Propofol increases pulmonary artery smooth muscle myofilament calcium sensitivity: role of protein kinase C

BACKGROUND: Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway. METHODS: Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i. RESULTS: The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with G? 6976 also attenuated propofol-induced increases in tension. CONCLUSION: These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.     

Propofol Attenuates β-Adrenoreceptor-mediated Signal Transduction via a Protein Kinase C-dependent Pathway in Cardiomyocytes

Background: Activation of [beta] adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. The objectives of the current study were to investigate the effects of propofol on [beta]-adrenoreceptor-mediated increases in cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i), cell shortening, L-type Ca2+ current (ICa) and cyclic adenosine monophosphate (cAMP) accumulation. The authors also investigated the site of action of propofol in the [beta]-adrenoreceptor signaling pathway, as well as the role of protein kinase C (PKC), and tested the hypothesis that propofol would inhibit the [beta]-adrenoreceptor signaling pathway via a PKC-dependent mechanism.

Methods: Freshly isolated ventricular myocytes were obtained from adult rat and guinea pig hearts. Myocyte shortening (video edge detection) and [Ca2+]i (fura-2, 340/380 ratio) were monitored simultaneously in individual cells. Conventional whole cell patch clamp analysis was used to measure the ICa in individual myocytes. cAMP production was assessed in suspensions of myocytes using an enzyme immunoassay kit.

Results: Propofol (0.1-10 [mu]m) had no effect on steady state [Ca2+]i, cell shortening, ICa, or cAMP production. In contrast, propofol caused dose-dependent decreases in isoproterenol-stimulated increases in [Ca2+]i, shortening, ICa, and cAMP. Forskolin-induced increases in [Ca2+]i, shortening, and cAMP production were not altered by propofol. PKC activation with phorbol myristate acetate attenuated isoproterenol-stimulated cAMP production. Inhibition of PKC with bisindolylmaleimide (broad range inhibitor) or Go 6976 (inhibitor of Ca2+-dependent PKC isoforms) abolished propofol-induced inhibition of isoproterenol-stimulated increases in [Ca2+]i, shortening, and cAMP production.     

Mechanisms of Direct Inhibitory Action of Ketamine on Vascular Smooth Muscle in Mesenteric Resistance Arteries

Background: Ketamine was previously suggested to relax vascular smooth muscle by reducing the intracellular Ca2+ concentration ([Ca2+]i). However, no direct evidence is available to indicate that ketamine reduces the [Ca2+]i in vascular smooth muscle of systemic resistance arteries.

Methods: Endothelium-intact or -denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and [Ca2+]i were measured simultaneously in the fura-2-loaded, endothelium-denuded strips. In some experiments, only isometric force was measured in either the endothelium-intact or [beta]-escin-treated, endothelium-denuded strips.

Results: In the endothelium-intact strips, lower concentrations (<= 30 [mu]m) of ketamine slightly enhanced norepine-phrine-induced contraction, whereas higher concentrations (>= 100 [mu]m) of ketamine inhibited both norepinephrine- and KCl-induced contractions. In the fura-2-loaded strips, ketamine (>= 100 [mu]m) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine or KCl. Ketamine also inhibited the norepinephrine-induced increase in [Ca2+]i after treatment with ryanodine. In the absence of extracellular Ca2+, ketamine notably inhibited the norepinephrine-induced increase in [Ca2+]i, whereas it only minimally inhibited caffeine-induced increase in [Ca2+]i. Ketamine had little influence on the [Ca2+]i-force relation during force development to stepwise increment of extracellular Ca2+ concentration during either KCl depolarization or norepinephrine stimulation. Ketamine did not affect Ca2+-activated contractions in the [beta]-escin membrane-permeabilized strips.     

Mechanisms of Direct Inhibitory Action of Isoflurane on Vascular Smooth Muscle of Mesenteric Resistance Arteries

Background: Isoflurane has been shown to directly inhibit vascular reactivity. However, less information is available regarding its underlying mechanisms in systemic resistance arteries.

