends of the vessel were cannulated with fire-polished glass pipettes fabricated from 1.2-mm OD borosilicate glass (tip diameters of 30 micro meter) and secured to the cannulae with 11–0 suture (Figure 1(B)).

The perfusate consisted of buffered physiologic saline solution (PSS) of the following composition (mM): NaCl 119; NaHCO³24; KCl 4.7; CaCl²1.6; KH²PO⁴1.18; MgSO⁴1.17;[ethylenedinitrilo]tetraacetic acid (EDTA) 0.025; glucose 5.5; and bubbled with 95% Oxygen²/5% CO²(pH = 7.35).

The perfusion system consisted of a series of glass bath reservoirs with valves to select the reservoir used for perfusion (Figure 1(C)). The perfusate exited through the bottom of the reservoir and was circulated to the vessel bath by a peristaltic pump. The equilibration gas (95% Oxygen²/5% Co²) was directed from parallel calibrated flowmeters and control valves to the bubbler for each reservoir. The reservoirs were tightly sealed at the top with a Teflon plug through which passed the equilibration gas inlet line connected to the glass bubbler, a gas outlet line directed to the gas-tight cover over the vessel bath, and a return line for recirculation of buffer from the vessel bath. Temperature was maintained at 37 degrees Celsius throughout the system by water-jacketing around the bath reservoirs and the vessel bath.

The intravascular system was closed, and transmural pressure was maintained with a pressure servo system (Figure 1(D)). After initially pressurizing the vessel to 30 mmHg and checking for leaks, the vessel bath was transferred to the stage of a Nikon Diaphot inverted microscope, and intravascular pressure was incrementally raised to 60 mmHg. A micrometer attached to one of the cannula was used to gently elongate the vessel to remove any redundancy in the vessel walls, and a gas-tight cover was placed over the vessel bath to provide an isolated environment through which the equilibration gas (95% Oxygen²/5% CO sub 2 plus/minus halothane) was circulated.

After a 30-min stabilization, the integrity of the vascular smooth muscle was tested by a brief application of a maximal dose of phenylephrine (10⁶M), which produced a sustained (tonic) constriction that was uniform along the length of the vessel. “Healthy” vessels responded to phenylephrine with at least a 50% decrease in inside diameter (ID), and vessels that did not respond in this manner were considered damaged and were not studied further. The integrity of the endothelium was tested by applying a maximal dose of acetylcholine (ACh, 10⁶M) to the phenylephrine-containing bath after the phenylephrine constriction had stabilized ([nearly equal] 3–5 min). ACh-induced vasodilation is well known to be endothelium-dependent, [36,37] and complete relaxation of the phenylephrine-induced constriction by ACh was taken as an indication of an intact endothelium. All the vessels that exhibited a healthy response to phenylephrine also met this criterion for an intact endothelium. Endothelial function is characteristically well preserved in this preparation because the endothelial cell surface (i.e., the vascular lumen) is undisturbed during the isolation and cannulation procedures. [33,35,38] To assess the endothelial dependence of the actions of halothane on these vessels, the endothelium was removed mechanically before cannulation in some vessels. This was accomplished by rubbing the intimal surface of the vessel with a 30–50 micro meter stainless steel wire or passing a 50–75-micro meter hair through the vessel lumen as described previously. [38] The endothelium denudation procedure was considered “successful” when less than 5% relaxation of the phenylephrine-induced constriction was observed with application of a supramaximal dose of ACh (10 sup -5 M). In the vessels in which the endothelial denuding procedure was attempted (n = 10), ACh-induced relaxation was successfully removed (greater or equal to 95%) in half of the vessels. In the other five vessels, significant endothelial function persisted or smooth muscle function was impaired, and these vessels were not studied further.

Halothane was added using a calibrated temperature and flow-compensated vaporizer (Ohio Medical) or, for the highest anesthetic concentrations (10–20 vol%), a copper kettle vaporizer. Halothane concentrations in the gas phase and in the perfusate were measured using gas chromatography. Gas and perfusate samples were taken from both the vessel bath and the bath reservoir. Gas samples were injected directly and compared to standard gas mixtures of halothane and air. Perfusate samples were extracted with heptane containing an internal standard (isoflurane) and compared to standard mixtures of halothane and isoflurane. In perfusion studies without vessels, halothane equilibrated rapidly (less or equal to 2 min) with the perfusate in the bath reservoir; both the gas and perfusate halothane concentrations remained stable throughout the perfusion system. The gas and perfusate halothane concentrations measured during dose-response experiments were all within 10% of the values predicted by the vaporizer setting and the published solubility (i.e., the gas: buffer partition coefficient at 37 degrees Celsius). [39] The halothane concentrations reported here (in mM) represent the mean perfusate concentrations measured at each vaporizer setting (n = 8–14).

