Journal of Phy8iology (1990), 426, pp. 1-18 With figures Printed in Great Britain

1

HALOTHANE INCREASES Ca2+ EFFLUX VIA Ca 2+ CHANNELS OF SARCOPLASMIC RETICULUM IN CHEMICALLY SKINNED RAT MYOCARDIUM

BY J. S. HERLAND, F. J. JULIAN AND D. G. STEPHENSON* From the Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Boston, MA 02115, USA and the *Department of Zoology, La Trobe University, Bundoora, Victoria 3083, Australia

(Received 5 July 1989) SUMMARY

1. A method has been developed to study Ca2+ fluxes across the sarcoplasmic reticulum (SR) of chemically (saponin) skinned myocardium without interference from the SR Ca2+ pump. 2. Exposure of rat cardiac trabeculae to a solution containing 50 jtg/ml saponin for 10 min or longer caused an SR Ca2+ efflux which was not blocked by Ruthenium Red (RRed) and did not require the presence of nucleotides. 3. Exposure of the saponin-treated cardiac preparation to 11 mM-AMP, when the SR Ca2+ pump was not active, enhanced Ca2+ release from the SR by a mechanism which was blocked by 10 /SM-RRed. 4. The amount of Ca2+ loaded by the 10 min saponin-treated trabeculae was maintained constant for at least 3 min when the preparations were transferred to low [Ca2+] solutions (0-1 mM-EGTA; pCa > 7 5) containing ATP. This indicated that the Ca2+ pump can efficiently recycle Ca2+ lost from the SR under these conditions. 5. Halothane (0 47 and 1-89 mM) reversibly increased the rate of Ca2+ release from the SR regardless of whether or not the SR Ca2+ pump was active. This effect was more marked at 1-89 mm than at 0-47 mm. RRed (10 ,tM) completely blocked the Ca2+ release induced by both concentrations of halothane. 6. The presence of nucleotide (11 mM-AMP) did not affect the halothane-induced Ca2+ release when the Ca2+ pump was inactive. 7. Exposure of cardiac preparations to solutions containing more than 5 mmhalothane irreversibly damaged the ability of the SR to load Ca2 . 8. The results suggest that at lower doses (0'47 and 1-89 mM) halothane specifically and reversibly stimulates Ca2+ efflux via the RRed-sensitive SR Ca2+-release channel by a mechanism which does not require the presence of nucleotides or relatively high [Ca2+]. The results also suggest that AMP and halothane act independently and nonsynergistically to increase Ca2+ efflux through the same SR Ca2+-release channel. At higher doses (>5 mM) halothane irreversibly damages the SR membrane, presumably by disrupting the lipid bilayer.

MS 7807 1

PHY 426

2

J. S. HERLAND, F. J. JULIAN AND D. G. STEPHENSON INTRODUCTION

Halothane is a commonly used volatile anaesthetic that is also a potent mvocardial depressant (Komai & Rusy, 1982; Housmans & Murat, 1988 a, b: Wheeler. Rice, Hansford & Lakatta, 1988). Several studies have shown that halothane reduces net Ca2+ accumulation in sarcoplasmic reticulum (SR) of cardiac muscle (Su & Kerrick, 1979; Malinconico & McCarl, 1982; Casella, Suite, Fisher & Blanck, 1987), but the precise mechanisms of action responsible for the negative inotropic effect of halothane are still unclear. In this work, we used rat ventricular trabeculae which were skinned with saponin (Kitazawa, 1977) to study release of Ca2± from the sarcoplasmic reticulum under conditions such that the ATP-dependent Ca2± pump was switched on or off by adding or removing ATP from the bathing solutions (Kakuta, 1984; Horiuti, 1986). The results described here have been presented in part to the Biophysical Society (Herland, Stephenson & Julian, 1989). METHODS

