Judy Y. Su

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca2+ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca2+, Mg2+, MgATP2−, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml−1 SR was loaded actively with 0.02 mM45CaCl2, resulting in >90 nmol·mg protein−1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca2+, (2) the absolute amount of Ca2+ released by caffeine increases with the Ca2+ load of the SR, (3) increasing the ionic strength (μ) from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca2+ release increases with increasing (a) external Ca2+ concentrations up to 5 μM total Ca2+ (or 3 μM free Ca2+) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca2+ release is not affected by changes of either the Mg2+ or the MgATP2− concentration.

Effects of halothane on caffeine-induced tension transients in functionally skinned myocardial fibers

The effects of halothane on caffeine-induced tension transients in functionally skinned myocardial fibers were investigated. Fiber bundles from mechanically disrupted rabbit right ventricular papillary muscles were mounted on a tension transducer. The fiber preparation was loaded with Ca2+; Ca2+ was then released by the use of caffeine (25 mM); and the area of the resulting tension transient was measured. Each preparation was sequentially transferred from control to test to control solution. The control solutions were equilibrated with 100% N2, and the test solutions with a mixture of N2 and various halothane concentrations. The preparation was exposed to halothane during the Ca2+ uptake or the release phase only, or during both Ca2+ uptake and release phases. The areas of the test tension transients were compared with those of the two control tension transients. It was found that halothane depressed the caffeine-induced tension transient either during the uptake phase or the combined-uptake-and-release phase but not during the release phase. The halothane-induced depression was dose-dependent, reversible, and comparable to the depression observed in intact isolated papillary muscles. We conclude that halothane could induce myocardial depression by inhibiting Ca2+ uptake by the sarcoplasmic reticulum.

Isoflurane Activates PKC and Ca2+-Calmodulin-dependent Protein Kinase II via MAP Kinase Signaling in Cultured Vascular Smooth Muscle Cells

Background Protein kinase C (PKC) and Ca2+–calmodulin-dependent protein kinase II (CaMKII) have been implicated in isoflurane-increased force in skinned femoral arterial strips. The extracellular signal–regulated kinases (ERK1/2) of mitogen-activated protein kinase have been shown to be target effectors of PKC and CaMKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells. Methods Vascular smooth muscle cells were prepared by cell migration from isolated rabbit femoral arterial segments. Growth of passage of vascular smooth muscle cells (80–90% confluence, passage 5–10) was arrested for 48 h before experiments, during which time phorbol 1,3-diaceylester treatment was used to down-regulate PKC. Cells were treated for 30 min with one of the inhibitors of mitogen-activated protein kinase kinase (PD98059), PKC (Go6976 and bisindolylmaleimide), or CaMKII (KN-93 and KN-62) at 10 &mgr;m. After administration of isoflurane, vascular smooth muscle cells were frozen rapidly, homogenized, and centrifuged. The homogenates were used for identification of phosphorylated ERK1/2 or for further centrifugation to separate the membrane from the cytosol for identification of PKC isoforms (&agr; and ϵ) by Western blotting. Results Isoflurane increased ERK1/2 phosphorylation in a dose-dependent manner and reached a plateau at 10 min. PD98059 or down-regulated PKC blocked the increase of phosphorylated ERK1/2 levels by isoflurane, and bisindolylmaleimide, KN-93, or KN-62, but not by Go6976 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKCϵ but not of PKC&agr; was increased by isoflurane. Conclusions ERK1/2 signaling is downstream of PKC and CaMKII activated by isoflurane in vascular smooth muscle cells.

Effects of halothane on Ca2+-activated tension development in mechanically disrupted rabbit myocardial fibers

The effect of halothane on maximal and submaximal Ca2+-activated tension in mechanically disrupted right ventricular papillary muscle from rabbits was studied. Steady-state isometric tension generation was measured in the muscle bundle. The relaxing solution contained (in mM) [mg2+]=1, [K+]=70, [MgATP¨-]=2, [creatine phosphate¨-]=15, [EGTA total]=7 and imidazole proprionate. The contracting solution contained in addition Ca2+ in various concentrations. In all solutions ionic strength was maintained at 0.15 and pH at 7.00±0.02 at 20°C. Each fiber bundle was immersed in control solutions equilibrated with 100% N2 and test solutions equilibrated with various concentrations of halothane-N2 mixture. Increasing doses of halothane (1–4%) significantly shifted the relationship between Ca2+ and tension towards higher [Ca2+] and depressed the maximum Ca2+-activated tension. The maximum tension generated atpCa=3.8 was depressed 5% per 1% increase in halothane concentration. The percentage of maximum tension at submaximum Ca2+ concentrations (pCa=5.6–5.0) was not significantly decreased until halothane concentration was greater than 2%. It is concluded that halothane slightly but significantly depressed the interactions of contractile proteins and to a lesser degree Ca2+-activation of the regulatory proteins. The halothane-induced depression was completely reversible.