Stock solutions for each of the drugs tested were mixed fresh on the day of the experiment and added directly to the volume-calibrated bath reservoirs. The high [Potassium sup +](40 mM) bath (i.e., KCl treatment) consisted of PSS with equimolar substitution of KCl for Sodium Chlorine. The low [Calcium²+](250 nM) bath consisted of nominally Calcium²⁴-free PSS with the addition of 2 mM ethylene glycolbis(beta-ami-noethylether) N,N,N',N'-tetraacetic acid and an amount of added Calcium²+(1.44 mM) calculated to bring the free [Calcium²+] to [nearly equal] 250 nM according to the method of Fabiato. [40].

We developed a video image analysis system that permits the ID and OD of the vessel to be continuously monitored and displayed in real-time at video rates (30 samples/s:Figure 1(E)). Images of the vessel are directed to the video port of the inverted microscope equipped with a digital video camera (Dage-MTI model CCD72) interfaced to a programmable video board (Truevision Targa-M8) in an 80386- or 80486-based computer. The algorithm for image analysis to determine ID and OD involved the initial application of user-adjustable “thresholding” to convert the eight-bit gray scale pixel values to either black (below the threshold) or white (above the threshold). These small vessels are translucent; however, when focused on the center of the vessel, the vessel walls on the “raw” image are relatively dense. Thus, when the threshold is properly adjusted, the two optically dense vessel walls on the “analyzed” vessel image (after thresholding) appear as two black stripes aligned vertically (Figure 1(E)). Simple algorithms are used to extract the pixel values corresponding to the inside and outside “edges” of the vessels on each video line, and ID and OD are calculated from precalibrated micrometer/pixel values for the microscope objective being used. The OD and ID values obtained for each video line are averaged, and these averaged OD and ID values are displayed on the computer video display terminal and recorded on computer hard disk data files in real-time. Direct measurements of vessel diameter from recorded images on video tape indicate that the ID and OD measurements made with this system are accurate.

Data files were analyzed using computer playback programs in which maximum, minimum, and average OD and ID values were extracted from user-defined intervals. To determine the vasoconstricting responses to halothane and caffeine, the average diameter of the pretreatment interval was compared to the minimum diameter (i.e., most constricted) in the posttreatment interval. The magnitude of the sustained constrictions produced by KCl and phenylephrine was determined by comparing the averaged diameter before treatment to that obtained during treatment. The marked oscillations in diameter observed during treatment with phenylephrine in endothelial-intact vessels (which occurred at a rate of [nearly equal] 0.2 Hz-vida infra) were effectively filtered out by averaging the diameters over several minutes. The vasodilation produced by halothane was determined by comparing the average diameter obtained during the sustained KCl or phenylephrine constrictions to the average diameter after the addition of halothane. OD changes were used for most analyses. ID changes generally gave identical results except that ID was more difficult to estimate during peak constrictions. Halothane vasoconstriction dose-response relationships were determined by normalizing the responses at each dose to the maximal response in each experiment. For the vasodilating dose-response relationships, the maximal response in each vessel was defined as complete (100%) relaxation of the KCl or phenylephrine constriction. The normalized responses from individual experiments at each dose were averaged and fit using Fig P software (Biosoft, Milltown, NJ) with a standard Hill equation where: response (%)= 100/(1 +(1/(([halothane]/EC⁵⁰) sup -P))). The half-maximal halothane concentrations (EC⁵⁰) and cooperativity values (P) were derived from these fits. STATA software was used for statistical comparisons (Computing Resource Center, Los Angeles, CA). One-way analysis of variance was used for comparing the vasoconstricting responses to halothane, caffeine, and KCl in the normal versus low Calcium²+ bath and before versus after ryanodine treatment. Two-way analysis of variance was used to compare the dose-response data for the-halothane-induced vasodilation with and without ryanodine and in the presence and absence of an intact endothelium. A P value of less or equal 0.05 was accepted as indicating a significant treatment effect. The Bonferonni method was used to correct for multiple comparisons in post hoc testing where appropriate.

