Propofol restores transient receptor potential vanilloid receptor subtype-1 sensitivity via activation of transient receptor potential ankyrin receptor subtype-1 in sensory neurons.

BACKGROUND: Cross talk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has been demonstrated recently. Moreover, the intravenous anesthetic propofol has directly activates TRPA1 receptors and indirectly restores sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity. METHODS: Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. The intracellular Ca concentration was measured in individual cells via fluorescence microscopy. After TRPV1 desensitization with capsaicin (100 nM), cells were treated with propofol (1, 5, and 10 µM) alone or with propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 µM), or the TRPA1 agonist, allyl isothiocyanate (AITC; 100 µM); capsaicin was then reapplied. RESULTS: In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC after desensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC was capable of restoring TRPV1 sensitivity. CONCLUSIONS: These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol's effects on sensory neurons may be clinically important and may contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue.

Propofol modulates agonist-induced transient receptor potential vanilloid subtype-1 receptor desensitization via a protein kinase Cepsilon-dependent pathway in mouse dorsal root ganglion sensory neurons.

BACKGROUND: The activity of transient receptor potential vanilloid subtype-1 (TRPV1) receptors, key nociceptive transducers in dorsal root ganglion sensory neurons, is enhanced by protein kinase C epsilon (PKCepsilon) activation. The intravenous anesthetic propofol has been shown to activate PKCepsilon. Our objectives were to examine whether propofol modulates TRPV1 function in dorsal root ganglion neurons via activation of PKCepsilon. METHODS: Lumbar dorsal root ganglion neurons from wild-type and PKC& epsilon;-null mice were isolated and cultured for 24 h. Intracellular free Ca concentration was measured in neurons by using fura-2 acetoxymethyl ester. The duration of pain-associated behaviors was also assessed. Phosphorylation of PKCepsilon and TRPV1 and the cellular translocation of PKCepsilon from cytosol to membrane compartments were assessed by immunoblot analysis. RESULTS: In wild-type neurons, repeated stimulation with capsaicin (100 nm) progressively decreased the transient rise in intracellular free Ca concentration. After desensitization, exposure to propofol rescued the Ca response. The resensitizing effect of propofol was absent in neurons obtained from PKCepsilon-null mice. Moreover, the capsaicin-induced desensitization of TRPV1 was markedly attenuated in the presence of propofol in neurons from wild-type mice but not in neurons from PKCepsilon-null mice. Propofol also prolonged the duration of agonist-induced pain associated behaviors in wild-type mice. In addition, propofol increased phosphorylation of PKCepsilon as well as TRPV1 and stimulated translocation of PKCepsilon from cytosolic to membrane fraction. DISCUSSION: Our results indicate that propofol modulates TRPV1 sensitivity to capsaicin and that this most likely occurs through a PKCepsilon-mediated phosphorylation of TRPV1.

Propofol activates and allosterically modulates recombinant protein kinase C epsilon.

BACKGROUND: Myocardial protection by anesthetics is known to involve activation of protein kinase C epsilon (PKC epsilon). A key step in the activation process is autophosphorylation of the enzyme at serine 729. This study's objectives were to identify the extent to which propofol interacts with PKC epsilon and to identify the molecular mechanism(s) of interaction. METHODS: Immunoblot analysis of recombinant PKC epsilon was used to assess autophosphorylation of PKC epsilon at serine 729 before and after exposure to propofol. An enzyme-linked immunosorbant assay kit was used for measuring PKC epsilon activity. Spectral shifts in fluorescence emission maxima of the C1B subdomain of PKC epsilon in combination with the fluorescent phorbol ester, sapintoxin D, was used to identify molecular interactions between propofol and the phorbol ester/diacylglycerol binding site on the enzyme. RESULTS: Propofol (1 microM) caused a sixfold increase in immunodetectable serine 729 phosphorylated PKC epsilon and increased catalytic activity of the enzyme in a dose-dependent manner. Dioctanoylglycerol-induced or phorbol myristic acetate-induced activation of recombinant PKC epsilon activity was enhanced by preincubation with propofol. Both propofol and phorbol myristic acetate quenched the intrinsic fluorescence spectra of the PKC epsilon C1B subdomain in a dose-dependent manner, and propofol caused a further leftward-shift in the fluorescence emission maxima of sapintoxin D after addition of the C1B subdomain. CONCLUSIONS: These results demonstrate that propofol interacts with recombinant PKC epsilon causing autophosphorylation and activation of the enzyme. Moreover, propofol enhances phorbol ester-induced catalytic activity, suggesting that propofol binds to a region near the phorbol ester binding site allowing for allosteric modulation of PKC epsilon catalytic activity.

Beta 1-adrenergic receptor autoantibodies mediate dilated cardiomyopathy by agonistically inducing cardiomyocyte apoptosis.