Methods: Endothelium-denuded smooth muscle strips were prepared from rat mesenteric resistance arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips, whereas only the force was measured in the [beta]-escin membrane-permeabilized strips.

Results: Isoflurane (3-5%) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine (0.5 [mu]m) or KCl (40 mm). These inhibitions were similarly observed after depletion of intracellular Ca2+ stores by ryanodine. Regardless of the presence of ryanodine, after washout of isoflurane, its inhibition of the norepinephrine response (both [Ca2+]i and force) was significantly prolonged, whereas that of the KCl response was quickly restored. In the ryanodine-treated strips, the norepinephrine- and KCl-induced increases in [Ca2+]i were both eliminated by nifedipine, a voltage-gated Ca2+ channel blocker, whereas only the former was inhibited by niflumic acid, a Ca2+-activated Cl- channel blocker. Isoflurane caused a rightward shift of the Ca2+-force relation only in the fura-2-loaded strips but not in the [beta]-escin-permeabilized strips.     

Droperidol Inhibits Intracellular Ca2+, Myofilament Ca2+ Sensitivity, and Contraction in Rat Ventricular Myocytes

Background: Droperidol has recently been associated with cardiac arrhythmias and sudden cardiac death. Changes in action potential duration seem to be the cause of the arrhythmic behavior, which can lead to alterations in intracellular free Ca2+ concentration ([Ca2+]i). Because [Ca2+]i and myofilament Ca2+ sensitivity are key regulators of myocardial contractility, the authors' objective was to identify whether droperidol alters [Ca2+]i or myofilament Ca2+ sensitivity in rat ventricular myocytes and to identify the cellular mechanisms responsible for these effects.

Methods: Freshly isolated rat ventricular myocytes were obtained from adult rat hearts. Myocyte shortening, [Ca2+]i, nitric oxide production, intracellular pH, and action potentials were monitored in cardiomyocytes exposed to droperidol. Langendorff perfused hearts were used to assess overall cardiac function.

Results: Droperidol (0.03-1 [mu]m) caused concentration-dependent decreases in peak [Ca2+]i and shortening. Droperidol inhibited 35 mm KCl-induced increase in [Ca2+]i, with little direct effect on sarcoplasmic reticulum Ca2+ stores. Droperidol had no effect on action potential duration but caused a rightward shift in the concentration-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. Droperidol decreased pHi and increased nitric oxide production. Droperidol exerted a negative inotropic effect in Langendorff perfused hearts.     

Propofol Increases Contractility during α1a-Adrenoreceptor Activation in Adult Rat Cardiomyocytes

Background: The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca2+ sensitivity, and contraction of individual cardiomyocytes during activation of [alpha]1a adrenoreceptors and to determine the cellular mechanism of action.

Methods: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine ([alpha]1b-adrenoreceptor antagonist) and BMY 7378 ([alpha]1d-adrenoreceptor antagonist). Data are reported as mean +/- SD.

Results: Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKC[alpha], PKC[delta], PKC[varepsilon], and PKC[zeta] reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+-H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001).     

Propofol decreases myofilament Ca2+ sensitivity via a protein kinase C-, nitric oxide synthase-dependent pathway in diabetic cardiomyocytes