Female rats (CD strain, Charles River Breeders, Wilmington, MA, USA), 226-250 g, were killed by cervical dislocation. The heart was immediately removed and rinsed several times, while beating, with several portions of an oxygenated physiological solution at room temperature (23 TC). The solution contained 144 mM-Na+, 4-2 mM-K+, 1-2 mM-Mg2", 153-2 mM-Cl-, 1-2 mM-SO42-, 7-4 mm-HEPES buffer, 5-6 mM-glucose, 2-5 mM-Ca2", and insulin (5 U/1000 ml), at pH 7-4. The heart was transferred to a silicone elastomer dish filled with physiological solution in which the right ventricular free wall was cut open and peeled back. Right ventricular trabeculae were examined under a binocular microscope for suitability, i.e. 80-120 ,ym wide and a minimum of 1-5 mm long. If a suitable muscle or muscle segment was found, individual strands teased from 10-0 twisted silk were used to gently tie off a 1-52-0 mm segment. The muscle segment was then removed from the heart and placed in an experimental chamber filled with relaxing solution B (described in the 'solutions' section), and gently tied to stainless-steel wires extending from a fixed arm and a force transducer. The segment was then aligned parallel to the wires and was securely attached with two double overhand knots of 10-0 monofilament nylon. The muscle preparation was then skinned by placing it into relaxing solution B containing 50 ,ug/ml saponin (Endo & Kitazawa, 1978) for 10 min unless otherwise stated. Force was measured using a Cambridge Technology model 400 force transducer with sensitivity set at 200 mV/mN. The wires from the transducer and fixed arm were mounted on separate micromanipulator arms allowing independent movement of each wire in any direction. The experimental muscle chamber contained a wide shallow well which facilitated mounting of the preparation and four other wells, each with a capacity of about 1 ml in which the preparation could be immersed. Changing wells could be accomplished in 1-2 s. After skinning, the preparation was stretched until resting force was just noticeable (< 5% of maximum Ca2±-activated force). Forces were recorded on a Nicolet model 4094B digital oscilloscope and stored on floppy discs. A small volume (0 5-10 0 1tl) of liquid halothane (Halocarbon Laboratories) was injected into a closed glass syringe containing a small stir bar and 5-10 ml of given solution. The syringe w as then placed on a magnetic stirrer and stirred for 60 min. In this way. the control and halothane solutions were always identical in composition except for the presence of the halothane. Halothane was used in concentrations of 0-47 and 1-89 mm unless otherwise stated. These concentrations were routinely verified by gas chromatography and were found to be within 5 % of the desired values. A PerkinElmer Sigma 1 gas chromatograph with Carbo pack B column (Supelco, Bellefonte. PA. USA) and a flame ionization detector was used for these measurements. Gas chromatographic measurements of the concentration of a standard 1 mm solution of halothane over the course of this study had a coefficient of variation of 10 %. In the concentrations used, halothane altered neither pH nor free Ca2+ in any of the solutions as judged from direct measurements with pH and Ca2+-sensitive (Orion model 93-20) electrodes. Although evaporation of halothane did occur, it was found that after

EFFECT OF HALOTHANE ON SARCOPLASMIC RETICU"LUMI 10 min at room temperature (23 'C), 80-85% of the original (syringe) concentration remained in solution in the tissue chamber. All experiments with halothane were completed with 5 min of filling the well with the respectively halothane-containing solutions. The composition of the bathing solutions used for the saponin-skinned pieces of ventricular trabeculae are shown in Table 1. Solutions of type A. B. H. were prepared using methods modified from those previously described by Ashley & Moisescu (1977). 'Moisescu & Thieleezek (1978) and TABLE 1. Bathing solutions for SR experiments with saponin-skinned trabeculae ATP AMP HDTA EGTA CaEGTA K Caffeine Nat Solution (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) A 8 50 16 136 B 8 50 16 136 H 8 50 16 136 SR Ca-load 8 49-9 * * 16 136 Relax 01 8 49-9 16 136 Rigor 0.1 49-9 146 11 AMP-Rigor 01 22 122 49-9 Caffeine 0.1 8 49-9 16 136 30 * Total EGTA in SR Ca-load solution was 0-1 mM; = All 05 /LM. contained solutions (mM): [Ca2"] Mg2 , 1; TES, 70; NaN3, 1; pH 7-10 +001 at 23 + 1 'C. The balance between negative and positive charges was made up by Cl-, which varied between 4-4 mm in AMP-Rigor solution and 27 mm in Rigor solution. The ionic strength of the solutions (F/2 = £c-Iz1I2) was between 200 and 217 mM, while the concentration of ionic equivalents (I = iXcilzil) was between 145 and 155 mm. t Does not include 1 mM-Na+ from NaN3. HDTA, 1, 6-diaminohexane-NNN',N'-tetraacetic acid; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid; EGTA, ethyleneglycol-bis-(,8-aminoethyl ether) NVN',V'-tetraacetic acid.