BACKGROUND: Antibodies to the beta1-adrenergic receptor (beta1AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby beta1AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM. METHODS AND RESULTS: We used the beta1AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the beta1AR. After transfer into naive male hosts, beta1AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of beta1AR IgG before transfer and by selective pharmacological antagonism of host beta1AR but not beta2AR. We found that beta1AR autoantibodies shifted the beta1AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by beta1AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK. CONCLUSIONS: Our data show how beta1AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Propofol modulates Na+-Ca2+ exchange activity via activation of protein kinase C in diabetic cardiomyocytes.

BACKGROUND: The authors' objective was to identify the role of the Na+-Ca2+ exchanger (NCX) in mediating the contractile dysfunction observed in diabetic cardiomyocytes before and after exposure to propofol. METHODS: Freshly isolated ventricular myocytes were obtained from normal and diabetic rat hearts. Intracellular concentration of Ca2+ and cell shortening were simultaneously measured in electrically stimulated, ventricular myocytes using fura-2 and video-edge detection, respectively. Postrest potentiation (PRP) and sarcoplasmic reticulum Ca2+ load were used to assess propofol-induced changes in the activity of the NCX. RESULTS: Propofol (10 microM) increased PRP in diabetic cardiomyocytes but had no effect on PRP in normal cardiomyocytes. Removal of sodium enhanced and KB-R7943 (reverse mode NCX inhibitor) blocked PRP in both normal and diabetic cardiomyocytes. In the absence of sodium, propofol enhanced PRP in diabetic cardiomyocytes but had no additional effect in normal cardiomyocytes. KB-R7943 completely blocked propofol-induced potentiation of peak intracellular concentration of Ca2+ and shortening in both cell types. Propofol increased sarcoplasmic reticulum Ca2+ load and prolonged removal of cytosolic Ca2+ in diabetic cardiomyocytes, but not in normal cardiomyocytes. Removal of sodium enhanced propofol-induced increases in sarcoplasmic reticulum Ca2+ load and further prolonged removal of cytosolic Ca2+, whereas KB-R7943 completely blocked propofol-induced increase in sarcoplasmic reticulum Ca2+ load. Protein kinase C inhibition with bisindolylmaleimide I prevented the propofol-induced increase in PRP and prolongation in Ca2+ removal. CONCLUSIONS: These data suggest that propofol enhances PRP via activation of reverse mode NCX, but attenuates Ca2+ removal from the cytosol via inhibition of forward mode NCX in diabetic cardiomyocytes. The actions of propofol are mediated via a protein kinase C-dependent pathway.

Propofol-induced activation of protein kinase C isoforms in adult rat ventricular myocytes.

BACKGROUND: Myocardial protection by anesthetics is known to involve activation of protein kinase C (PKC). The authors' objective was to identify the PKC isoforms activated by propofol in rat ventricular myocytes. They also assessed the intracellular location of individual PKC isoforms before and after treatment with propofol. METHODS: Freshly isolated ventricular myocytes were obtained from adult rat hearts. Immunoblot analysis of cardiomyocyte subcellular fractions was used to assess translocation of individual PKC isoforms before and after exposure to propofol. An enzyme-linked immunosorbent assay kit was used for measuring PKC activity. Immunocytochemistry and confocal microscopy were used to visualize the intracellular location of the individual PKC isoforms. RESULTS: Under baseline conditions, PKC-alpha, PKC-delta, and PKC-zeta were associated with both the cytosolic and membrane fractions, whereas PKC-epsilon was exclusively located in the cytosolic fraction. Propofol (10 microM) caused translocation of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from cytosolic to membrane fraction and increased total PKC activity (211 +/- 17% of baseline; P = 0.003) in a dose-dependent manner. Immunocytochemical localization of the individual PKC isoforms demonstrated that propofol caused translocation of PKC-alpha to the intercalated discs and z-lines; PKC-delta to the perinuclear region; PKC-epsilon to sites associated with the z-lines, intercalated discs, and the sarcolemma; and PKC-zeta to the nucleus. CONCLUSIONS: These results demonstrate that propofol causes an increase in PKC activity in rat ventricular myocytes. Propofol stimulates translocation of PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta to distinct intracellular sites in cardiomyocytes. This may be a fundamentally important cellular mechanism of anesthesia-induced myocardial protection in the setting of ischemia-reperfusion injury.

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.

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. CONCLUSION: These results demonstrate that temperature and stimulation frequency alter the inhibitory effect of propofol on cardiomyocyte [Ca2+]i and contraction. In isolated cardiomyocytes, the inhibitory effects of propofol are more pronounced during hypothermia and at higher stimulation frequencies and involve activation of protein kinase C. In Langendorff perfused hearts at constant heart rate, the inhibitory effects of propofol at clinically relevant concentrations are more pronounced during normothermic conditions.