Propofol attenuates capacitative calcium entry in pulmonary artery smooth muscle cells.

BACKGROUND: Depletion of intracellular Ca2+ stores results in capacitative Ca2+ entry (CCE) in pulmonary artery smooth muscle cells (PASMCs). The authors aimed to investigate the effects of propofol on CCE and to assess the extent to which protein kinase C (PKC) and tyrosine kinases mediate propofol-induced changes in CCE. METHODS: Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration was measured using fura 2 in PASMCs using a dual-wavelength spectrofluorometer. Thapsigargin (1 microM), a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was activated when extracellular Ca2+ (2.2 mM) was restored. RESULTS: Thapsigargin caused a transient increase in intracellular Ca2+ concentration (182+/-11%). Restoring extracellular calcium (to induce CCE) resulted in a peak (246+/-12% of baseline) and a sustained (187+/-7% of baseline) increase in intracellular Ca2+ concentration. Propofol (1, 10, 100 microM) attenuated CCE in a dose-dependent manner (peak: 85+/-3, 70+/-4, 62+/-4%; sustained: 94+/-5, 80+/-5, 72+/-5% of control respectively). Tyrosine kinase inhibition (tyrphostin 23) attenuated CCE (peak: 67+/-4%; sustained: 74+/-5% of control), but the propofol-induced decrease in CCE was still apparent after tyrosine kinases inhibition. PKC activation (phorbol 12-myristate 13-acetate) attenuated CCE (peak: 48+/-1%; sustained: 53+/-3% of control), whereas PKC inhibition (bisindolylmaleimide) potentiated CCE (peak: 132+/-11%; sustained: 120+/-4% of control). Moreover, PKC inhibition abolished the propofol-induced attenuation of CCE. CONCLUSION: Tyrosine kinases activate and PKC inhibits CCE in PASMCs. Propofol attenuates CCE primarily via a PKC-dependent pathway. CCE should be considered a possible cellular target for anesthetic agents that alter vascular smooth muscle tone.

Propofol increases myofilament Ca2+ sensitivity and intracellular pH via activation of Na+-H+ exchange in rat ventricular myocytes.

BACKGROUND: The objectives were to determine the extent and mechanism of action by which propofol increases myofilament Ca2+ sensitivity and intracellular pH (pHi) in ventricular myocytes. METHODS: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. Myocyte shortening (video edge detection) and pHi (2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, 500/440 ratio) were monitored simultaneously in individual cells field-stimulated (0.3 Hz) and superfused with HEPES-buffered solution (pH 7.4, 30 degrees C). RESULTS: Propofol (100 microM) reduced the Ca2+ concentration required for activation of myofibrillar actomyosin ATPase from pCa 5.7 +/- 0.01 to 6.6 +/- 0.01. Increasing pHi (7.05 +/- 0.03 to 7.39 +/- 0.04) with NH4Cl increased myocyte shortening by 35 +/- 12%. Washout of NH4Cl decreased pHi to 6.82 +/- 0.03 and decreased myocyte shortening to 52 +/- 10% of control. Propofol caused a dose-dependent increase in pHi but reduced myocyte shortening. The propofol-induced increase in pHi was attenuated, whereas the decrease in myocyte shortening was enhanced after pretreatment with ethylisopropyl amiloride, a Na+-H+ exchange inhibitor, or bisindolylmaleimide I, a protein kinase C inhibitor. Propofol also attenuated the NH4Cl-induced intracellular acidosis, increased the rate of recovery from acidosis, and attenuated the associated decrease in myocyte shortening. Propofol caused a leftward shift in the extracellular Ca2+-shortening relation, and this effect was attenuated by ethylisopropyl amiloride. CONCLUSIONS: These results suggest that propofol increases the sensitivity of myofibrillar actomyosin ATPase to Ca2+ (ie., increases myofilament Ca2+ sensitivity), at least in part by increasing pHi via protein kinase C-dependent activation of Na+-H+ exchange.

AKAP-mediated targeting of protein kinase a regulates contractility in cardiac myocytes.

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.

Changes in calcium cycling precede cardiac dysfunction during autoimmune myocarditis in mice.