In pressurized vessels, application of buffer pre-equilibrated with halothane had a marked vasoconstricting action (Figure 2(A)). The response was concentration-dependent over the range of 0.1–5 mM (0.25–20 vol%) and produced a maximum reduction in internal diameter of 53–76%(n = 8). In most vessels, the vasoconstriction was largely transient, although a small sustained constriction (usually less or equal 10% of peak) often was observed (e.g., Figure 2(D) and Figure 8(A)). The concentration dependence of halothane's peak vasoconstricting action is shown in Figure 2(B). Decreases in OD (plus/minus SEM) at each halothane dose were normalized to the maximum response in each vessel, averaged, and fit with a Hill equation (solid line) yielding an EC⁵⁰of 0.46 mM (1.5 vol% or 1.3 MAC).

Reducing the bath [Calcium²+] from 1.8 to 250 nM had no effect on the vasoconstricting action of halothane (Figure 2(C)) or the transient vasoconstriction produced by caffeine, which releases Calcium²+ from intracellular stores [41,42](Figure 2(C)). In contrast, reducing the bath [Calcium²+] significantly attenuated the vasoconstricting response resulting from raising the bath [Potassium sup +](“KCl”), which is due to extracellular Calcium²+ influx through depolarization-activated sarcolemmal Calcium²+ channels. [43,44] The peak constricting responses to halothane, caffeine, and KCl were reduced by 3.2 plus/minus 4.5%, 4.0 plus/minus 5.1%, and 84.4 plus/minus 7.2%(mean plus/minus SEM, n = 5), respectively, in the low Calcium²+ bath (P < 0.05). Similar results were obtained when Calcium²+ influx with blocked with nifedipine (1 micro Meter) or Cadmium²+(100 micro Meter; data not shown). The vasoconstricting actions of halothane, KCl, and caffeine were not affected by bath additions of the alpha-adrenergic antagonist, phentolamine (1 micro Meter; n = 3), indicating that the vasoconstricting responses to these agonists were not due to sympathetic transmitter from residual nerve endings in the preparation. The vasoconstricting action of halothane thus appeared to be due to a direct action on vascular tissue independent of Calcium²+ influx. The results suggested that halothane, like caffeine, produced constriction by releasing Calcium²+ from intracellular stores.

To further explore the possibility that halothane constrictions resulted from Calcium²+ release from the same intracellular Calcium²+ store(s) as caffeine, we examined the effects of ryanodine—a sarcoplasmic reticulum (SR) Calcium²+ channel agonist that depletes caffeine-sensitive intracellular Calcium sup 2+ stores [41,42]—on halothane constrictions (Figure 2(D)). As shown in Figure 2(D), ryanodine (10 micro Meter) had little effect on the sustained KCl constriction but eliminated the vasoconstricting responses to both caffeine and halothane (P < 0.05, n = 4). As ryanodine is an open channel blocker, which depletes the Calcium²+ store by locking the channel in an open state, [42] caffeine was initially applied to open the channels and thereby deplete the store. Thus, the vasoconstricting response to the initial application of caffeine after ryanodine was largely unaffected (Figure 2(D)), whereas the responses to subsequent application of either caffeine or halothane were blocked completely. We also tested whether halothane, like caffeine, was able to initiate the ryanodine-induced depletion of the store. As with caffeine, the first response to halothane after ryanodine was not blocked, whereas subsequent responses to halothane or caffeine were blocked completely (not shown).

These data indicated that the vasoconstricting effect of halothane was dependent on Calcium²+ release from the caffeine/ryanodine-sensitive intracellular Calcium²+ store. The effect of halothane on the integrity of this store was further examined by applying caffeine during continuous halothane (Figure 3). Halothane produced vasoconstriction, and when caffeine was applied 5 min later during continuous halothane, the magnitude of the caffeine constrictions was blocked in a concentration-dependent manner (Figure 3(A)). The fitted data yielded an IC⁵⁰of 1.62 mM ([nearly equal] 5.4 vol% at 37 degrees Celsius;Figure 3(B)). The reduction in the magnitude of the caffeine constrictions produced by halothane was stable after 5 min of continuous halothane and was not affected by increasing the duration of the halothane application. In addition, repeated caffeine applications 10 min apart in 2.5% halothane (0.74 mM) showed no additional or cumulative inhibition (not shown), suggesting that the caffeine-releasable store(s) was able to refill to the same steady-state level during continuous halothane.

