Halothane has been reported to affect the integrity of intracellular Ca2+ stores in a number of tissues including vascular smooth muscle. However, the actions of halothane on intracellular Ca2+ stores are not yet fully understood.
Employing the isometric tension recording method, the action of halothane in isolated endothelium-denuded rat mesenteric arteries under either intact or beta-escinmembrane-permeabilized conditions was investigated.
Halothane (0.125-5%) produced concentration-dependent contractions in Ca2+ free solution in both intact and membrane-permeabilized muscle strips. Ryanodine treatment or repetitive application of phenylephrine eliminated both caffeine-and halothane-induced contractions in the Ca2+ free solution. When either halothane and caffeine, caffeine and halothane, phenylephrine and halothane, or inositol 1,4,5-triphosphate and halothane were applied consecutively in the Ca2+ free solution in either intact or membrane-permeabilized muscle strips, the contraction induced by application of the second agent of the pair was inhibited compared to application of that agent alone. However, when procaine was applied before and during application of the first agent, the contraction induced by the first agent was inhibited and the contraction induced by the second agent was restored. Heparin inhibited the inositol 1,4,5-triphosphate-mediated contraction, but not contractions induced by halothane or caffeine. Halothane (0.125-5%), applied during Ca2+ loading, produced concentration-dependent inhibition of the caffeine contraction (used to estimate the amount of Ca2+ in the store) in both intact and membrane-permeabilized muscle strips. In contrast, halothane applied with procaine during Ca2+ loading produced concentration-dependent enhancement of the caffeine contraction. This enhancement was observed only in the intact but not in the membrane-permeabilized condition.
Halothane has two distinct actions on the intracellular Ca2+ stores of vascular smooth muscle, a Ca2+ releasing action and a stimulating action on Ca2+ uptake. Halothane releases Ca2+ from the stores that are sensitive to both caffeine/ryanodine and phenylephrine/inositol 1,4,5-triphosphate through a procaine-sensitive mechanism. The observed inhibitory effect on Ca2+ uptake is probably caused by the Ca2+ uptake after blockade of Ca2+ release may be membrane-mediated.
IT has been well recognized that cytosolic free calcium concentration ([Calcium2+]c) is a major determinant for cellular function in a variety of cell types. Intracellular Calcium2+ stores play an important role in the regulation of [Calcium2+]c and subsequent cellular activity. It has been suggested that volatile anesthetics, including halothane, cause an alteration in cellular homeostasis by affecting the integrity of intracellular Calcium2+ stores in a variety of cell types including cardiac, [1–3]skeletal, [4–6]and vascular smooth muscles. [7–11].
It has been suggested that halothane stimulates Calcium2+ release or leakage from the intracellular Calcium2+ stores of cardiac, [1,2,4]skeletal, [4–6]and vascular smooth muscle cells. [7–9,11]In isolated sarcoplasmic reticulum (SR) vesicles from cardiac or skeletal muscle, halothane has been shown to increase [sup 3 Hydrogen]ryanodine binding, an index for the open state probability of the SR Calcium2+ release channel, suggesting that halothane increases activity of ryanodine-sensitive SR Calcium2+ release channels and thereby releases Calcium2+ from SR. [2,4]Halothane also was shown previously to modulate the effects of caffeine and ryanodine on SR in isolated aorta, implying that halothane may have an effect on the ryanodine-sensitive SR Calcium2+ stores in vascular smooth muscle as well. In addition, recent studies in isolated mesenteric arteries have shown that halothane induces Calcium2+ release from ryanodine-sensitive stores. [8,9,11]However, studies in smooth muscle have suggested that the ryanodine/caffeine-sensitive SR also has receptors for inositol 1,4,5-triphosphate (IP3), a second messenger for receptor agonist-induced Calcium2+ release, [12–14]and recent studies have shown that halothane can increase IP3formation through stimulation of G-protein-dependent phospholipase C activity in erythrocyte membranes or vascular smooth muscle under certain conditions. ,* Thus, IP3formation potentially may be involved in the halothane-induced Calcium2+ release. In addition, halothane also has been shown to cause Calcium2+ leakage from IP3-sensitive stores independent of IP3formation in clonal pituitary cells, in which caffeine (or ryanodine)-sensitive stores do not exist. [16,17]Therefore, the mechanisms behind the Calcium2+ releasing action (or leakage) of halothane remain debatable.