Effects of enflurane on functionally skinned myocardial fibers from rabbits

Enflurane, at clinical concentrations, decreases the contractility of isolated intact cardiac muscle. The authors investigated the intracellular mechanism(s) of this depression by examining the Ca2+ activation of the contractile proteins and Ca2+ uptake and release from the sarcoplasmic reticulum (SR) using functionally skinned fibers from right ventricular papillary muscle of rabbits. This preparation permits control of intracellular ionic composition (pH 7.0, 20 C). The [Ca2+]-tension relationship and caffeine-induced tension transient (as a measure of the amount of Ca2+ release) were analyzed. Enflurane significantly but only slightly depressed the maximum Ca2+-activated tension (10 per cent decrease at 5 per cent enflurane) and did not change the [Ca2+] required for half-maximal activation of the fibers. In contrast, enflurane markedly inhibited the Ca2+ uptake by the SR (30–85 per cent decrease at 2.5–7.5 per cent enflurane). The inhibition was dose-dependent. Ca2+ release from the SR with 25 mM caffeine was not changed at low concentrations of enflurane (1–5 per cent), but was decreased at high concentration (25 per cent decrease at 7.5 per cent enflurane). Enflurane (1–7.5 per cent), however, increased (13–44 per cent) the submaximum caffeine (2 mM)-induced Caa+ release from the SR, and the effect was not dose-dependent. The aforementioned effects were reversible. These results are similar to those previously reported for halothane. It is concluded that enflurane may induce myocardial depression mainly by inhibiting Ca2+ uptake by the SR.

Role of PKC in isoflurane-induced biphasic contraction in skinned pulmonary arterial strips.

Background Activation or inhibition of protein kinase C (PKC) has been implicated in the anesthetic-induced contraction or relaxation of different types of arteries. In skinned pulmonary arterial strips, the initial halothane-induced contraction has been attributed to PKC activation, but the subsequent relaxation has not. Whether isoflurane has a similar biphasic effect is not known. This study examined the role of PKC and its isoforms in the effect of isoflurane on pulmonary artery. Methods Rabbit pulmonary arterial strips were mounted on force transducers and treated with saponin to make the sarcolemma permeable (“skinned” strips). Skinned strips were activated by low Ca2+ (pCa 6.5 or pCa 6.3 buffered with 7 mm EGTA) or the PKC activator phorbol-12,13-dibutyrate (1 &mgr;m) until force reached a steady state (control). Various concentrations of isoflurane (test) were administered, and force was observed at time intervals up to 60 min. The PKC inhibitors (bisindolylmaleimide and Go6976 from 0.1 to 10 &mgr;m) were preincubated in a relaxing solution and the subsequent contracting solutions. The results were expressed as a percentage of control, with P < 0.05 considered significant for statistical comparison between the tests and time controls. Results In a dose-dependent fashion, isoflurane (1–5%) initially increased (5–40%) and then decreased (3–70%) the Ca2+- or phorbol-12,13-dibutyrate (pCa 6.7 buffer)–activated force. The increased in force caused by isoflurane was partially reduced by 3 and 10 &mgr;m bisindolylmaleimide, but not by Go6976. Isoflurane-induced relaxation was partially prevented by both inhibitors at 0.1 and 0.3 &mgr;m. Conclusions Isoflurane causes biphasic effects in skinned pulmonary arterial strips that may be in part mediated by different isoforms of PKC.

Mechanisms of isoflurane-increased submaximum Ca2+-activated force in rabbit skinned femoral arterial strips.