Increasing the bath [Potassium sup +] to 40 mM produced sustained vasoconstriction that is well known to be dependent on Calcium sup 2+ influx through voltage-activated Calcium²+ channels [43,44]. Consistent with this explanation, KCl constrictions in these vessels were dependent on bath [Calcium²+](Figure 2(C)) and blocked by the Calcium²+ channel blockers nifedipine or Cadmium²+(not shown). KCl-induced constrictions were largely unaffected by ryanodine (Figure 2(D)), indicating that Calcium²+ release does not play a major role in the response to KCl. After the sustained KCl constriction had stabilized, halothane (0.1–2.6 mM, [nearly equal] 0.25–10 vol%) was added sequentially to the high [Potassium sup +]-containing bath (Figure 4). Halothane produced vasoconstriction in the KCl-constricted vessels, as had been observed in the unstimulated vessels (Figure 2), but this was followed by concentration-dependent vasodilation (Figure 4(B)). The effect of halothane on KCl-constricted vessels treated with ryanodine was tested to determine the relationship of the vasoconstricting and the vasodilating actions of halothane (Figure 4(C)). As noted in unstimulated vessels, ryanodine eliminated the vasoconstricting effect of halothane (Figure 4(C)). However, ryanodine treatment had no obvious effect on the vasodilating action of halothane.

Phenylephrine (10 sup -6 M) produced sustained vasoconstriction similar to that produced by KCl except that phenylephrine constrictions were characterized by marked oscillations in diameter (Figure 5(A)). Phenylephrine-induced constrictions, like those produced by KCl, were dependent on extracellular Calcium²+ and blocked completely by nifedipine (1 micro Meter; not shown). The effects of halothane on phenylephrine-constricted vessels were similar to those observed in KCl-constricted vessels (Figure 5(B)). Halothane produced initial constriction followed by concentration-dependent vasodilation. Ryanodine did not significantly affect the vasodilating action of halothane (Figure 5(C)) or the magnitude of the phenylephrine constriction (Figure 5(D)).

Fits to the averaged concentration response data indicated that halothane abolished the sustained KCl constriction with an EC⁵⁰of 0.36 mM ([nearly equal] 1.2 vol%) before ryanodine and 0.25 mM ([nearly equal] 0.8 vol% at 37 degrees Celsius) after ryanodine (Figure 6(A)). For the phenylephrine-constricted vessels, fits to the mean concentration response data yielded EC⁵⁰values of 0.75 mM and 0.59 mM ([nearly equal] 2.5 and 1.95 vol% at 37 degrees Celsius) for complete vasodilation of these vessels by halothane before and after ryanodine treatment, respectively (Figure 6(B)). Halothane also attenuated the phenylephrine-induced oscillations in diameter, which were abolished by ryanodine as shown in Figure 5(B and C). We did not systemically study the concentration dependence of this effect, however, halothane abolished these oscillations at relatively low concentrations (less or equal to 0.3 mM, n = 5).

Initial experiments were directed at removal of endothelial function pharmacologically with the endothelium-dependent relaxant factor (EDRF) pathway blockers N-nitro-L-arginine (100 micro Meter), a nitric oxide synthase inhibitor, and/or methylene blue (10 micro Meter), a guanylate cyclase inhibitor. Although these treatments alone or in combination did not substantially affect the vasoconstricting or vasodilating actions of halothane, the EDRF pathway blockers also failed to abolish ACh-induced vasorelaxation (not shown). This observation was similar to that recently reported by other investigators [45] and is consistent with the suggestion that endothelial factors other than EDRF (i.e., nitric oxide) are responsible for endothelium-mediated relaxation in rat mesenteric resistance arteries. The effect of pretreatment with indomethacin (5 micro Meter), a cyclooxygenase inhibitor, on halothane vasoconstriction and vasorelaxation also was studied. Similar to the EDRF pathway inhibitors, indomethacin did not substantially affect the vasoconstricting and vasodilating actions of halothane, nor did it abolish the vasorelaxing effect of ACh (not shown). Because we were therefore unable to pharmacologically remove ACh-induced endothelium-dependent relaxation, which appeared to be multifactorial, we attempted to physically remove the endothelium before cannulation in some vessels to determine whether the vasoconstricting and vasodilating actions of halothane were endothelium-dependent. Physical removal of the endothelium had the added advantage of removing the actions of all endothelial-dependent vasoconstricting and vasodilating factors that might be involved in the actions of halothane. [13,46] Removal of the endothelium was accomplished as described in Methods, and the success of the procedure was based on elimination (greater or equal to 95%) of ACh-induced relaxation (Figure 7). In addition, the vessels had to display a “healthy” vasoconstricting response to phenylephrine (see Methods). Successful removal of the endothelium, however, did result in attenuation or elimination of the oscillations in diameter typically observed during application of phenylephrine (Figure 7).