In addition to the Calcium2+ releasing action, halothane also may affect Calcium2+ uptake into intracellular Calcium2+ stores. Previous studies have yielded conflicting results about the action of halothane on Calcium2+ uptake. Under various experimental conditions, the adenosine triphosphate (ATP)-dependent Calcium2+ uptake has been reported to be inhibited, [1,3,18–20]unaffected, or enhanced [18,21,22]by halothane in cardiac, skeletal, or vascular smooth muscle. While these studies may suggest an action on SR Calcium2+-ATPase, the involvement of SR Calcium2+-ATPase activity in the action of halothane on ATP-dependent Calcium2+ uptake remains unclear. Although SR Calcium2+-ATPase activity has been reported to be inhibited by halothane, recent studies with isolated SR vesicles from cardiac or skeletal muscle have demonstrated a stimulating action of halothane on the SR Calcium2+-ATPase activity in addition to a stimulating action on Calcium2+ release (or leakage). [1,5]However, in spite of the observed increased SR Calcium sup 2+-ATPase activity, halothane did not increase the net Calcium2+ uptake rate (or total uptake of Calcium2+) in these experiments. [1,5]Because accumulation of Calcium2+ in SR is known to cause feedback inhibition of the SR Calcium2+-ATPase activity, [24–26]the halothane-induced stimulation of SR Calcium2+-ATPase activity might therefore have been secondary to depressed Calcium2+ uptake owing to enhanced Calcium2+ leakage by halothane. Finally, recent studies have suggested that halothane may increase [Calcium2+]c, a major determinant for SR Calcium2+-ATPase activity and Calcium2+ uptake into stores, by inhibiting Calcium2+ extrusion by plasma membrane Calcium2+-ATPase or the Sodium 2+/Calcium2+ exchanger. [27–30]Thus, halothane may stimulate Calcium2+ uptake by increasing [Calcium2+]c.
The current study represents the first description of the effects of halothane on SR in small resistance arteries. In the current study, we were, for the first time, successful in separating two distinct actions of halothane on SR in vascular smooth muscle under physiologic conditions, that is, a Calcium2+ releasing action and a stimulating action on Calcium2+ uptake. We demonstrated that halothane stimulates Calcium2+ release from both caffeine/ryanodine-sensitive and phenylephrine/IP3-sensitive stores, which appear to completely overlap in these vessels. In this study, we demonstrated that the Calcium sup 2+ releasing action can be effectively blocked by procaine, but not by heparin, a specific inhibitor of IP3-induced Calcium2+ release. In addition, when the Calcium2+ releasing action was blocked with procaine, halothane actually had a stimulating effect on Calcium2+ uptake. This indicates that the inhibitory effect of halothane on Calcium sup 2+ uptake observed in the absence of procaine both in this study and previously in rabbit aorta, was a result of the Calcium2+ releasing action of halothane. Finally, the stimulating action of halothane on Calcium2+ uptake was eliminated after membrane-permeabilization, suggesting that stimulation of Calcium2+ uptake by halothane is membrane-mediated. This stimulating action, therefore, may be caused by inhibition of membrane-mediated Calcium2+ clearance rather than stimulation of SR-Calcium2+ ATPase.
Materials and Methods
Tension Measurement Experiments
After receiving institutional approval, Sprague-Dawley rats breathed 100% Oxygen2for 2–3 min and were then anesthetized with halothane (halothane/oxygen). The methods of our tension recording experiments with endothelium-denuded strips were described in detail previously. [9,10,31]Briefly, the mesenteric arteries were exposed, excised rapidly, and placed immediately in a dissecting chamber filled with 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)-buffered physiologic salt solution (PSS, room temperature [nearly equal] 22 degrees Celsius). The distal portion of the third or fourth order branch (0.15–0.20 mm in diameter) of the artery were dissected free of surrounding tissues, and endothelium-denuded transverse strips (150–200 micro meter wide, 250–400 micro meter long) were prepared for isometric tension measurement. The muscle chamber (0.9 ml) was mounted on a microscope stage, and the strip was stretched to [nearly equal] 1.1 times its resting length to obtain a maximal contractile response to high Potassium sup +. The solution was changed by perfusing it rapidly from one end while aspirating it simultaneously from the other end. Intact muscle experiments were conducted at both 22 degrees C (room temperature) and 35 degrees C, whereas membrane-permeabilized muscle experiments were performed only at 22 degrees C to prevent early deterioration of the strips. Functional removal of the endothelium was confirmed by lack of acetylcholine (10 micro Meter)-induced endothelium-dependent relaxation. The membrane permeabilization was achieved by incubating the strips with 50 micro Meter beta-escin for 22–24 min at 22 degrees C in relaxing solution after measuring steady contractions induced by high Potassium sup +. In some experiments, 0.3 micro Meter ionomycin was used to functionally deplete the intracellular Calcium2+ store as reported previously. Because receptor coupling has been reported to be retained in beta-escin-permeabilized smooth muscle, receptor coupling was examined by the response to phenylephrine (10–100 micro Meter) in the presence of guanosine 5'-triphosphate (50 micro Meter) after permeabilization.