Stephenson & Williams (1981). The SR Ca-load solution was made from solution H with 0-1 mmtotal EGTA. Free Ca2+ in the SR Ca-load solution was adjusted to about 0 5 /SM as measured with the Ca2+-sensitive electrode. This Ca2+ concentration was subthreshold for force generation but did allow ATP-dependent Ca2+ loading into the sarcoplasmic reticulum (Fabiato & Fabiato, 1975; Endo, 1977). The ionized Ca2+ in solutions was not modified by the presence of up to 10 mMhalothane, as measured with the Ca2+-sensitive electrode. The Relax solution was made from solution H by adding 0-1 mM-EGTA from relaxing solution B. In this solution the final pCa was greater than 7-5. The Rigor solution lacked ATP in order to make the Ca2+ pump inactive (Kakuta, 1984; Horiuti, 1986). Total Mg2+ (added as MgCl2) concentration was reduced to 1-2 mm so that [Mg2+] was about 1 mm, and the KCl concentration was increased to make the concentration of ionic equivalents similar to that in the other solutions. The AMP-Rigor solution lacked ATP but contained 11 mm-AMP in order to activate the sarcoplasmic reticulum Ca2+-release channel (Horiuti, 1986). Total MgCl2 was reduced to 2-2 mm in this solution so that [Mg2+] was about 1 mM as in all other solutions. The caffeine-containing Ca2+-release solution (Caffeine) was made from Relax solution by adding 30 mM-caffeine. All solutions contained 1 mM-NaN3 to prevent Ca2+ accumulation by mitochondria. In some experiments 10 /M-Ruthenium Red (Sigma) was added to aliquots of the above solutions without changing the concentrations of their constituents including that of ionized Ca2+ concentration by more than 1 %. The amount of releasable Ca2+ from the sarcoplasmic reticulum was estimated from the area under the caffeine-induced for transients (Endo. 1977; Kitazawa. 1977; Kakuta. 1984; Horiuti. 1986; Ohta, Endo, Nakamo, Morohoshi, Wanikawa & Ohga, 1989). Since the release of Ca2+ by caffeine from the sarcoplasmic reticulum is a relatively slow process. lasting many seconds (Stephenson, 1981), the area under the caffeine-induced force transients, rather than the peak force response. provides a more appropriate estimate of the total amount of Ca2± released. An integration programme was used to calculate the area under the caffeine-induced force transients as illustrated in Figs 1-8. A dose-response experiment was performed in order to determine the caffeine concentration required to produce the maximal caffeine-induced Ca2+-release response under our conditions. 1-2

4

J. S. HERLAND, F. J. JULIAN AND D. G. STEPHENSON

Saturation was achieved at 20 mM-caffeine, so 30 mM-caffeine was chosen to assure maximal response. Addition of 2% Triton X-100 in the Caffeine solution to completely disrupt the sarcoplasmic reticulum and release all stored Ca2+ produced only a small increase of 10-15% in the area under the force transient. This suggests that 30 mM-caffeine in the Caffeine solution causes release of most Ca2+ in the sarcoplasmic reticulum of cardiac preparations. Prior to loading Ca2+ in the SR Ca-load solution the SR was always emptied of Ca2+ by exposure to the Caffeine solution for at least 1 min and then to the 50 mM-EGTA solution B for another minute. The preparation was then transferred for 10-15 s from solution B to solution H. This was sufficient to remove most of the EGTA in the preparation because increasing the period of time in solution H to 2 min did not affect the time course of Ca2+ loading in the SR Ca-load solution. For experiments in which the preparation was exposed to halotharle during the loading period, the preparation was moved, at the appropriate moment, to an identical loading solution mixed with halothane. At the end of the loading period the preparation was moved into caffeine-containing solution for assay of Ca2+ releasable from SR. Results for individual preparations were expressed as relative areas compared with the mean of the area for controls before and after. For studies of SR Ca2+ with the Ca2+ pump 'on', the preparation was loaded with Ca2+ in the SR Ca-load solution as described above. At the end of the loading period, the preparation was moved from loading solution to the Relax solution for a period of 3 min and then into the Caffeine solution to assay Ca2+ release. For studies of SR Ca2+ with the Ca2+ pump 'off', the preparation was loaded with Ca2+ in the SR Ca-load solution as described above. At the end of the loading period, the preparation was moved to either Rigor or AMP-Rigor solution for a period of 2 min. The preparation developed a rigor contracture in either of these solutions. The preparation was then moved to the Relax solution for 1 min in order to cause relaxation prior to immersion into Caffeine solution for assay of Ca2+ release. RESULTS