As shown in Figure 8, removal of the endothelium had little effect on the magnitude of the vasoconstricting and vasodilating actions of halothane. Comparison of the concentration-response data for the vasoconstricting and vasodilating actions of halothane in the endothelial-intact and endothelial-denuded vessels (Figure 8(D-F)) suggested that endothelium potentially had some modulating effect on the concentration response relationship for the vasoconstricting action, shifting the EC⁵⁰to 0.81 mM, compared to 0.48 mM in endothelium-intact vessels (Figure 8(D); P < 0.05). However, there were no significant difference between the endothelial-denuded and the intact vessels at any halothane dose. Endothelial removal had no significant effect on the vasodilating action of halothane in KCl- or phenylephrine-constricted vessels at any halothane dose, with EC⁵⁰values derived from the fits to the mean data of 0.52 and 0.67 mM, respectively, after endothelial removal, compared to 0.36 and 0.75 mM in endothelial-intact vessels (Figure 8(E and F)).

In this study, vasoconstricting and vasodilating effects of halothane on intact mesenteric resistance blood vessels are described. The vessels were studied in vitro, thereby eliminating the potentially confounding effects of halothane on hemodynamics, organ metabolism, or sympathetic output that may secondarily influence vascular tone in vivo. [1–11] In addition, these effects of halothane on isolated vessels were not substantially influenced by physical removal of the endothelium, thereby indicating that they were due to direct actions on vascular smooth muscle. Under conditions of a physiologic temperature and transmural pressure load, the vasoconstricting action of halothane was largely transient and was followed by a vasodilating action in phenylephrine- or KCl-constricted vessels. In contrast to effects previously reported in large conductance arteries, the vasoconstricting and vasodilating actions in resistance vessels occurred at clinically relevant concentrations (i.e., 0.5–2.5 MAC) and were of sufficient magnitude to indicate that they contribute significantly to the cardiovascular effects of halothane in vivo.

Because volatile anesthetics, including halothane, generally are viewed as vasodilators, [12–26] the striking vasoconstricting action of halothane—which was clearly evident at clinically relevant (i.e., anesthetic) concentrations (EC⁵⁰[nearly equal] 1.5 MAC)—was somewhat unexpected. Muldoon et al. previously reported on a constricting effect of halothane in dog femoral artery rings, but that effect was comparatively small and was sustained. [27] Stone and Johns reported on a sustained constricting action of halothane in rat aortic rings, as well as comparable effects of isoflurane and enflurane. [13] However, that effect also was small and, in contrast to the femoral constricting action reported by Muldoon et al., was endothelium-dependent. The constricting action of halothane in that study was only obvious in endothelial intact preparations after treatment with indomethacin. The vasoconstricting action in the mesenteric resistance vessels reported here was largely transient and endothelium-independent and did not depend on indomethacin pretreatment.

The transient vasoconstricting effect of halothane reported by Su and Zhang in KCl-constricted rat aortic rings, [19] although relatively small, was most similar to that which we observed in the mesenteric resistance vessels. In addition, in permeabilized rabbit aortic smooth muscle, they demonstrated that halothane both enhanced caffeine constrictions and potentiated ryanodine-induced block of caffeine responses, suggesting an effect of halothane on Calcium²+ release. [19] Our data are consistent with those findings and more directly demonstrate that halothane induces transient constrictions due to Calcium²+ release from caffeine/ryanodine-sensitive Calcium²+ stores. In the mesenteric vessels, halothane alone produced constrictions as large as those produced by maximal doses of caffeine, and halothane was as effective as caffeine at accelerating ryanodine-induced depletion of the caffeine-sensitive store. After ryanodine treatment, the constricting responses to both caffeine and halothane were eliminated.