Solutions and Drugs
The ionic concentrations of the HEPES-buffered PSS for the intact muscle experiments were as follows (in mM): NaCl 138, KCl 5.0, MgCl21.2, CaCl21.5, HEPES 10, glucose 10. The pH was adjusted with NaOH to 7.35 at either 22 degrees C or 35 degrees C. The high Potassium sup + solutions were prepared by replacing NaCl with KCl isoosmotically. The Calcium2+ free solution was prepared by removing CaCl2, and adding 2 mM ethyleneglycol-bis-(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA).
The composition of relaxing or activating solutions used in the membrane-permeabilized muscle experiments were determined by solving multiequilibrium equations using a hydrogen ion activity coefficient of 0.75 and association constants for the various ions as detailed by us elsewhere. The pH level was adjusted with KOH to 7.00 at 22 degrees Celsius, and the ionic strength was kept constant at 0.2 M by adjusting the concentration of potassium Methanesulfonate. [9,31]Guanosine 5'-triphosphate (50 micro Meter) was present throughout the experimental periods to prevent rundown of contractile responses in the permeabilized strips. [33,34]The concentration of EGTA used in the loading or activating solution was 4 mM, while either 4 or 0.05 mM EGTA was used in Calcium2+ free solution depending on the protocol used. (see Experimental Design).
HEPES, beta-escin, ionomycin, procaine, guanosine 5'-triphosphate, inositol 1,4,5-triphosphate (IP3, I-9766), and low molecular weight heparin (porcine intestinal mucosa, average molecular weight [nearly equal] 6,000) were obtained from Sigma Chemical (St. Louis, MO). The ethyleneglycol-bis-(beta-aminoethyl ether) N,N',N'-tetraacetic acid, piperazine-1,4-bis-(2-ethanesulfonic acid)(PIPES-K2) and methanesulfonic acid were obtained from Fluka Chemie AG (Buchs, Switzerland). Ryanodine was purchased from Agri Systems International (Wind Gap, PA). Halothane was obtained from Ayerst Laboratories (Philadelphia, PA). All other reagents were of the highest grade available commercially.
It has been well established that [Calcium2+]c is a major determinant of vascular smooth muscle contraction and contraction can therefore be used as an estimate of [Calcium2+]c. Increases in [Calcium2+]c resulting from Calcium2+ release from intracellular stores or mobilization from the extracellular space results in vasoconstriction; and decreases in [Calcium2+]c due to sequestration of Calcium2+ in stores or membrane clearance results in vasorelaxation. In these studies, the magnitude of the contraction in Calcium2+ free extracellular solution was used to estimate the amount of Calcium2+ in the intracellular stores and to investigate the action of halothane on intracellular Calcium2+ stores. In addition, because contraction may be influenced by changes in Calcium2+ sensitivity, potential effects of each agent on Calcium2+ sensitivity were considered and controlled for in our experiments as well.
We first examined the effects of removal of extracellular Calcium2+ on halothane-induced contractions, and found that halothane-induced vasoconstriction is mainly caused by Calcium2+ release from intracellular stores (see Results). To characterize the halothane-sensitive intracellular Calcium2+ stores, we next examined the effects of ryanodine on caffeine-, phenylephrine-, and halothane-induced contractions, and the effects of repetitive application of phenylephrine on caffeine- or halothane-induced contractions in Calcium sup 2+ free solution in intact muscle. Ryanodine (10 micro Meter) was applied to the strips with caffeine (20 micro Meter) for [nearly equal] 1–2 min, and the strips were then treated with ryanodine (10 micro Meter)-containing PSS for 25 min as reported previously. In the latter experiment, phenylephrine was applied repetitively until it became unable to evoke any contraction, and then the effects of this repetitive application of phenylephrine on caffeine- or halothane-induced contractions were assessed by comparing the results with the time control data.