SR Ca2+ loading in the presence and absence of ATP Trace A of Fig. 1 shows a response obtained from a trabecula after a 10 min loading period in the SR Ca-load solution. After equilibration for 1 min in Relax solution with 0.1 mM-EGTA (Ca2+ < 0 03 ,UM), exposure to 30 mM-caffeine (Caffeine) elicited a typical caffeine-induced force transient. The very small caffeine-induced force responses in traces B and C show that without ATP in the loading solution, essentially no Ca2+ was stored in the SR. (The small transient responses to caffeine in B and C are most probably due to some Ca2+ being loaded by the SR immediately after transferring the preparation from the Rigor solutions to the Relax solution which contained ATP.) This was true regardless of whether or not AMP was included in the loading Rigor solutions (traces B and C). Note that a rigor contracture developed in the ATP-free loading solution. Before testing for force generation by exposure to the caffeine solution, it was necessary to produce relaxation by soaking for 1 min in the Relax solution. The large response shown in trace D indicates that the rigor contractures did not damage the preparation. The results from Fig. 1 show that Ca2" loading by the SR can be reversibly prevented by omitting ATP in the SR loading solution. Results similar to those shown in Fig. 1 were obtained with five preparations.

Ca2" efflux from SR in the presence and absence of nueleotides The area under the 30 mM-caffeine-induced force responses differed by less than 5% following exposure to the Relax solution for 30, 60, 120 and 180 s after a typical 10 min loading period in the SR Ca-load solution. This observation obtained with five preparations, suggests that the amount of Ca2+ releasable by caffeine from the SR is not greatly changed by incubation in the Relax solution for up to 3 min. The area

EFFECT OF HALOTHAIVE ON SARCOPLASMIC RETICULUM 5 under the caffeine-induced force response was higher (by about 20 %) when the preparation was transferred to the Caffeine solution directly from the SR Ca-load rather than from the Relax solution (see e.g. Fig. 7A, B and F, G). This was entirely expected because of the higher Ca2+ concentration in the SR Ca-load than in the Relax solution. A

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Fig. 1. Effect of removing ATP from the loading environment. Sequential force response obtained with one trabecula. In A and D the SR has been initially depleted of (Ca2d and then the preparation was loaded in the SR Ca-load solution ([Ca2+J = 0-5 /M) for 10 min and then was exposed to 30 mM-caffeine solution after equilibration for 1 min in the Relax solution ([Ca21] < 003 yM) as indicated by arrows. In B and C the preparation was exposed for 10 min to Rigor and AMP-Rigor solutions, respectively, each of which contained additional Ca2+ such that [Ca2+] was 0 5 /M, and then treated with Relax and Caffeine solutions as in A and D. The area under the caffeine-induced responses is delineated by a dashed line.

Incubation in the Rigor solution for 2 min after loading the SR with Ca2+. and before equilibration in the Relax solution (Fig. 2, traces B and D), show that Ca21 was lost from the SR as indicated by the smaller caffeine-induced responses compared with the controls (traces A and E). The caffeine-induced response was further reduced when AMP was present in the Rigor solution (compare trace C with traces B and D). The area of the caffeine-induced response of trace C normalized to the mean of the areas of the caffeine-induced responses of traces B and D was 0-49. These results show that there was substantial Ca2+ efflux from the SR in the ATP-free solutions, and that this Ca2+ efflux was further stimulated by AMP. The results obtained with fifteen preparations are summarized in Table 2.