Myocardial inflammation contributes to the development of dilated cardiomyopathy, as well as other cardiac diseases. We have previously shown decreased left ventricular function in mice with autoimmune myocarditis. To test the hypothesis that decreased function is mediated by changes in contractility and/or Ca2+ cycling, we isolated cardiac myocytes from mice with myocarditis and age-matched controls at two time points: day 18 (prior to cardiac dysfunction) and day 35 (during cardiac dysfunction). We measured cell shortening and the Ca2+ transient simultaneously at 28 degrees C and 0.3 Hz. We also quantified proteins which regulate contractility and [Ca2+](i), using Western blot analysis. Results showed no change in cell shortening or systolic Ca2+ on day 18, despite a significant reduction in diastolic Ca2+. By day 35, the decrease in diastolic Ca2+ was accompanied by significantly reduced cell shortening and a decrease in the systolic Ca2+ transient. Protein levels of the sarcoplasmic reticulum Ca2+ ATPase were unchanged at both time points, while phospholamban and the sodium/calcium exchanger were significantly reduced in myosin-immunized mice at both time points. Calsequestrin was unchanged at day 18, but was significantly reduced in the myosin-immunized mice on day 35. Results of this study suggest that decreased diastolic Ca2+, as well as protein levels of phospholamban and the sodium/calcium exchanger, may actually contribute to disease progression in autoimmune myocarditis, while changes in calsequestrin may be related to systolic dysfunction in this model.

Calcium mobilization in ovarian cancer cells in response to lysophospholipids.

Intracellular free Ca(2+)concentration ([Ca(2+)](i)) plays a critical role in regulating many diverse cellular functions including cell proliferation and programmed cell death (apoptosis) (1). An elevation in [Ca(2+)](i) activates enzymes (phospholipase A(2), phospho- lipase D and some isoforms of protein kinase C) associated with the liberation of bioactive lipids such as arachidonic acid (AA) and lysophosphatidic acid (LPA) (1,2). AA and its metabolites have been implicated in multiple steps of carcinogenesis (3,4). LPA as well as several other bioactive lipids stimulate release of Ca(2+)from intracellular stores and regulate proliferation of ovarian cancer cells I5-7).

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle.

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.

Sphingosylphosphorylcholine is a ligand for ovarian cancer G-protein-coupled receptor 1.

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.

Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.

Binding, partial agonism, and potentiation of alpha(1)-adrenergic receptor function by benzodiazepines: A potential site of allosteric modulation.

Benzodiazepines, a class of drugs commonly used to induce anesthesia and sedation, can attenuate intracellular calcium oscillations evoked by alpha(1)-adrenergic receptor (alpha(1)-AR) stimulation in pulmonary artery smooth muscle cells. We postulated a direct action of benzodiazepines in modulating alpha(1)-AR function at the receptor level. Benzodiazepines bound to each of the cloned alpha(1)-AR subtypes (alpha(1a)-, alpha(1b)-, or alpha(1d)-AR) on COS-1 cell membranes transiently transfected to express a single population of alpha(1)-AR subtype. The ability of benzodiazepines to alter alpha(1)-AR signal transduction was investigated by measuring total inositol phosphate generation in rat-1 fibroblast cells, stably transfected to express a single alpha(1)-AR subtype. By themselves, benzodiazepines displayed partial agonism. At alpha(1b)-ARs and alpha(1d)-ARs, the maximal inositol phosphate response to phenylephrine was potentiated almost 2-fold by either midazolam or lorazepam (100 microM). At alpha(1a)-ARs, diazepam, lorazepam, and midazolam all increased the maximal response of the partial agonist clonidine at these receptors, whereas the response to the full agonist phenylephrine was unaltered or inhibited. The potentiating actions of midazolam and its partial agonism at alpha(1)-ARs was blocked by the addition of 1 microM prazosin, an alpha(1)-AR antagonist, and not by a gamma-aminobutyric acid(A)-receptor antagonist. These studies show that benzodiazepines modulate the function of alpha(1)-ARs in vitro, and this is the first report of a potential allosteric site on alpha(1)-ARs that may be therapeutically useful for drug design.

Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.