To further characterize the Calcium2+ releasing action of halothane, we consecutively applied either halothane and caffeine, caffeine and halothane, phenylephrine and halothane, IP3and caffeine, IP3and halothane, or caffeine and caffeine in Calcium2+ free solution in either intact or beta-escin-permeabilized muscle. In some experiments, the effects of procaine, a proposed inhibitor of Calcium sup 2+-induced Calcium2+ release, [35–37]or heparin, believed to specifically inhibit IP3-induced Calcium2+ release, [31,35,37]on the Calcium2+ releasing actions of these agents were examined. After depletion of the stores with caffeine, the stores were loaded with Calcium2+ by incubating the muscle in 1.5 mM Calcium2+ containing PSS solution for 8 min or pCa 6.5 solution for 5 min under the intact or permeabilized conditions, respectively. After Calcium2+ loading, the above Calcium2+ releasing agents were applied consecutively in 2 mM or 0.05 mM EGTA-containing Calcium2+ free solutions in the intact or permeabilized strips, respectively. The intervals for consecutive applications of these agents were based on the results of control experiments, in which each Calcium2+-releasing agent was applied to vascular strips precontracted with high Potassium sup + or 3 micro Meter Calcium2+ in the intact or membrane-permeabilized condition, respectively. The observed time necessary for regaining the control level of contraction after washout of these Calcium2+-releasing agents was used in our protocols to allow a sufficient interval for vascular smooth muscle to recover from the effects of the first agent at the time of application of the second agent. Periods of 2, 8, and 3 min were found to be sufficient for vascular smooth muscle to almost fully (> 95%) recover from the effects of halothane (5%), caffeine (20 mM), and phenylephrine (10 micro Meter) on high Potassium sup +-induced contractions, respectively, under the intact condition; whereas 3 or 5 min was sufficient for vascular smooth muscle to recover from the effects of halothane (0.5–5.0%), caffeine (20 micro Meter) or IP3on 3 micro Meter Calcium2+-induced contraction under the permeabilized condition. In addition, 2 or 3 min was considered sufficient for vascular smooth muscle to almost recover from the effects of inhibitors of Calcium sup 2+ release (i.e., procaine and heparin) based on the results of control experiments similar to the aforementioned control experiments for the Calcium2+-releasing agents. The experiments examining the effects of these inhibitors alone on the contraction induced by the subsequently applied agent were conducted with each experimental paradigm (see Results). In the experiments using consecutive application of two agents, the effect of the first agent on the Calcium2+ stores sensitive to the second agent was determined from the changes in the amplitude of contraction induced by the second agent administered alone.
Finally, we investigated the effects of halothane on Calcium sup 2+ uptake into the stores in either the intact or permeabilized conditions. For these experiments, halothane was applied during the Calcium2+ loading phase with or without procaine, which inhibited the halothane-induced Calcium2+ release in the above experiments. Calcium sup 2+ loading was performed by incubating the muscle in PSS (1.5 min) for 8 min or in pCa 6.5 solution for 5 min in the intact or membrane-permeabilized condition, respectively. The amount of Calcium2+ in the store was then estimated from the amplitude of contraction induced by caffeine 2 min or 5 min after removal of Calcium2+ loading solution in the intact or membrane-permeabilized conditions, respectively. In the control experiments, procaine alone (10 or 30 mM) was applied during the Calcium2+ loading phase.
Halothane Delivery and Analysis
Halothane was delivered via a halothane vaporizer (Ohio Medical Products, Madison, WI) in line with the air gas aerating the solutions in both the intact and membrane-permeabilized muscle experiments. Each solution was equilibrated with halothane for at least 10 min before introduction to the chamber, which was covered with a thin glass plate to prevent the equilibration gas from escaping into the atmosphere. We previously reported the concentrations of halothane in the solutions under the same experimental conditions determined by gas chromatography. The mean concentrations produced by 0.5% and 2% halothane in the PSS solution at 22 degrees C were 0.28 and 1.14 mM, respectively, whereas those produced by 1.5% and 3% halothane at 35 degrees C were 0.45 and 0.89 mM, respectively. The mean concentrations produced by 0.5% and 2% halothane in the piperazine-1,4-bis-(2-ethanesulfonic acid)-buffered solutions used for the permeabilized muscle experiments were 0.31 and 1.23 mM, respectively. Because the relationship between actual concentrations of halothane in the solutions and anesthetic concentrations (vol %) in the gas mixture is theoretically linear, the anesthetic concentrations on the X-axis are displayed as vol % for the halothane concentration-response relationships.
Calculation and Statistical Analysis
Data were expressed as mean+/-SEM. The n denotes the number of strips; the number of animals was noted only when it differed from the number of strips.
The concentration-response data for the halothane-induced contractions were fitted according to a four parameter logistic model as described by De Lean et al., and the EC50(the concentration that produced 50% of the maximal response) values were derived from the least-squares fit using the above model.
Statistical analysis was made by a one- or two-factor analysis of variance and Student's t test (paired or unpaired), when appropriate. A P level of < 0.05 was considered significant.
Characterization of the Calcium sup 2+ Releasing Action of Halothane
Halothane, in a concentration-dependent manner (0.125–5%), generated transient vasoconstriction both in the presence (n = 6) and absence (n = 7) of extracellular Calcium2+ in the intact strips with EC50values of 0.32%(22 degrees C) and 0.39%(22 degrees C), respectively. The removal of extracellular Calcium2+ did not reduce significantly the magnitude of the halothane-induced contraction, suggesting that the halothane contraction is a result of Calcium2+ release from intracellular Calcium2+ stores.