Background Isoflurane enhances contraction in isolated intact arterial rings by a protein kinase C (PKC) activator and also causes contracture in skinned arterial strips. This study investigated the mechanisms of this isoflurane activation of the contractile proteins of skinned strips. Methods The skinned strips, mounted on photodiode force transducers, were prepared from rabbit femoral arteries treated with saponin. The strips were activated by 1 [micro sign]M Ca2+ (buffered with 7 mM EGTA) with or without inhibitors for PKC and calmodulin-dependent protein kinase II (CaM kinase II). When force reached steady state, isoflurane was administered and changes in force were observed. Another group of the strips was frozen to assay myosin light chain phosphorylation (MLC-p) using two-dimensional electrophoresis and immunoblotting. Analysis of variance was used to compare the results from test and control groups. Probability values <0.05 were significant. Results Isoflurane (1-5%) dose dependently increased (24-81%) the Ca (2+-activated) force. At 1% and 5% isoflurane, MLC-p did not change either as the force increased or reached a new steady state level. However, with 3% isoflurane, MLC-p transiently decreased (29.1% and 17.1% of total MLC for 0% and 3% isoflurane, respectively). The 3% isoflurane-increased force was blocked by 10 [micro sign]M bisindolymaleidmide, an inhibitor of PKC, but not by 10 [micro sign]M Go-6976, an inhibitor of Ca2+-dependent PKC, and was enhanced 50% by 0.1 mM KN-62, an inhibitor of CaM kinase II. Conclusions Isoflurane increased submaximum Ca2+-activated force in skinned femoral arterial strips by activating Ca2+-independent PKC, possibly [Greek small letter epsilon] isoenzyme. The isoflurane-decreased MLC-p may be caused by activation of CaM kinase II.

Mechanisms of action of enflurane on vascular smooth muscle. Comparison of rabbit aorta and femoral artery.

Background:This study was performed to elucidate the mechanisms of action of enflurane by comparing the vascular smooth muscle responses of conduit arteries of larger (aorta) and smaller (femoral artery) diameter to enflurane using isolated rings and skinned strips. Methods:Isolated intact rings (endothelium denuded) of aorta and femoral artery from rabbits were activated by various concentrations of norepinephrine (NE) and the effects of enflurane were examined at the steady-state force. In a separate study, the rings were pretreated with verapamil before the NE activation and tested with enflurane. In the saponintreated arterial strips (“skinned”), the effects of enflurane on Ca2+ uptake or release from the sarcoplasmic reticulum were studied using caffeine-induced tension transients. Results:In isolated aortic rings, enflurane (0.9%-5%) enhanced tension development at low NE concentrations (5 and 30 nM) but depressed it at highest concentration (10 µm). In contrast, enflurane depressed tension development in the femoral artery at all NE concentrations. Enflurane caused significant increase in the NE-activated force in rings pretreated with verapamil. In skinned strips, enflurane (l%-3%) decreased Ca2+ uptake (concentration resulting in 50% depression: 1.8% for aorta and 2.5% for femoral artery) and increased Ca2+ release from the sarcoplasmic reticulum (59%-208% for aorta and 10%-55% for femoral artery). These effects were dose-dependent. Enflurane potentiated ryanodine depression of caffeine-induced tension transients. Conclusions:Enflurane has similar mechanisms of action in aorta and femoral artery: blocking Ca2+ influx, and causing, at least in part, Ca2+ release from the sarcoplasmic reticulum through the ryanodine-receptor channel. These cellular actions of enflurane account for the depression in femoral artery and enhancement in aorta of NE-activated force in isolated rings.

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 μM, ryanodine slightly but significantly increased the submaximal Ca2+-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine ≥1 μM decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and “activity”-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 μM and 5 μM for SL and AM, respectively, following ryanodine administration in the release phase, and 100 μM and 30 μM, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca2+ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca2+ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.

Mechanisms of ryanodine-induced depression of caffeine-induced tension transients in skinned striated rabbit muscle fibers.

Evidence suggests that ryanodien affects ligandgated calcium channels in the sarcoplasmic reticulum (SR) resulting in depressed muscle contraction. In skinned fibers from striated muscle the effects of ryanodine were examined (1) on Ca2+ uptake and on Ca2+ release to differentiate whether the effects are on the pump or channel, and (2) during the tension transient, with ryanodine exposure at various times either simultaneous with or directly after exposure to caffeine. Of total calcium content in the SR, 25 mM caffeine released>90% in papillary muscle (PM), ≃25% in soleus (SL), and ≃20% in adductor magnus (AM). Ryanodine (100 μM for 1–3 s for AM and SL; 1 μM for 7–10 s for PM), in the initial loading phase, did not significantly change, and in the initial release phase, markedly depressed the subsequent control caffeine-induced tension transients (C2) in all three muscle types. The depression increased with increasing time of exposure to ryanodine (10 μM) in the order of PM>AM>SL. Upon introduction of ryanodine after caffeine-induced tension transients, maximal depression was observed at half-maximum rise of the tension transient, followed by recovery of depression to completion in SL, and only partially in AM and PM at steady state of relaxation. The extent of recovery was in the order of SL>AM>PM. The data suggest that ryanodine affects Ca2+ releasing channel as a result of its binding to open channels.