Effects ofRuthenium Red on Ca2+ efflux from SR In the presence of the specific SR Ca21 channel blocker Ruthenium Red (RRed; 10 /tM), the caffeine-induced force responses were of similar magnitude regardless of whether or not AMP was included in the ATP-free solutions. This is shown in Fig. 3, traces B. C and D, and is summarized in Table 2. This suggests that AMIN stimulates Ca2+ release via the RRed-sensitive Ca2±-release channel. The area of the caffeine-induced response of trace C normalized to the mean of the areas of the

J. S. HERLAND, F. J. JULIAN AND D. G. STEPHENSON

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TABLE 2. Effect of AMP on releasable Ca2+ from sarcoplasmic reticulum

At/AC 10 ,um-Ruthenium Red Treatment Control No in SR Ca-load, Rigor Statistical condition condition Ruthenium Red and AMP-Rigor solutions significance Incubation Incubation 0-64 + 013 0-58 + 012 n.s. in Rigor in Relax N= 10 N= 7 solution solution Incubation Incubation 0-57 +014 0-94+0 15 P < 0 002 N= 5 in AMP-Rigor in Rigor N= 6 solution solution The incubation protocols were the same as shown in Figs 2 and 3. Normalized area (At/AC) of the caffeine-induced force transient responses after treatment (At) versus mean (Aj) of areas for controls before the after (mean s.D.).

EFFECT OF HALOTHANE ON SARCOPLASMIC RETIl('TLU(71 caffeine-induced responses of traces B and D is 0 82. The average value from six experiments is 0 94 (Table 2). Without RRed. this value is 0 49 for Fig. 2. while the average for five experiments is 0(57 (Table 2). It should be noted that 10 ,am-RRed produced a shift of less than 003 pCa units towards higher pCa values of the A

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Fig. 3. Effects of ATP removal and AMIP addition on the amount of releasable (a21 inl the SR in the presence of Ruthenium Red (RRed). Sequential responses from one trabecula where 10 ,uM-RRed was present in all solutions with the exception of the Caffeine solution. For each trace the SR has been initially depleted of Ca2+ and then the preparation has been loaded for 10 min in the SR Ca-load solution with 10lM-RRed. The results are similar to those shown in Fig. 2 except that exposure to A.MP-Rigor solution ill the presence of 10 ItM-RRed did not appear to decrease the amount of releasable Ca2+ as much as in the absence of RRed (Fig. 2 and Table 2). The area under the caffeine-inlduced force responses is delineated by a dashed line.

isometric force-pCa curve of cardiac preparations activated in heavily Ca21-buffered (50 mM-total EGTA) solutions obtained by mixing solutions A and B in various proportions. This result was obtained with three cardiac preparations treated with 2% v/v Triton X-100 to remove any possible contribution from intracellular Ca 2 compartments to the myofibrillar Ca + level. From Table 2 one could also draw the conclusion that RRed did not block the efflux of Ca21 from the SR in the absence of nucleotides (Rigor solution), indicating that this Ca2± efflux was not via the RRed-sensitive SR Ca2±-release channel, but rather through a non-specific pathway. This pathway is most probably produced by saponin. since increasing the exposure time to saponin increased the magnitude of this efflux.

Effects of prolonged saponin treatment The effects of exposure of the cardiac preparation to saponin for different lengths of time is shown in Fig. 4, where traces A, B and C w ere obtained from a preparation

8

J. S.

HERLAND, F. J. JULIAN AND D. G. STEPHENSON

treated for 10 min in saponin (50 fg/ml) solution, while traces D, E and F were obtained from the same preparation after a further 20 min soak in the saponin solution. Note that the caffeine-induced force responses obtained immediately after loading in the SR Ca-load solution were not greatly reduced after additional saponin Caffeine

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Caffeine Fig. 4. Effect of duration of saponin (50 Iug/ml) treatment upon net Ca21 loading and Caa21 efflux from SR. Caffeine-induced responses with a trabecula exposed for 10 min (traces A-C) and then for an additional 20 min (traces D-F) to 50 ,ug/ml saponin. Note the small caffeine-induced response in trace E compared with B after exposure to Rigor solution, which indicates a markedly higher Ca2' efflux from the SR after 30 min than after 10 min exposure to saponin. For each trace the SR has been initially depleted of Ca2' and then the preparation has been loaded for 10 min in the SR Ca-load solution. The area under the caffeine-induced force responses is delineated by a dashed line.

treatment (average area under the force transients in D and F is 0 75 of that in A and C). However, the caffeine-induced response was greatly diminished after the additional saponin treatment following exposure to the Rigor solution for 1 min (area under force transient in E is only 0-23 of that in B). This diminished response indicates that there was a much higher net Ca21 loss from the SR in the Rigor solution after the preparation was exposed for an additional 20 min to saponin. One could argue that the removal of ATP from the preparation in the Rigor solution was faster in Fig. 4E than in Fig. 4B due to a faster rate of diffusion of all ionic species in and out of the preparation after prolonged exposure to saponin. Then, assuming the same Ca2+ leak from the SR, more Ca2+ would have leaked out from the SR in the better skinned preparation during the 1 min long exposure to the Rigor solution. In agreement with this is the slightly more prolonged state of rigor in trace E than in trace B in Fig. 4. However, the relative response in trace E compared with controls