Although we did not directly measure intracellular Calcium sup 2+ release by halothane, halothane-induced transient increase in [Ca sup 2+]ⁱhas been demonstrated in cardiac muscle, [47] skeletal muscle, [48] and vascular smooth muscle cells. [49,50] In both cardiac and skeletal muscle SR preparations, halothane induced Calcium sup 2+ efflux that was sensitive to ruthenium red, a specific ryanodine receptor antagonist. [51–53] Thus, the Calcium²+-releasing action may be due to a direct effect on ryanodine-sensitive channels. In addition, however, ruthenium red-insensitive Calcium²+ efflux has been demonstrated in cardiac and skeletal muscle SR, [52–54] and the sensitivity of halothane-induced constrictions or Calcium²+ release to ruthenium red has not been studied. Although the molecular mechanism(s) involved in the halothane-induced constrictions in mesenteric resistance vessels thus remains unclear, the similarities of the halothane and caffeine responses in these vessels—including the sensitivity of both to ryanodine, the insensitivity of both to extracellular Calcium²+ influx, and the ability of both to induce ryanodine block—clearly indicate an effect of halothane that involves the caffeine/ryanodine-sensitive Calcium²+ store that may involve either a direct or an indirect effect(s) on ryanodine-sensitive channels.

The fact that caffeine constrictions were abolished by halothane in a concentration-dependent manner is consistent with a direct effect of halothane on the caffeine/ryanodine-sensitive Calcium sup 2+ store. However, the difference in the EC⁵⁰for the constricting effect of halothane (0.46 mM [nearly equal] 1.5 vol% at 37 degrees Celsius) and inhibition of caffeine constrictions by halothane (1.6 mM [nearly equal] 5.3 vol% at 37 degrees Celsius) was substantial. This difference may reflect the fact that caffeine induces supramaximal amounts of Calcium²+ release, making it necessary to substantially reduce the caffeine-releasable pool before producing a measurable effect on caffeine constrictions. Alternatively, the discrepancy between the EC sub 50 for the vasoconstricting actions and the effect on caffeine constriction may reflect multiple effects on the caffeine-sensitive Calcium²+ store. Su and Zhang proposed that halothane may decrease refilling of the caffeine-releasable store in vascular smooth muscle based on their finding that halothane treatment during Calcium²+ loading of the SR resulted in attenuated caffeine constrictions. [19] However, that effect is similar to the attenuating effect of continuous halothane on caffeine constriction that we observed, which may be more readily explained by the Calcium 2+-releasing effect of halothane. Most recent evidence suggests that SR Calcium²+-ATPase activity is enhanced rather than inhibited by halothane. [52–54] Perhaps such halothane-induced increases in ATPase activity or the potentiating effect of halothane on caffeine constriction observed by Su and Zhang [19] contribute to the relative preservation of caffeine responses during continuous halothane. It is also conceivable that the ryanodine-sensitivity of halothane constrictions is not the result of a direct effect on the caffeine/ryanodine-sensitive store. Halothane, for example, could indirectly activate the Calcium²+-induced Calcium sup 2+-release mechanism of the caffeine/ryanodine-sensitive store in vascular smooth muscle. [55] These and other possible mechanism(s) for the vasoconstricting action of halothane should be investigated further in vascular smooth muscle SR preparations.

Although the constricting effect of halothane was largely transient, the magnitude of this effect at clinical concentrations suggests that halothane could produce brief increases in vascular resistance during anesthetic induction or after increases in concentration. After transient constriction, halothane produced sustained vasodilation of KCl- or phenylephrine-constricted vessels, suggesting that the vasodilating action would predominate in stimulated vessels. However, in unstimulated vessels, halothane had a small sustained constricting effect. Halothane has been reported to produce an increase in mesenteric resistance in vivo [1–3,5,55] that has been attributed to increased sympathetic tone [56] or to metabolic auto-regulatory vasoconstriction due to halothane-induced decreases in oxygen consumption by the gut. [57] Our findings suggest that a direct vasoconstricting action could be involved in mesenteric vasoconstriction during halothane anesthesia.