BACKGROUND: The authors' objective was to assess the role of protein kinase C (PKC) and nitric oxide synthase (NOS) in mediating the effects of propofol on diabetic cardiomyocyte contractility, intracellular free Ca2+ concentration ([Ca2+]i), and myofilament Ca2+ sensitivity. METHODS: Freshly isolated ventricular myocytes were obtained from normal and diabetic rat hearts. [Ca2+]i and cell shortening were simultaneously measured in electrically stimulated, ventricular myocytes using fura-2 and video-edge detection, respectively. Actomyosin adenosine triphosphatase activity and troponin I (TnI) phosphorylation were assessed in [32P]orthophosphate-labeled myofibrils. Western blot analysis was used to assess expression of PKC and NOS. RESULTS: Propofol (10 microM) decreased peak shortening by 47 +/- 6% with little effect on peak [Ca2+]i (92 +/- 5% of control) in diabetic myocytes. Maximal actomyosin adenosine triphosphatase activity was reduced by 43 +/- 7% and TnI phosphorylation was greater (32 +/- 6%) in diabetic myofibrils compared with normal. Propofol reduced actomyosin adenosine triphosphatase activity by 17 +/- 7% and increased TnI phosphorylation in diabetic myofibrils. PKC inhibition prevented the propofol-induced increase in TnI phosphorylation and decrease in shortening. Expression of PKC-alpha, PKC-delta, PKC-epsilon, and constitutive NOS were up-regulated and inducible NOS was expressed in diabetic cardiomyocytes. NOS inhibition attenuated the propofol-induced decrease in shortening. CONCLUSION: Myofilament Ca2+ sensitivity and, to a lesser extent, peak [Ca2+]i are decreased in diabetic cardiomyocytes. Increases in PKC and NOS expression in combination with TnI phosphorylation seem to contribute to the decrease in [Ca2+]i and myofilament Ca2+ sensitivity. Propofol decreases [Ca2+]i and shortening via a PKC-, NOS-dependent pathway.     

Propofol Decreases Myofilament Ca2+ Sensitivity via a Protein Kinase C-, Nitric Oxide Synthase-dependent Pathway in Diabetic Cardiomyocytes

Background: The authors' objective was to assess the role of protein kinase C (PKC) and nitric oxide synthase (NOS) in mediating the effects of propofol on diabetic cardiomyocyte contractility, intracellular free Ca2+ concentration ([Ca2+]i), and myofilament Ca2+ sensitivity.

Methods: Freshly isolated ventricular myocytes were obtained from normal and diabetic rat hearts. [Ca2+]i and cell shortening were simultaneously measured in electrically stimulated, ventricular myocytes using fura-2 and video-edge detection, respectively. Actomyosin adenosine triphosphatase activity and troponin I (TnI) phosphorylation were assessed in [32P]orthophosphate-labeled myofibrils. Western blot analysis was used to assess expression of PKC and NOS.

Results: Propofol (10 [mu]m) decreased peak shortening by 47 +/- 6% with little effect on peak [Ca2+]i (92 +/- 5% of control) in diabetic myocytes. Maximal actomyosin adenosine triphosphatase activity was reduced by 43 +/- 7% and TnI phosphorylation was greater (32 +/- 6%) in diabetic myofibrils compared with normal. Propofol reduced actomyosin adenosine triphosphatase activity by 17 +/- 7% and increased TnI phosphorylation in diabetic myofibrils. PKC inhibition prevented the propofol-induced increase in TnI phosphorylation and decrease in shortening. Expression of PKC-[alpha], PKC-[delta], PKC-[varepsilon], and constitutive NOS were up-regulated and inducible NOS was expressed in diabetic cardiomyocytes. NOS inhibition attenuated the propofol-induced decrease in shortening.     

Propofol Increases Phosphorylation of Troponin I and Myosin Light Chain 2 via Protein Kinase C Activation in Cardiomyocytes

Background: Troponin I (TnI) and myosin light chain 2 (MLC2) are important myofibrillar proteins involved in the regulation of myofilament calcium (Ca2+) sensitivity and cardiac inotropy. The objectives of this study were to determine the role of protein kinase C (PKC) in mediating propofol-induced changes in actomyosin adenosine triphosphatase activity in cardiac myofibrils and to examine the extent to which propofol alters the phosphorylation of TnI and MLC2 in cardiomyocytes.

Methods: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of actomyosin adenosine triphosphatase activity and phosphorylation of TnI and MLC2. Western blot analysis for PKC-[alpha] was performed on cardiomyocyte subcellular fractions. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and myocyte shortening was assessed using fura-2 and video edge detection, respectively.