Ryanodine treatment, which eliminated the contraction induced by caffeine (20 mM; P < 0.05), also eliminated both the maximal contractions induced by phenylephrine (10 micro Meter) and halothane (5%) in the Calcium2+ free solution (22 degrees C; P < 0.05, n = 3), indicating that both phenylephrine-and halothane-sensitive stores are sensitive to ryanodine in this artery. In addition, the repetitive application of phenylephrine (10 micro Meter) nearly completely eliminated contractions induced by subsequently applied caffeine (20 mM) or halothane (5%) in the Calcium2+ free solution (P < 0.05, n = 3), indicating that both caffeine- and halothane-sensitive stores are sensitive to phenylephrine in this artery.
When halothane and caffeine were applied consecutively in the Calcium2+ free solution, halothane generated contraction and inhibited the contraction induced by subsequently applied caffeine in either intact or permeabilized strips (Figure 1, Figure 2, Figure 3) in a concentration-dependent manner. When procaine (10–30 mM) was applied before and during application of halothane, the halothane-induced contractions were inhibited strongly or eliminated as was the halothane-induced inhibition of the caffeine contraction (Figure 1, Figure 2, Figure 3). Procaine (10 or 30 mM) applied alone (without halothane) did not significantly affect the contraction induced by caffeine applied subsequently in either the intact or permeabilized condition (P > 0.05, Figure 1(A), Figure 2(B), and Figure 3); the amplitude of the caffeine contractions after prior treatment with procaine (30 mM) in the intact strips at 22 degrees C and 35 degrees C were 0.97+/-0.02 (n = 3) and 1.02+/-7.5 (n = 4) times control, respectively (Figure 1(A)); and the amplitude of caffeine contraction after prior treatment with procaine (10 mM) in the permeabilized strips was 1.00+/-0.04 times (n = 4, 3 animals) control (Figure 2(B)).
In the experiments in which caffeine and halothane (n = 4) or phenylephrine and halothane (n = 3) were applied consecutively in the Calcium2+ free solution in the intact strips (22 degrees C), both caffeine and phenylephrine almost completely inhibited the contraction induced by subsequently applied halothane (Figure 4); and procaine, applied with either caffeine or phenylephrine, nearly eliminated both the caffeine-and phenylephrine-induced contractions (Figure 4), and also eliminated the inhibition by caffeine or phenylephrine of the contraction induced by subsequently applied halothane (Figure 4).
After membrane permeabilization with beta-escin, phenylephrine (up to 100 micro Meter) did not evoked any contraction in the presence of guanosine 5'-triphosphate (50 micro Meter), indicating that receptor coupling is not retained in our beta-escin-permeabilized strips. However, IP3reproducibly evoked contraction in the beta-escin strips. When IP sub 3 and caffeine were applied consecutively in the Calcium2+ free solution in the permeabilized strips, preapplication of IP3significantly inhibited the contraction induced by subsequently applied caffeine (Figure 5(A)). Heparin (0.9–2.7 mg/ml), applied with IP3, inhibited both the IP3-induced contraction and the IP3-induced inhibition of caffeine contraction (Figure 5(A)) in a concentration-dependent fashion. Preapplication of heparin alone did not significantly affect the caffeine contraction (Figure 5(A)) and a concentration of heparin (2.7 mg/ml) that strongly inhibited the IP3contraction did not significantly affect either halothane- or caffeine-induced contractions (Figure 5(B)). Procaine, applied with IP sub 3, eliminated both the IP3contraction and the IP3-induced inhibition of caffeine contraction (Figure 6(A)). When IP3and halothane were applied consecutively in the Calcium2+ free solution in the permeabilized strips, preapplication of IP3significantly inhibited the contraction induced by halothane (Figure 6(A)).
Finally, when caffeine was applied repetitively in the Calcium sup 2+ free solution in the permeabilized muscle, the second application of caffeine failed to evoke any significant contraction. However, procaine, applied with the first application of caffeine, eliminated the contraction induced by the first application of caffeine and completely restored the contraction induced by the second application of caffeine (Figure 6(B)).
Effects of Halothane on Calcium sup 2+ Uptake into the Intracellular Stores
Halothane, applied during the Calcium2+ loading phase, inhibited the caffeine contraction in a concentration-dependent manner, indicating a decrease in the amount of Calcium2+ in the intracellular stores, in both intact (22 degrees C and 35 degrees C) and permeabilized (22 degrees C) strips (Figure 7and Figure 8). However, halothane, applied during the Calcium2+ loading phase with procaine, enhanced the caffeine contraction in intact muscle in a concentration-dependent manner (Figure 7and Figure 8). In the permeabilized muscle, however, halothane applied with procaine (10 mM) did not enhance the caffeine-induced contraction (Figure 8). A higher concentration of procaine (30 mM) or a longer Calcium2+ loading time (8 min), did not alter the finding that halothane could not enhance the caffeine-induced contraction in the permeabilized muscle (n = 3). No significant differences were found among the data obtained with 10 mM procaine and 5 min Calcium2+ loading time, 30 mM procaine and 5 min Calcium2+ loading time, and 30 mM procaine and 8 min Calcium2+ loading time.