Differential effects of fentanyl and morphine on intracellular Ca2+ transients and contraction in rat ventricular myocytes.

BACKGROUND: Our objective was to elucidate the direct effects of fentanyl and morphine on cardiac excitation-contraction coupling using individual, field-stimulated rat ventricular myocytes. METHODS: Freshly isolated myocytes were loaded with fura-2 and field stimulated (0.3 Hz) at 28 degrees C. Amplitude and timing of intracellular Ca2+ concentration (at a 340:380 ratio) and myocyte shortening (video edge detection) were monitored simultaneously in individual cells. Real time Ca2+ uptake into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. RESULTS: The authors studied 120 cells from 30 rat hearts. Fentanyl (30-1,000 nM) caused dose-dependent decreases in peak intracellular Ca2+ concentration and shortening, whereas morphine (3-100 microM) decreased shortening without a concomitant decrease in the Ca2+ transient. Fentanyl prolonged the time to peak and to 50% recovery for shortening and the Ca2+ transient, whereas morphine only prolonged the timing parameters for shortening. Morphine (100 microM), but not fentanyl (1 microM), decreased the amount of Ca2+ released from intracellular stores in response to caffeine in intact cells, and it inhibited the rate of Ca2+ uptake in isolated sarcoplasmic reticulum vesicles. Fentanyl and morphine both caused a downward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on the Ca2+ transient. CONCLUSIONS: Fentanyl and morphine directly depress cardiac excitation-contraction coupling at the cellular level. Fentanyl depresses myocardial contractility by decreasing the availability of intracellular Ca2+ and myofilament Ca2+ sensitivity. In contrast, morphine depresses myocardial contractility primarily by decreasing myofilament Ca2+ sensitivity.

Thiopental alters contraction, intracellular Ca2+, and pH in rat ventricular myocytes.

BACKGROUND: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2'],) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes. METHODS: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+], (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+l, were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied. RESULTS: Field stimulation increased [Ca2+]i from 85 + 10 nM to 355 + 22 nM (mean + SEM). Myocytes shortened by 10% of resting cell length (127 + 5 tlm). Times to peak [Ca2+], and shortening were 139 + 6 and 173 + 7 msec, respectively. Times to 50% recovery for [Ca2+], and shortening were 296 + 6 and 290 + 6 ms, respectively. Addition of thiopental (30-1,000 /lM) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+], and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 uM) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+],. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+-H+ exchange. CONCLUSION: Thiopental has a direct negative inotropic effect on cardiac excitation-contraction coupling at the cellular level, which is mediated by a decrease in [Ca2+],. Thiopental also increases myofilament Ca2+ sensitivity via alkalinization of the cell, which may partially offset its negative inotropic effect.

Benzodiazepines differentially inhibit phenylephrine-induced calcium oscillations in pulmonary artery smooth muscle cells.

BACKGROUND: Modulation of intracellular free calcium is a critical determinant of vasomotor tone. The authors investigated the effects of three benzodiazepines on alpha-adrenergic-induced oscillations in intracellular free calcium in individual pulmonary artery smooth muscle cells. METHODS: Pulmonary artery smooth muscle cells were cultured from explants of canine intrapulmonary artery. Fura-2-loaded pulmonary artery smooth muscle cells were continuously superfused with phenylephrine (10 microM) at 37 degrees C on the stage of an inverted fluorescence microscope. Intracellular free calcium was measured using a dual wavelength spectrofluorometer. After establishment of steady-state intracellular free calcium oscillations induced by phenylephrine, lorazepam, diazepam, or midazolam was added to the superfusate. The amplitude and frequency of the intracellular free calcium oscillations were compared before and after addition of each agent. RESULTS: Resting mean +/- SEM values of intracellular free calcium were 68 +/- 8 nM. Phenylephrine stimulated dose-dependent oscillations in intracellular free calcium, which reached a peak concentration of 676 +/- 35 nM and a frequency of 1.08 +/- 0.1 transients/min. Addition of lorazepam (1 microM) inhibited (P < 0.05) the amplitude (591 +/- 32 nM) but not the frequency (0.97 +/- 0.1 transients/min) of the oscillations. Conversely, diazepam (1 microM) decreased (P < 0.05) the frequency (0.79 +/- 0.1 transients/min) but not the amplitude (663 +/- 37 nM) of the oscillations. These effects were dose-dependent. In contrast, midazolam (1-30 microM) had no effect on the amplitude or frequency of intracellular free calcium oscillations. At concentrations higher than 100 microM, however, all three benzodiazepines inhibited both the amplitude and frequency of the intracellular free calcium oscillations. CONCLUSION: Lorazepam and diazepam but not midazolam exerted differential inhibitory effects on phenylephrine-induced intracellular free calcium oscillations. Benzodiazepines may alter the pulmonary vascular response to sympathetic alpha-adrenoreceptor activation by direct inhibition of intracellular free calcium signaling in pulmonary artery smooth muscle cells.