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severalfold higher after a 30 min treatment with saponin than after a 10 min treatment with saponin when the Ca2+ pump is not active. The similarity in magnitude of the caffeine-induced responses shown in traces A and D indicate that the Ca2± pump can cope reasonably well in the presence of ATP with an increased Ca2+ efflux caused by the longer saponin treatment. The faster decay time of the caffeine response in Fig. 4D and F compared with that in A and C is probably due to the faster rate of Ca2+ diffusion out of the preparation after prolonged exposure to saponin.

Effects of halothane on Ca2+ efflux from SR When the saponin-skinned cardiac preparations were exposed to halothane in the Rigor solution (Fig. 5, B) the subsequent caffeine-induced force transients were clearly smaller than controls (A and C) showing that halothane depleted the SR store of Ca2+. The summary of results obtained with halothane is shown in Table 3. Halothane's effect was greater at 1P89 mm than at 0-47 mm. implying a dosedependent effect. For these doses of halothane, the effect was fully reversible. When 10 /uM-RRed was included in the SR Ca-load, Rigor and Relax solutions the sizes of the caffeine-induced force transients were essentially not changed by exposure to halothane (Fig. 5D-G and Table 3), showing that halothane promotes Ca2+ release from the SR via the RRed-sensitive Ca2+ release channel. When halothane was added to the AMP-Rigor solution (Fig. 6B and Table 3), the subsequent caffeine-induced force transient was smaller than controls (Fig. 6A and

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14

J. S. HERLAN.rD, K J. JULIAN A.NVD D. G. STEPHE41V=NO

C and Table 3). The decrease of releasable Ca2± in the presence of 0 47 mM-halothane and 11 mM-AMP as shown in Fig. 6B did not appear to be quantitatively different from that caused by 0-47 mM-halothane in the Rigor solution (Table 3 and Fig. 5) showing that halothane does not release Ca2+ via a mechanism which is activated by nucleotides. Halothane's effect upon SR releasable Ca2+, with the Ca2+ pump active. was studied by including halothane in the Relax solution subsequent to loading in the SR Ca-load solution and prior to caffeine exposure (Fig. 7 C and D). The caffeine-induced force transients became progressively smaller as the halothane dose was increased. Halothane's effect was fully reversible as shown in Fig. 7E. The ability of halothane to release Ca2+ is more directly demonstrated by results shown in Fig. 71), where simple exposure to halothane in a relaxing solution caused a force transient. Both the appearance of the halothane-induced force transient and the reduction in area of the subsequent caffeine-induced force transient were blocked when 10 /ImRRed was included in both the SR Ca-load and Relax solution (Fig. 7F-J). A summary of results of this type of experiment is also included in Table 3. Halothane effects on net SR Ca2+ loading Halothane's effect upon net Ca2+ loading by the SR was studied by exposing the preparation to halothane for the last 3 min of a 10 min loading period in the SR Caload solution. This was done by moving the preparation from a well with SR Ca-load solution into another well with Ca-load solution in which halothane had been previously dissolved. The subsequent caffeine-induced force transient was smaller after exposure to halothane (Fig. 8B and C, Table 3) compared with controls (A and D). Halothane's effect was again reversible (Fig. 8D) and was greater at 1t89 mM (C) than at 0-47 mm (B). Also, 10 jtM-RRed effectively blocked the SR effects of halothane. In two experiments the cardiac preparations were exposed to either 5 or 9 4 m.mhalothane. In both cases, the preparations rapidly and irreversibly lost the ability to either load Ca2+ or release it when exposed to 30 mM-caffeine. This shows that these high concentrations of halothane irreversibly damage the SR.