Procaine (up to 30 mM) alone did not significantly affect the caffeine contraction in the intact strips (Figure 7(A)). Interestingly, in the permeabilized strips, a transient increase in tension was observed after the Calcium2+ loading with procaine, and procaine (10 mM) itself slightly ([nearly equal] 10%), but significantly increased the caffeine contraction (Figure 8(A)). Therefore, the enhanced caffeine contraction after procaine alone was used as the control for the analysis of effects of halothane and procaine on the Calcium2+ uptake after permeabilization. The transient increase in tension after washout of procaine was not significantly affected by 0.3 micro Meter ionomycin, which eliminated the caffeine contraction (Figure 8(A); 1.02+/- 0.12 [n = 3] times control [before ionomycin]). This suggests that the procaine-induced contraction was not caused by a Calcium2+-releasing effect but an increase in Calcium2+ sensitivity.
Halothane's Dual Actions on Sarcoplasmic Reticulum
Halothane appears to have two distinct actions on SR Calcium2+ stores of vascular smooth muscle in this resistance artery: a Calcium2+ releasing action and a stimulating action on Calcium2+ uptake. Halothane caused vasoconstriction, which was little affected by removal of extracellular Calcium2+, and the halothane contraction was eliminated after ryanodine treatment, known to deplete caffeine-sensitive SR Calcium sup 2+ stores by binding to SR ryanodine receptors and thereby locking the SR Calcium2+-releasing channel into an open state. [12,40,41]Because our experiments were performed in the absence of endothelium, these findings indicate that halothane stimulates Calcium2+ release from SR of vascular smooth muscle cells directly, thereby causing vasoconstriction. Involvement of increases in Calcium2+ sensitivity in the halothane contraction appears to be ruled out by its inability to increase tension in the Calcium2+-activated permeabilized muscle after depletion of the intracellular Calcium2+ stores by ionomycin demonstrated in our previous study with the same artery. [9,42]Halothane, when added to the Calcium2+ loading solution, inhibited the caffeine-induced contraction, indicating a decrease in the amount of stored Calcium2+, and suggesting that halothane inhibits Calcium2+ uptake. However, when halothane was applied during the Calcium2+ loading phase with procaine, which inhibited the Calcium2+ releasing action of halothane, the caffeine contraction was enhanced, suggesting that halothane may actually enhance Calcium2+ uptake. Although it was suggested previously that procaine itself may increase Calcium2+ uptake owing to inhibition of Calcium2+ leakage from the stores, [43,44]procaine applied alone during the Calcium2+ loading phase did not affect the caffeine contraction (Figure 7(A)), suggesting that procaine was not responsible for the increase in Calcium2+ uptake observed in our experiments. In addition, the clearly demonstrated concentration dependence in the stimulating effect of halothane on Calcium sup 2+ uptake in the presence of a fixed concentration of procaine further indicates that the observed effect was unrelated to the action of procaine. Therefore, these findings suggest that halothane has a stimulating action on Calcium2+ uptake in addition to its Calcium2+-releasing action, and that the inhibitory effect of halothane on Calcium2+ uptake in the absence of procaine was due to its Calcium sup 2+ releasing action.
Mechanisms of the Stimulating Action of Halothane on Calcium sup 2+ Uptake
Interestingly, halothane increased Calcium2+ uptake only under the intact, but not the membrane-permeabilized condition, suggesting that the action is membrane-mediated, and not caused by an increase in the SR Calcium2+ ATPase activity. Although halothane has been reported to increase SR Calcium2+ ATPase activity in isolated SR vesicles from cardiac or skeletal muscle, [1,5]the net Calcium2+ uptake rate or total uptake of Calcium2+ was not increased in those studies. Because an increase in intravesicular Calcium2+ concentration is known to inhibit SR Calcium2+ ATPase activity, [24–26]the increase in SR Calcium2+ ATPase activity observed in cardiac SR vesicles might have been secondary to halothane-induced Calcium2+ release (or leakage) from the SR, which also was documented in the same SR vesicles. In other words, this Calcium2+-releasing action could have depleted the SR Calcium2+ stores and thereby attenuated the feedback inhibition on the SR Calcium2+ ATPase by stored Calcium2+. The blockade of the Calcium2+-releasing action of halothane by procaine in our study should have prevented this secondary stimulation of SR Calcium2+ ATPase activity.