Results: Propofol (30 [mu]m) reduced the Ca2+ concentration required for activation of actomyosin adenosine triphosphatase activity, and this effect was abolished by bisindolylmaleimide I. In addition, propofol stimulated dose-dependent phosphorylation of TnI and MLC2. PKC activation with phorbol myristic acetate also stimulated an increase in phosphorylation of TnI and MLC2. The actions of propofol and phorbol myristic acetate together on phosphorylation of TnI and MLC2 were not additive. PKC inhibition with bisindolylmaleimide I attenuated phorbol myristic acetate- and propofol-induced phosphorylation of TnI and MLC2. Propofol stimulated translocation of PKC-[alpha] from cytosolic to membrane fraction. Propofol caused a shift in the extracellular Ca2+-shortening relationship, and this effect was abolished by bisindolylmaleimide I.     

Propofol increases contractility during alpha1a-adrenoreceptor activation in adult rat cardiomyocytes

BACKGROUND: The objective of this study was to identify the extent to which propofol alters intracellular free Ca2+ concentration ([Ca2+]i), myofilament Ca sensitivity, and contraction of individual cardiomyocytes during activation of alpha1a adrenoreceptors and to determine the cellular mechanism of action. METHODS: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Myocyte shortening and [Ca2+]i were simultaneously monitored in individual cardiomyocytes exposed to phenylephrine after treatment with chloroethylclonidine (alpha1b-adrenoreceptor antagonist) and BMY 7378 (alpha1d-adrenoreceptor antagonist). Data are reported as mean +/- SD. RESULTS: Phenylephrine increased myocyte shortening by 124 +/- 9% (P = 0.002), whereas peak [Ca2+]i only increased by 8 +/- 3% (P = 0.110). Inhibition of phospholipase A2 and phospholipase C attenuated the phenylephrine-induced increase in shortening by 84 +/- 11% (P = 0.004) and 15 +/- 6% (P = 0.010), respectively. Inhibition of protein kinase C (PKC) and Rho kinase attenuated the phenylephrine-induced increase in shortening by 17 +/- 8% (P = 0.010) and 74 +/- 13% (P = 0.006), respectively. In the presence of phenylephrine, propofol increased shortening by 40 +/- 6% (P = 0.002), with no concomitant increase in [Ca2+]i. PKC inhibition prevented the propofol-induced increase in shortening. Selective inhibition of PKCalpha, PKCdelta, PKCepsilon, and PKCzeta reduced the propofol-induced increase in shortening by 12 +/- 5% (P = 0.011), 36 +/- 8% (P = 0.001), 32 +/- 9% (P = 0.007), and 19 +/- 5% (P = 0.008), respectively. Na+ - H+ exchange inhibition reduced the propofol-induced increase in shortening by 56 +/- 7% (P = 0.001). CONCLUSION: Activation of alpha1a adrenoreceptors increases cardiomyocyte shortening primarily via a phospholipase A2-dependent, Rho kinase-dependent increase in myofilament Ca2+ sensitivity. Propofol further increases myofilament Ca2+ sensitivity and shortening via a PKC-dependent pathway and an increase in Na+ - H+ exchange activity.     

Propofol attenuates beta-adrenoreceptor-mediated signal transduction via a protein kinase C-dependent pathway in cardiomyocytes