DISCUSSION

Effects of saponin on Ca2t efflux from SR Results obtained from this study show that exposure to 50 jug/ml saponin produces a significant pathway for Ca2+ efflux from the SR which does not depend on the presence of nucleotides and is not blocked by RRed. Assuming that the area under the caffeine-induced force response provides a reliable index for the total amount of releasable Ca2+ from the SR (Endo, 1977; see Methods), then the results in which the SR pump was inactive (Table 2, Figs 2 and 3) suggest that up to 40 'S of the SR-releasable Ca2+ can be lost in 2 min via this pathway from preparations skinned for 10 min in 50 jig/ml saponin. Earlier observations by Endo & Kitazawa (1978) suggested that 20 min exposure to 50 /tg/ml saponin selectively destroys the surface membrane of cardiac preparations and that higher concentrations of saponin

EFFECT OF HALOTHANE ON SARCOPLAsSMWIC RETl TL11MI

1.5

were necessary to make the SR leaky. Increasing the total duration of the saponin treatment to 30 min markedly increased the magnitude of this Ca2+ efflux as indicated by the 4-fold reduction in the size of the caffeine-induced response (Fig. 4B and E). The mechanism for this Ca2± efflux is likely to involve small perforations in the SR membrane similar to those reported to occur in the sarcolemma after exposure to saponin (Miller. Elder & Smith. 1985; Kargacin & Fav, 1987). When the Ca2+ pump was active, the amount of Ca2+ in the SR decreased only slightly. particularly in the preparations treated for 10 min with saponin. This indicates that the Ca2+ pump is capable of preventing net Ca2+ loss from the SR even in the presence of a substantial Ca2+ efflux.

Effects of AMP on Ca2+ efflux from SR The net Ca2+ efflux from cardiac SR was increased when AMP was present in the ATP-free solution termed 'AMP-Rigor'; that is, when the Ca2+ pump was inactive (Kakuta, 1984; Horiuti, 1986). This efflux component was effectively blocked by 10 /tM-RRed, indicating that this nucleotide-dependent Ca2+ efflux was via the RRed-sensitive Ca2+-release channel. Similar properties were observed with single SR Ca2+-release channels from cardiac muscle incorporated in lipid bilayers (Rousseau, Smith & Meissner, 1987).

Effects of halothane and the mechanism of action on SR Halothane in concentrations of 047 and 1P89 mmx reversibly increased the Ca2+ efflux from the SR in a dose-dependent fashion. However, exposure to halothane in concentrations above 5 mm resulted in the irreversible loss of ability to load Ca2+ by the SR. The effect of halothane at concentrations of 047 and 1P89 mm on the maximum Ca2+-activated force response was minimal (Su & Kerrick, 1978; Herland, Stephenson & Julian, 1988) and in our hands tended to make the contractile apparatus more sensitive to Ca2+ (Herland et al. 1988). From the results of Kissin, Morgan & Smith (1983), we estimate that 0 47 and 1[89 mm halothane in the blood of the rat would correspond under our conditions to about 07 and 2-8 MAC (minimum alveolar concentration) respectively. One MAC in animals is the alveolar concentration of anaesthetic vapour mid-way between the concentrations required to block and allow gross movement in response to a supramaximal painful stimulus (Eger, Saidman & Brandstater, 1965). Therefore, our results show that halothane has a marked effect on the Ca2+ efflux from the cardiac SR and on the net Ca2+ loading of the cardiac SR at concentrations below 1 MAC. Thus, 047 mM-halothane (0-7 MAC) decreased the area of the caffeine-induced force response by a factor of 3 when the Ca2+ pump was inactive and by a factor 1[9 when it remained active (Table 3) despite the increase in the sensitivity of the contractile apparatus to Ca2+ in the presence of halothane (Herland et al. 1988). The entire Ca2+ efflux induced by halothane was through the RRed-sensitive Ca2+ channel because the halothane-induced component was completely blocked by 10 jtM-RRed (Table 3). From our experiments it can also be concluded that halothane in concentrations as high as 1[89 mm is unlikely to have a marked effect on the Ca2+ pump because the amount of caffeine-releasable Ca2+ in the presence of halothane was not different from controls when 10 ItM-RRed was present in the solutions