The vasodilating action of halothane on KCl- and phenylephrine-constrictions was notable in resistance vessels. Halothane completely and reversibly blocked KCl constrictions with an EC⁵⁰essentially identical to the anesthetic MAC in this species (i.e., 0.36 mM [nearly equal] 1.2 vol% at 37 degrees Celsius), and phenylephrine constrictions were blocked with an EC⁵⁰[nearly equal] 2 MAC (i.e., 0.75 mM [nearly equal] 2.5 vol% at 37 degrees Celsius). In contrast, the direct vasodilating action of halothane previously demonstrated in isolated conductance arteries from rat [12,13,15,17] were considerably smaller than those reported here. In the original study of Sprague et al., halothane attenuated phenylephrine constrictions in isolated rat aorta with an EC⁵⁰of approximately 1.2 mM ([nearly equal] 4% at 37 degrees Celsius). [12] Similarly, in more recent studies, 2–2.3% halothane (at 37 degrees Celsius [nearly equal] 0.6–0.75 mM) produced only [nearly equal] 20% relaxation of phenylephrine- or norepinephrine-constricted rat aortic rings [15] or strips. [17] Stone and Johns demonstrated that the vasodilating action in rat aortic rings was endothelium-independent, with [nearly equal] 1.5 mM halothane (5% at 37 degrees Celsius) producing [nearly equal] 30% relaxation. [13] The effect of halothane on KCl constrictions in rat vessels has not been studied, although KCl-constricted rabbit aortic rings were relaxed only [nearly equal] 20–30% by [nearly equal] 0.9 mM halothane (3% at 37 degrees Celsius), [19] and Hatano et al. found no effect of [nearly equal] 0.7 mM halothane (2.3% at 37 degrees Celsius) on KCl-constricted canine coronary strips from “small” coronary arteries (890 plus/minus 10 micro meter), whereas strips from “large” coronary arteries (2,730 plus/minus 80 micro meter) were relaxed by [nearly equal] 10%. [24] Although this latter result suggested the possibility that vasorelaxation may depend on vessel size, Bollen et al. found no difference in the vasorelaxation produced by halothane in small (0.5–1.0 mm OD) and medium-sized (1.0–1.5 mm OD) porcine coronary artery rings constricted with KCl, [22] and these vessels were more sensitive to halothane, with [nearly equal] 50% relaxation by 0.6 mM halothane (2% at 37 degrees Celsius). Although the actions of volatile anesthetics on conductance and resistance vessels have not been compared directly, these results suggest that mesenteric resistance vessels may be more sensitive than conductance vessels to the vasodilating actions of halothane. Moreover, the more potent vasodilating action of halothane reported here in resistance vessels, rather than that previously reported in conductance vessels, is more likely to contribute to decreases in resistance in vivo. The overall effect of halothane on the mesenteric vascular bed in vivo, whether vasoconstricting or vasodilating, would be expected to depend on the magnitude of autonomic or metabolic influences and the effects of halothane on these influences, as well as the direct actions of halothane reported here.