In the intact muscle, another major determinant for Calcium2+ uptake, cytoplasmic Calcium2+ concentration ([Calcium2+]c), may have been affected by halothane-induced changes in the Calcium2+ extrusion systems such as plasma membrane Calcium2+-ATPase or the Sodium sup+/Calcium2+ exchanger. Recent studies have demonstrated inhibitory action of volatile anesthetics including halothane on plasma membrane Calcium2+ ATPase activity in erythrocyte or brain synaptosomes and on Sodium sup +/Calcium2+ exchanger in cardiac cells. [27–30]Therefore, it is conceivable that halothane stimulates Calcium2+ uptake by increasing [Calcium2+]c as a result of inhibition of plasma membrane Calcium2+ ATPase activity. An effect on the Sodium sup +/Calcium2+ exchanger also is possible, but the Sodium sup +/Calcium2+ exchanger does not appear to play a major role in Calcium2+ extrusion in vascular smooth muscle under the conditions in which the Calcium2+ loading was performed in the current study. [45,46]Although either stimulation of sarcolemmal Calcium2+ influx or inhibition of Calcium2+ leakage from SR could also lead to enhancement of Calcium2+ uptake, there have been no reports, to our knowledge, that demonstrated a stimulating action of halothane on sarcolemmal Calcium2+ influx or an inhibitory action of halothane on Calcium2+ leakage from SR.
Characterization of Halothane-induced Calcium sup 2+ Release
The halothane-induced contraction in Calcium2+ free solution was eliminated after either ryanodine treatment or repetitive application of phenylephrine in the intact muscle. In addition, when either halothane and caffeine, caffeine and halothane, phenylephrine and halothane, or IP3and halothane were applied consecutively, contraction induced by the latter agent was consistently inhibited. These findings indicate that halothane stimulates Calcium2+ release from SR Calcium2+ stores sensitive to both caffeine (or ryanodine) and phenylephrine (or IP3) in this resistance artery. The elimination of the phenylephrine contraction in Calcium2+ free solution by ryanodine treatment and the elimination of the caffeine contraction by repetitive applications of phenylephrine both suggest that the caffeine (or ryanodine)-sensitive stores and the phenylephrine (or IP3)-sensitive stores are overlapped in this artery as has been suggested previously in other smooth muscle. [32,47,48]Thus, we were unable to more specifically distinguish the effects of halothane on the caffeine/ryanodine- versus phenylephrine/IP sub 3 -sensitive stores in this artery. A recent study demonstrating that halothane increases Calcium2+ leakage from IP3-sensitive stores in clonal pituitary cells, where the caffeine-sensitive stores are absent, suggests that halothane may directly affect IP3-sensitive stores. In addition, previous studies have demonstrated volatile anesthetic effects on [sup 3 Hydrogen]ryanodine binding to isolated SR vesicles from both skeletal and cardiac cells, suggesting direct interactions between volatile anesthetics including halothane and caffeine/ryanodine-sensitive Calcium2+ stores. [2,4].
The current study has provided new information about the procaine sensitivity of the Calcium2+-releasing action of halothane. It has been suggested that procaine is an inhibitor of Calcium2+-induced Calcium2+ release. [35,37]However, our data indicate that procaine inhibits the Calcium2+ releasing actions of caffeine, phenylephrine and IP3, suggesting that procaine may be a nonspecific inhibitor of Calcium2+ release as reported previously. .
Heparin inhibited only the IP3-induced contraction, and not halothane- or caffeine-induced contractions, indicating that heparin inhibits IP3-induced Calcium2+ release and not halothane- or caffeine-induced Calcium2+ release. The observed inhibition of IP3-induced Calcium2+ release by heparin is in agreement with recent studies demonstrating specific inhibition of IP3-induced Calcium2+ release by heparin in vascular smooth muscle. [32,36,38]Heparin has been suggested to inhibit IP3-induced Calcium2+ release by binding to the specific binding sites for IP3or the IP3receptor and thereby competitively inhibiting responses to IP3. [50,51]Although recent studies have suggested that halothane can stimulate G-protein-dependent phospholipase-C activity and increase IP3formation within the concentration range we studied (e.g., IC50[nearly equal] 2.8 mM in erythrocyte membranes), ,* the lack of an inhibitory effect of heparin on Calcium2+ release by 2 and 5% halothane in the permeabilized muscle (i.e., 1.14 and 2.85 mM) suggests that the halothane-induced Calcium2+ release observed in our experiments was not mediated by IP3. However, IP3production after phospholipase-C-mediated hydrolysis of phosphatidylinositol biphosphate may not occur under the permeabilized condition in our study, and we cannot exclude that IP3production contributes to the halothane-induced Calcium2+ release we observed in the intact condition.