16

J. S. HERLAND, F. J. JULIAN AND D. G. STEPHENSON

(Table 3). This was irrespective of whether or not the Ca21 pump was active. This is an important conclusion because from earlier results (Su & Kerrick, 1979) it was not possible to distinguish between a halothane effect on the Ca2+ pump or on the Ca2+ efflux. Another important observation from this study is that halothane stimulates Ca2+ release from the SR in the absence of nucleotides. This result alone strongly suggests that halothane does not promote Ca2+ release from the cardiac SR by enhancing the Ca2+-induced Ca2+ release mechanism as suggested from experiments with SR vesicles (Ogawa & Kurebayashi, 1982; Ohnishi, Taylor & Gronert, 1983; Beeler & Gable, 1985; Ohnishi, 1987; Katsuoka & Ohnishi, 1988). This is because the presence of nucleotides in millimolar concentration is considered essential in order for Ca2+_ induced Ca2+ release to take place under physiological conditions (Fabiato & Fabiato, 1975; Endo, 1977; Kaktita, 1984; Horiuti, 1986; Ohta et al. 1989). Moreover, work on the Ca2+-release channel in lipid bilayers indicates that the Ca2+_ dependent activation of the Ca2+-release channels is greatly potentiated in the presence of nucleotides (Rousseau et al. 1987). We therefore suggest that halothane stimulates Ca2+ release from the cardiac SR through the specific Ca2+-release channels, which are blocked by RRed (Nagasaki & Fleischer, 1988), but not via the Ca2+-induced Ca2+-release mechanism which is greatly potentiated in the presence of nucleotides. This implies that Ca2+ may be released via the cardiac RRed-sensitive Ca2+-release channel without involving the Ca2+-induced Ca2+-release mechanism. The suggestion that Ca2+ release promoted by halothane is through the specific Ca2+release channels in the SR is further supported by indirect evidence. Thus, if the Ca2+ release promoted by halothane was through a difference population of RRedsensitive Ca2+-release channels other than the nucleotide-sensitive Ca2+-induced Ca2+-release channels, or if AMP and halothane interacted synergistically to activate Ca2+ release from SR, then one would expect that proportionally more Ca2+ should be released by halothane in the presence of AMP than in the absence of AMP. As shown in Table 3 (first two conditions) this was clearly not the case. On the contrary, the almost identical normalized results obtained under the two conditions (0-32 and 034 respectively) strongly suggest that halothane and AMP act non-synergistically and upon the same Ca2+-release channels. In addition, these results appear to rule out the possibility that AMP interferes with the mechanism of action of halothane on the SR Ca2+ channel. Therefore, halothane most probably activates the SR Ca2+-release channels directly regardless of whether nucleotides are present or not. These results may be of considerable interest with regard to the syndrome of malignant hyperthermia (see e.g. Ohnishi, 1987; Ohta et al. 1989) if halothane stimulates calcium release through the Ca2+-release channels in the same way in skeletal muscle SR as repeated here for cardiac muscle SR. Halothane could act directly on the Ca2+ channel protein subunits as suggested by the reversible change in the conformation of SR proteins observed at dose levels similar to those used in this study (Augustin & Hasselbach, 1973). Alternatively, the mode of action of halothane on the Ca2+-release channel could be by modifying the lipid-Ca2+-release channel interaction. Halothane is known to be lipid soluble and has been shown to have a disordering effect upon the lipid bilayer of skeletal muscle SR (Ohnishi, Waring, Fang, Horinchi, Flick, Sadanaga & Ohnishi, 1986). Therefore,

EFFECT OF HALOTHANE ON SARCOPLASMIC RETICULUM 1M17 it could change the conformation of the Ca2+ channels allowing for greater Ca2+ efflux. Our results further suggest that at higher concentrations (> 5 mM) halothane disrupts the lipid bilayer causing irreversible damage to the SR membrane. Concluding remarks These results show that at least part of halothane's myocardial depressant effect is caused by a specific action on the RRed-sensitive, AMP-activated Ca2+-release channel in the SR and can explain the reduction in intracellular free [Ca21] changes (Wheeler et al. 1988) and in the ability of the SR to accumulate Ca2+ (Lain, Hess, Gertz & Briggs, 1968; Su & Kerrick, 1979; Casella et al. 1987). The results also demonstrate the potential use of halothane as a tool to open specifically the Ca2+release channels of the SR when used in lower concentrations, or to disrupt cellular membranes when used in higher concentrations. The authors appreciate the efforts of Mary Gioiosa in typing the manuscript. We also appreciate the advice of Dr G. Richard Arthur as well as the use of his gas chromatograph for measurement of halothane concentrations of our solutions. This study was supported by NIH grant HL35032 (J.S.H. and F.J.J.) and NH & MRC (D.G.S.).

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