BACKGROUND: Activation of beta adrenoreceptors by catecholamines is an important mechanism for increasing the inotropic state of the heart. The objectives of the current study were to investigate the effects of propofol on beta-adrenoreceptor-mediated increases in cardiomyocyte intracellular Ca2+ concentration ([Ca2+]i), cell shortening, L-type Ca2+ current (ICa) and cyclic adenosine monophosphate (cAMP) accumulation. The authors also investigated the site of action of propofol in the beta-adrenoreceptor signaling pathway, as well as the role of protein kinase C (PKC), and tested the hypothesis that propofol would inhibit the beta-adrenoreceptor signaling pathway via a PKC-dependent mechanism. METHODS: Freshly isolated ventricular myocytes were obtained from adult rat and guinea pig hearts. Myocyte shortening (video edge detection) and [Ca2+]i (fura-2, 340/380 ratio) were monitored simultaneously in individual cells. Conventional whole cell patch clamp analysis was used to measure the ICa in individual myocytes. cAMP production was assessed in suspensions of myocytes using an enzyme immunoassay kit. RESULTS: Propofol (0.1-10 mum) had no effect on steady state [Ca2+]i, cell shortening, ICa, or cAMP production. In contrast, propofol caused dose-dependent decreases in isoproterenol-stimulated increases in [Ca2+]i, shortening, ICa, and cAMP. Forskolin-induced increases in [Ca2+]i, shortening, and cAMP production were not altered by propofol. PKC activation with phorbol myristate acetate attenuated isoproterenol-stimulated cAMP production. Inhibition of PKC with bisindolylmaleimide (broad range inhibitor) or G? 6976 (inhibitor of Ca2+-dependent PKC isoforms) abolished propofol-induced inhibition of isoproterenol-stimulated increases in [Ca2+]i, shortening, and cAMP production. CONCLUSIONS: Clinically relevant concentrations of propofol attenuate beta-adrenergic signal transduction in cardiac myocytes via inhibition of cAMP production. The inhibitory site of action of propofol is upstream of adenylyl cyclase and involves activation of PKC alpha.     

Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries

BACKGROUND: Isoflurane has been shown to directly inhibit vascular reactivity. However, less information is available regarding its underlying mechanisms in systemic resistance arteries. METHODS: Endothelium-denuded smooth muscle strips were prepared from rat mesenteric resistance arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips, whereas only the force was measured in the beta-escin membrane-permeabilized strips. RESULTS: Isoflurane (3-5%) inhibited the increases in both [Ca2+]i and force induced by either norepinephrine (0.5 microM) or KCl (40 mM). These inhibitions were similarly observed after depletion of intracellular Ca2+ stores by ryanodine. Regardless of the presence of ryanodine, after washout of isoflurane, its inhibition of the norepinephrine response (both [Ca2+]i and force) was significantly prolonged, whereas that of the KCl response was quickly restored. In the ryanodine-treated strips, the norepinephrine- and KCl-induced increases in [Ca2+]i were both eliminated by nifedipine, a voltage-gated Ca2+ channel blocker, whereas only the former was inhibited by niflumic acid, a Ca2+-activated Cl- channel blocker. Isoflurane caused a rightward shift of the Ca2+-force relation only in the fura-2-loaded strips but not in the beta-escin-permeabilized strips. CONCLUSIONS: In mesenteric resistance arteries, isoflurane depresses vascular smooth muscle reactivity by directly inhibiting both Ca2+ mobilization and myofilament Ca2+ sensitivity. Isoflurane inhibits both norepinephrine- and KCl-induced voltage-gated Ca2+ influx. During stimulation with norepinephrine, isoflurane may prevent activation of Ca2+-activated Cl- channels and thereby inhibit voltage-gated Ca2+ influx in a prolonged manner. The presence of the plasma membrane appears essential for its inhibition of the myofilament Ca2+ sensitivity.     

The action of sevoflurane on vascular smooth muscle of isolated mesenteric resistance arteries (part 2): mechanisms of endothelium-independent vasorelaxation

BACKGROUND: The precise mechanisms behind the direct inhibitory action of sevoflurane on vascular smooth muscle have not been fully elucidated. METHODS: Endothelium-denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips. In another series of experiments, only isometric force was measured in the beta-escin-membrane-permeabilized strips. RESULTS: Sevoflurane (3-5%) inhibited the increases in both the [Ca2+]i and the force induced by either norepinephrine (0.5-10 microm) or 40 mm K+. Sevoflurane still inhibited the increase in [Ca2+]i induced by norepinephrine after depletion of intracellular Ca2+ stores with ionomycin, although it little influenced the increase in [Ca2+]i induced by norepinephrine after treatment with verapamil. In the fura-2-loaded membrane-intact muscle, sevoflurane caused a rightward shift of Ca2+-force relation during force development to stepwise increment of extracellular Ca2+ concentration during 40-mm K+ depolarization in either the presence or the absence of norepinephrine. In contrast, sevoflurane did not influence Ca2+-activated contraction in the beta-escin-permeabilized muscle, in which alpha-adrenergic receptor coupling was not retained. CONCLUSIONS: The inhibitory effects of sevoflurane on both norepinephrine- and potassium chloride (KCl)-induced contractions are caused by reduction of [Ca2+]i in vascular smooth muscle and inhibition of the myofilament Ca2+ sensitivity. The [Ca2+]i-reducing effect of sevoflurane observed in both the norepinephrine- and the K+-stimulated muscle is mainly caused by inhibition of voltage-gated Ca2+ influx. The inhibitory effect of sevoflurane on Ca2+ activation of contractile proteins seems to be mediated by the cell membrane or by some diffusible substances that are lost in the beta-escin-permeabilized cells.     