A previous study suggested that receptor-G-protein coupling is retained in beta-escin (20 micro Meter)-membrane permeabilized vascular smooth muscle. We have confirmed the preservation of receptor coupling in beta-escin (20 micro Meter)-permeabilized muscle from rabbit epicardial coronary artery (unpublished data). However, we saw no contractile responses to phenylephrine in the presence of guanosine 5'-triphosphate after permeabilization, suggesting that receptor coupling was not retained in the beta-escin (50 micro Meter)-permeabilized muscle in this study. The difference could be due to the higher concentration of beta-escin used in this study (20 micro Meter vs. 50 micro Meter), however, we found the higher concentration to be necessary for satisfactory permeabilization in our study.
Significant decreases in EC50values for the halothane contraction after permeabilization (2.43% vs. 0.52% at 22 degrees C) may suggest involvement of a membrane-mediated component in the halothane-induced Calcium2+ release. However, it is difficult to compare the EC50values between the intact and permeabilized conditions. In particular, there is no evidence that the intracellular Calcium2+ stores are preserved completely in the permeabilized muscle, and a part of halothane-sensitive stores might be damaged during permeabilization. In addition, the sensitivity of the intracellular stores to halothane or the sensitivity of SR Calcium2+-ATPase to Calcium sup 2+ might have been altered during permeabilization.
In conclusion, halothane stimulates Calcium2+ release from SR sensitive to both caffeine and phenylephrine in this artery through a procaine-sensitive mechanism. The halothane-induced Calcium2+ release may be due, at least in part, to a direct action on the SR. However, the nonspecific nature of the blocking action of procaine on Calcium2+ release, the lack of a specific inhibitor of Calcium2+ release modulated by activation of ryanodine receptors and the overlapping of caffeine/ryanodine- and phenylephrine/IP3-sensitive Calcium2+ stores did not allow us to definitively characterize the halothane-induced Calcium2+ release or the halothane-sensitive Calcium2+ stores. However, the lack of effect of heparin on halothane-induced Calcium2+ release in permeabilized muscle does indicate that the halothane-induced Calcium2+ release can occur independent of IP3production.
The Overall Effects of Halothane on the Intracellular Calcium sup 2+ Stores
Intracellular Calcium2+ stores (or SR) probably are important in the regulation of cytosolic free calcium concentration, which is believed to play a primary role in the regulation of vascular smooth muscle contraction or relaxation. Besides its well-recognized role as a source of Calcium2+, it has been suggested recently that the SR also functions as a buffer against vascular smooth muscle activation by Calcium sup 2+. The amount of Calcium2+ in the stores therefore is an important determinant of both the Calcium2+-releasing and Calcium sup 2+-buffering capacities. Although our data suggest opposing actions of halothane on the SR Calcium2+ stores, the overall effect of halothane appears to be to decrease the amount of Calcium2+ in the SR, which could alter vascular homeostasis both by decreasing Calcium2+ availability for vascular smooth muscle contraction as well as by affecting the Calcium2+-buffering capacity of SR. In the current study, no further decrease in the amount of Calcium2+ in SR was observed by prolonging the incubation time for halothane (1.5%, 35 degrees Celsius) from 8 to 12 min, suggesting that the effect of this clinically relevant concentration of halothane had reached a steady state within 8 min.
Halothane has dual actions on the SR of vascular smooth muscle in rat small mesenteric arteries, i.e., a Calcium2+-releasing action and a stimulating action on Calcium2+ uptake. Halothane stimulates Calcium sup 2+ release from SR Calcium2+ stores sensitive to both caffeine and phenylephrine through a procaine-sensitive, but not a heparin-sensitive, mechanism. The ability of halothane to cause vasoconstriction in the Calcium2+ free solution in the permeabilized muscle, where no receptor coupling was observed, suggests that the halothane-induced Calcium2+ release is due, at least in part, to a direct effect on the SR. The inhibitory effect of halothane on Calcium2+ uptake observed in the absence of procaine is probably due to its Calcium2+-releasing action. The disappearance of stimulating action of halothane on the Calcium2+ uptake after permeabilization suggests that the effect of halothane on Calcium2+ uptake may be membrane-mediated. These actions of halothane on vascular smooth muscle SR Calcium2+ stores may play a role in halothane-induced alterations of cardiovascular homeostasis.
The authors thank Professor Alex S. Evers, Chairman, Department of Anesthesiology, Washington University, and Professor Shosuke Takahashi, Chairman, Department of Anesthesiology, Kyushu University, for their encouragement and help; and Dr. Masamitsu Hatakenaka, Department of Clinical Pharmacology, Kyushu University, for his critical guidance regarding the calculations of the composition of solutions used in the permeabilized muscle experiments.
*Fehr DM, Larach DR, LaBelle EF, McCann RL, Zangari KA, Schuller HG: Differential halothane stimulation of inositol phosphate production in pulmonary, coronary and peripheral vascular smooth muscle (abstract). ANESTHESIOLOGY 1993; 79:A649.