Propofol and ketamine only inhibit intracellular Ca2+ transients and contraction in rat ventricular myocytes at supraclinical concentrations.

BACKGROUND: The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation-contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field-stimulated ventricular myocytes. METHODS: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field-stimulated at 0.3 Hz. RESULTS: Baseline [Ca2+]i (mean +/- SEM) was 80 +/- 12 nM, and resting cell length was 112 +/- 2 microm. Field stimulation increased [Ca2+]i to 350 +/- 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose-dependent decreases in peak [Ca2+]i and shortening. At 300 microM, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. CONCLUSIONS: These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.

Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II.

Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha, delta, epsilon, zeta, iota/lambda, and mu) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha, delta, zeta, iota/lambda, and mu) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-zeta was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The alpha and zeta isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.

Intravenous anesthetics attenuate phenylephrine-induced calcium oscillations in individual pulmonary artery smooth muscle cells.

BACKGROUND: The authors investigated the effects of intravenous anesthetics on alpha-adrenergic-induced oscillations in intracellular free calcium concentration ([Ca2+]i) in individual pulmonary artery smooth muscle cells (PASMCs). METHODS: PASMCs were cultured from explants of canine intrapulmonary artery. Fura-2-loaded PASMCs were continuously superfused with phenylephrine (10 microM) at 37 degrees C on the stage of an inverted fluorescence microscope. Measurement of [Ca2+]i was via a dual wavelength spectrofluorometer. Intravenous anesthetics were added to the superfusate to assess their effects on the phenylephrine-induced [Ca2+]i oscillations. RESULTS: Resting [Ca2+]i was 103 +/- 6 nM. Phenylephrine stimulated [Ca2+]i oscillations, reaching a peak concentration of 632 +/- 20 nM and a frequency of 1.53 +/- 0.14 transients/min. The effects of phenylephrine were dose-dependent. The effects of intravenous anesthetics on phenylephrine-induced [Ca2+]i oscillations were dose-dependent. Ketamine (100 microM) reduced the amplitude (221 +/- 22 nM) but not the frequency (1.48 +/- 0.11/min) of the oscillations, whereas thiopental (100 microM) decreased the amplitude (270 +/- 20 nM) and the frequency (1.04 +/- 0.10/min). Propofol (100 microM) and the Intralipid vehicle inhibited the amplitude (274 +/- 11 nM) but not the frequency (1.39 +/- 0.11/min) of the oscillations. The effects of ketamine and thiopental, but not propofol, were evident at clinically relevant concentrations. CONCLUSION: Ketamine, thiopental, and propofol exerted differential effects to inhibit the amplitude or the frequency of phenylephrine-induced [Ca2+]i oscillations in individual PASMCs. Thus, intravenous anesthetics may alter the pulmonary vascular response to alpha-adrenoreceptor activation by directly inhibiting [Ca2+]i signaling in PASMCs.

Phenylephrine-induced Ca2+ oscillations in canine pulmonary artery smooth muscle cells.