The Action of Sevoflurane on Vascular Smooth Muscle of Isolated Mesenteric Resistance Arteries (Part 2): Mechanisms of Endothelium-independent Vasorelaxation

Background: The precise mechanisms behind the direct inhibitory action of sevoflurane on vascular smooth muscle have not been fully elucidated.

Methods: Endothelium-denuded smooth muscle strips were prepared from rat small mesenteric arteries. Isometric force and intracellular Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded strips. In another series of experiments, only isometric force was measured in the [beta]-escin-membrane-permeabilized strips.

Results: Sevoflurane (3-5%) inhibited the increases in both the [Ca2+]i and the force induced by either norepinephrine (0.5-10 [mu]m) or 40 mm K+. Sevoflurane still inhibited the increase in [Ca2+]i induced by norepinephrine after depletion of intracellular Ca2+ stores with ionomycin, although it little influenced the increase in [Ca2+]i induced by norepinephrine after treatment with verapamil. In the fura-2-loaded membrane-intact muscle, sevoflurane caused a rightward shift of Ca2+-force relation during force development to stepwise increment of extracellular Ca2+ concentration during 40-mm K+ depolarization in either the presence or the absence of norepinephrine. In contrast, sevoflurane did not influence Ca2+-activated contraction in the [beta]-escin-permeabilized muscle, in which [alpha]-adrenergic receptor coupling was not retained.     

Effect of Propofol on Norepinephrine-induced Increases in [Ca2+] (i) and Force in Smooth Muscle of the Rabbit Mesenteric Resistance Artery

Background: Propofol (2,6-diisopropylphenol) possesses vasodilating activity in vivo and in vitro. The propofol-induced relaxation of agonist-induced contractions in small resistance arteries has not been clarified.

Methods: The effect of propofol was examined on the contractions induced by norepinephrine and high K+ in endothelium-denuded rabbit mesenteric resistance artery in vitro. The effects of propofol on the [Ca2+]i mobilization induced by norepinephrine and high K+ were studied by simultaneous measurement of [Ca2+]i using Fura 2 and isometric force in ryanodine-treated strips.

Results: Propofol attenuated the contractions induced by high K+ and norepinephrine, the effect being greater on the high K+-induced contraction than on the norepinephrine-induced contraction. In Ca2-free solution, norepinephrine produced a transient contraction resulting from the release of Ca2+ from storage sites that propofol attenuated. In ryanodine-treated strips, propofol increased the resting [Ca2+]i but attenuated the increases in [Ca2+]i and force induced by both high K+ and norepinephrine. In the presence of nicardipine, propofol had no inhibitory action on the residual norepinephrine-induced [Ca2+]i increase, whereas it still modestly increased resting [Ca2+]i, as in the absence of nicardipine.     

Effects of Primary Alcohols on Airway Smooth Muscle

Background: The investigation examined whether primary alcohols could be used as tools to explore the mechanism of anesthetic actions in airway smooth muscle (ASM). The hypothesis was that, like volatile anesthetics, the primary alcohols relax intact ASM by decreasing intracellular Ca2+ concentration ([Ca2+]i) and by inhibiting agonist-induced increases in the force developed for a given [Ca2+]i (Ca2+ sensitivity).