Both the phenylephrine- and the KCl-induced sustained constrictions were dependent on Calcium²+ influx through dihydropyridine-sensitive Calcium²+ channels. Ryanodine-sensitive Calcium²+ stores appear to be involved in the phenylephrine-induced oscillations, but depletion of these stores with ryanodine had little effect on the magnitude of the KCl- or phenylephrine-induced constrictions or the vasodilating action of halothane. Thus, the vasodilating effect of halothane appeared to be unrelated to the vasoconstricting effect of halothane, which was eliminated by ryanodine. Because activation of the contractile machinery involves Calcium²+ signaling (i.e., increased [Ca²+]ⁱ) followed by Calcium²+ activation of contractile proteins, [43,44] halothane-induced vasodilation must involve one of these two processes. Volatile anesthetics have been shown to block Calcium²+ currents through voltage-gated dihydropyridine-sensitive Calcium²+ channels in cardiac cells [58,59] and vascular smooth muscle cells, [60,61] suggesting that the vasodilation might be due in part to depressed Calcium²+ influx. In contrast, halothane (3% at 23 degrees Celsius [nearly equal] 1.8 mM) was reported to have only a small effect on maximal Calcium²+-activated tension in skinned rabbit aortic smooth muscle, [19] suggesting that halothane has little effect on Calcium²+ activation of contractile proteins. Our experiments do not provide further illumination regarding the likely mechanism(s) of the vasodilating action, although an effect on Calcium²+ activation of contractile proteins would have been expected to block the constricting responses to cytoplasmic Calcium²+([Ca²+]ⁱ) increases without regard to whether the [Ca²+]ⁱincreases were produced by KCl or caffeine. The fact that caffeine constrictions were relatively less sensitive to block by halothane argues in favor of an effect on Calcium²+ signaling (influx) rather than a primary effect on Calcium²+ activation of contractile proteins. The reasons for the relative insensitivity of phenylephrine-induced constrictions compared to KCl constrictions is unclear. Phenylephrine and KCl constrictions may differ with respect to the types of channels involved in membrane depolarization and the increases in membrane Calcium²+ conductance, [43,44] as well as the contribution of protein kinase Carbon-mediated increases in contractile protein sensitivity to Calcium sup 2+. [62,63] Because phenylephrine is well known to activate protein kinase C, [62,63] the smaller vasodilating effect of halothane on phenylephrine-constricted compared to KCl-constricted vessels suggests that protein kinase Carbon inhibition produced by halothane may be less important than inhibition of Calcium²+ channels. However, the mechanisms involved in KCl and phenylephrine constrictions, as well as the actions of halothane, may be complex. Additional studies will be necessary to more precisely define the mechanisms of halothane-induced vasodilation as well as the reasons for the difference in effectiveness of halothane in phenylephrine- or KCl-constricted mesenteric resistance blood vessels.

The vasoconstricting and vasodilating actions of halothane were not substantially affected by inhibitors of the EDRF pathway or indomethacin in endothelium-intact vessels. However, treatment with these agents did not eliminate ACh-induced endothelium-dependent vasodilation, and physical removal of the endothelium was necessary to abolish ACh responses. Endothelial removal had little effect on the vasoconstricting and vasodilating actions of halothane in these vessels, thus more clearly indicating that the vascular actions of halothane were largely due to direct effects on vascular smooth muscle. Similarly, the vasodilating action of halothane in agonist-constricted rat aorta [13] and canine [25] or porcine coronary arteries [21] was not affected by endothelium removal. These findings contrast with the endothelium-dependent vasodilating action of isoflurane in canine coronary arteries reported by Blaise et al. [23] However, other studies indicate that the vasodilating action of isoflurane in rat aorta [14] and canine cerebral arteries [64] is endothelium-independent. Halothane has been well documented to have an inhibitory effect on endothelium-dependent relaxation [15,16,27] that may contribute to halothane-induced vasoconstriction in rat aorta, [13] as discussed above. However, our data suggest that the prevailing effects are due to direct effects on smooth muscle in mesenteric resistance vessels. Halothane attenuated or eliminated the phenylephrine-induced oscillations in these vessels at clinically relevant halothane concentrations even below those associated with vasodilation. These oscillations also were eliminated by endothelial removal, which could reflect an endothelium-dependent effect. Halothane has been reported to have a similar attenuating effect on norepinephrine-induced oscillations in mesenteric veins. [65,66] Those oscillations, like ours, were ryanodine-sensitive, but they were not blocked by nitric oxide synthase inhibitors. [66] This suggests that the oscillations may be unrelated to endothelial nitric oxide production, although they could be related to other non-EDRF but endothelial-dependent process(es). The importance of the endothelial effect(s) of halothane remains uncertain at this time, although volatile anesthetic effects on the endothelium may have an important modulating role under some conditions. [46].

In summary, endothelium-independent vasoconstricting and vasodilating actions of halothane were demonstrated in rat mesenteric resistance blood vessels at clinically relevant concentrations. The magnitude of these actions, in addition to their concentration dependence, suggest that they can contribute significantly to the cardiovascular actions of halothane in vivo. The vasoconstricting action was dependent on Calcium²+ release from the ryanodine-sensitive intracellular Calcium²+ store. The vasodilating actions on phenylephrine- or KCl-constricted vessels were independent of the effect of halothane on intracellular Calcium²+ stores and appeared to be the result of either altered Calcium²+ signaling or depressed Calcium²+ activation of contractile proteins in vascular smooth muscle.

The authors thank Dr. Alex Evers, Dr. Joe Henry Steinbach, and Dr. Takashi Akata for discussions regarding this work.