Method: The effects of butanol, hexanol, and octanol on isometric force in canine tracheal smooth muscle were examined. The effects of hexanol on [Ca2+]i (measured with fura-2) and the relationship between force and [Ca2+]i were studied during membrane depolarization provided by KCl and during muscarinic stimulation provided by acetylcholine.

Results: The primary alcohols relaxed ASM contracted by KCl or acetylcholine in a concentration-dependent manner, with potency increasing as chain length increased. The alcohols could completely relax the strips, even during maximal stimulation with 10 [mu]m acetylcholine (median effective concentrations of 28 +/- 12, 1.3 +/- 0.4, and 0.14 +/- 0.05 mm [mean +/- SD] for butanol, hexanol, and octanol, respectively). Hexanol decreased both [Ca2+]i and force in a concentration-dependent manner. Hexanol decreased Ca2+ sensitivity during muscarinic stimulation but had no effect on the force-[Ca2+]i relationship in its absence.     

Intravenous Anesthetics Inhibit Capacitative Calcium Entry in Pulmonary Venous Smooth Muscle Cells

Background: The authors have previously demonstrated that propofol attenuates capacitative calcium entry (CCE) via the protein kinase C signaling pathway in pulmonary artery smooth muscle cells (PVSMCs). The current goals were to determine whether CCE exists in PVSMCs; to assess the roles of the protein kinase C, tyrosine kinase (TK), and [rho]-kinase signaling pathways in regulating CCE; and to investigate the extent and cellular mechanisms by which intravenous anesthetics (thiopental, midazolam, ketamine, and propofol) alter CCE.

Methods: Primary cultures of fura-2-loaded canine PVSMCs were placed in a dish (37[degrees]C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration ([Ca2+]i) was measured as the 340/380 fluorescence ratio in individual PVSMCs. Thapsigargin, a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was then activated by restoring extracellular Ca2+ (2.2 mm).

Results: Thapsigargin caused a transient increase in [Ca2+]i (160 +/- 6%). Restoring extracellular Ca2+ caused a rapid peak increase in [Ca2+]i (155 +/- 7% of baseline), followed by a sustained increase in [Ca2+]i (129 +/- 5% of baseline), i.e., CCE was stimulated in PVSMCs. Neither protein kinase C activation nor inhibition had an effect on CCE. [rho]-Kinase inhibition also had no effect on CCE, whereas TK inhibition attenuated both peak and sustained CCE. Thiopental, midazolam, ketamine, and propofol each attenuated both peak and sustained CCE. TK inhibition abolished the thiopental-, midazolam-, and ketamine-induced, but not the propofol-induced, decreases in CCE.     

Direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction.

Halothane directly relaxes airway smooth muscle. To determine the direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction, the effects of this anesthetic on the levels of several intracellular second messengers were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), Ca2+/phospholipid-dependent protein kinase (PKC) translocation, and intracellular cyclic adenosine monophosphate concentration ([cAMP]i). When carbachol (1 microM) was used to increase [Ca2+]i to the same concentration as that induced by high-K+ (72.7 mM), the carbachol-induced contraction was more than twice as great, indicating that carbachol enhances the sensitivity of contractile elements to Ca2+ or activates a Ca(2+)-independent mechanism. Similarly, 12-deoxyphorbol 13-isobutylate, a potent PKC activator, markedly potentiated high-K(+)-induced muscle contraction without an increase of [Ca2+]i. The addition of halothane (0.33, 0.75, 1.15, and 1.47 mM) decreased [Ca2+]i and the muscle tension induced by carbachol. However, the decrease of muscle tension was more marked than that of [Ca2+]i at the higher concentrations. Although [Ca2+]i in the presence of verapamil and carbachol was not affected by halothane, the anesthetic markedly decreased muscle force by decreasing the "Ca2+ sensitization" or the Ca(2+)-independent enhancement of tension observed with carbachol. Halothane (0.75 and 1.47 mM) significantly released the membrane-associated PKC to cytosol, which decreased PKC activity. [cAMP]i of the smooth muscle stimulated by carbachol was moderately but significantly increased by halothane. However, when equivalent relaxation was induced with forskolin, which acts via adenylate cyclase activation, a much higher [cAMP]i was observed, which suggests that halothane acts via an additional pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental Conditions Are Important Determinants of Cardiac Inotropic Effects of Propofol

Background: The rationale for this study is that the depressant effect of propofol on cardiac function in vitro is highly variable but may be explained by differences in the temperature and stimulation frequency used for the study. Both temperature and stimulation frequency are known to modulate cellular mechanisms that regulate intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity in cardiac muscle. The authors hypothesized that temperature and stimulation frequency play a major role in determining propofol-induced alterations in [Ca2+]i and contraction in individual, electrically stimulated cardiomyocytes and the function of isolated perfused hearts.

Methods: Freshly isolated myocytes were obtained from adult rat hearts, loaded with fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening were simultaneously measured in individual cells at 28[degrees] or 37[degrees]C at various stimulation frequencies (0.3, 0.5, 1, 2, and 3 Hz) with and without propofol. Langendorff perfused hearts paced at 180 or 330 beats/min were used to assess the effects of propofol on overall cardiac function.

Results: At 28[degrees]C (hypothermic) and, to a lesser extent, at 37[degrees]C (normothermic), increasing stimulation frequency increased peak shortening and [Ca2+]i. Times to peak shortening and rate of relengthening were more prolonged at 28[degrees]C compared with 37[degrees]C at low stimulation frequencies (0.3 Hz), whereas the same conditions for [Ca2+]i were not altered by temperature. At 0.3 Hz and 28[degrees]C, propofol caused a dose-dependent decrease in peak shortening and peak [Ca2+]i. These changes were greater at 28[degrees]C compared with 37[degrees]C and involved activation of protein kinase C. At a frequency of 2 Hz, there was a rightward shift in the dose-response relation for propofol on [Ca2+]i and shortening at both 37[degrees] and 28[degrees]C compared with that observed at 0.3 Hz. In Langendorff perfused hearts paced at 330 beats/min, clinically relevant concentrations of propofol decreased left ventricular developed pressure, with the effect being less at 28[degrees]C compared with 37[degrees]C. In contrast, only a supraclinical concentration of propofol decreased left ventricular developed pressure at 28[degrees]C at either stimulation frequency.     

Low-temperature Modification of the Inhibitory Effects of Volatile Anesthetics on Airway Smooth Muscle Contraction in Dogs

Background: Because exposure to low temperature can modify the effect of volatile anesthetics on airway smooth muscle contraction, this study was conducted to investigate low-temperature modifications of the inhibitory effects of isoflurane and sevoflurane on canine tracheal smooth muscle tone by simultaneously measuring the muscle tension and intracellular concentration of Ca2+ ([Ca2+]i) and by measuring voltage-dependent Ca2+ channel activity.

Methods: [Ca2+]i was monitored by the 500-nm light emission ratio of preloaded fura-2, a Ca2+ indicator. Isometric tension was measured simultaneously. Whole cell patch clamp recording techniques were used to observe voltage-dependent Ca2+ channel activity in dispersed muscle cells. Isoflurane (0-3.0%) or sevoflurane (0-3%) was introduced to a bath solution at various temperatures (37, 34, or 31[degrees]C).

Results: Low temperature (34 or 31[degrees]C) reduced high-K+-induced (72.7 mm) muscle contraction and increased [Ca2+]i, but it enhanced carbachol-induced (1 [mu]m) muscle contraction with a decrease in [Ca2+]i. The volatile anesthetics tested showed significant inhibition of both high-K+-induced and carbachol-induced airway smooth muscle contraction, with a concomitant decrease in [Ca2+]i. The inhibition of the carbachol-induced muscle contraction by volatile anesthetics was abolished partially by exposure to low temperature. Volatile anesthetics and low-temperature exposure significantly inhibited voltage-dependent Ca2+ channel activity of the smooth muscle.