Modulation of [Ca2+]i in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of alpha 1-adrenoceptors with phenylephrine (PE) on [Ca2+]i in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs pretreated with propranolol (5 mumol/L) were continuously superfused with PE at 37 degrees C on the stage of an inverted fluorescence microscope, and [Ca2+]i was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca2+]i were 96 +/- 4 nmol/L. PE (10 mumol/L) stimulated oscillations in [Ca2+]i at a frequency of 1.35 +/- 0.07/min, which reached a peak [Ca2+]i of 650 +/- 26 nmol/L (n = 69 cells). The oscillations lasted for > 30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca2+]i oscillations increased in a dose-dependent (3 x 10(-8) to 10(-4) mol/L) manner. Pretreatment with the alpha 1-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca2+ abolished the repetitive [Ca2+]i oscillations induced by PE. The voltage-operated Ca2+ channel blockers nifedipine (1 mumol/L) and verapamil (1 mumol/L) had no effect on the [Ca2+]i oscillations. In contrast, inhibition of phospholipase C with U73122 (10(-7) to 10(-5) mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10(-9) to 10(-7) mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 mumol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 mumol/L) had no effect. In freshly dispersed PASMCs, PE (10 mumol/L) induced oscillations in [Ca2+]i similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca2+]i oscillations in PASMCs via stimulation of alpha 1-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca2+ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca2+ and intracellular Ca2+ stores indicates that both Ca2+ influx and intracellular Ca2+ release play a role in the maintenance of the oscillations.

Arachidonic acid enhances contraction and intracellular Ca2+ transients in individual rat ventricular myocytes.

Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by inotropic stimuli alters contractility in cardiac muscle. Arachidonic acid (AA), a precursor for eicosanoid formation, is released in response to receptor activation and myocardial ischemia and has been demonstrated to alter K+ and Ca2+ channel activity. We investigated the effects of AA on contractility by simultaneously measuring [Ca2+]i and shortening in single field-stimulated rat ventricular myocytes. [Ca2+]i transients were measured using fura 2, and myocyte shortening was assessed using video edge detection. AA stimulated a doubling in the amplitude of the [Ca2+]i transient and a twofold increase in myocyte shortening. In addition, AA stimulated a 30% increase in the time to 50% diastolic [Ca2+]i and a 35% increase in the time to 50% relengthening. These effects of AA were mediated by AA itself (56 +/- 5%) and by cyclooxygenase metabolites. Pretreatment with the protein kinase C inhibitors staurosporine and chelerythrine nearly abolished (> 90% inhibition) these AA-induced effects. Inhibition of voltagegated K+ channels with 4-aminopyridine mimicked the effects of AA. Addition of AA to the 4-aminopyridine-treated myocyte had no additional effect on parameters of contractile function. These data indicate that AA alters the amplitude and duration of Ca2- transients and myocyte shortening via protein kinase C-dependent inhibition of voltage-gated K+ channels. Release of AA by phospholipases in response to receptor activation by endogenous mediators or pathological stimuli may be involved in mediating inotropic responses in cardiac muscle.

Activation of purinergic receptors triggers oscillatory contractions in adult rat ventricular myocytes.

Extracellular ATP is an important neurotransmitter that modulates cardiac function by activation of purinergic receptors. In this study, the effect of P2 purinergic receptor activation on contractions and on [Ca2+]i was investigated in adult rat ventricular myocytes. Fura 2 was used to measure [Ca2+]i, and video edge detection was used to measure contraction. Superfusion of 2-methylthio-adenosine-5'-triphosphate (2-M-S-ATP) over quiescent myocytes induced oscillations in contraction and in [Ca2+]i. The frequency of the oscillatory contractions increased with increasing concentrations of 2-M-S-ATP, but the amplitude of contractions varied from cell to cell and was independent of the concentration of 2-M-S-ATP. During electrical stimulation, activation of purinergic receptors in myocytes potentiated the amplitude of contraction and induced arrhythmias. In populations of quiescent myocytes, the plateau phase of the [Ca2+]i signal evoked by 2-M-S-ATP could be shown to represent summed oscillations in [Ca2+]i in individual cells. Pretreatment of quiescent myocytes with thapsigargin or caffeine reduced or abolished the oscillations in contractions and in [Ca2+]i triggered by 2-M-S-ATP, indicating a dependence of the oscillations on uptake and release of Ca2+ by the sarcoplasmic reticulum. These data demonstrate the novel phenomenon that activation of purinergic receptors in quiescent myocytes stimulates oscillations in [Ca2+]i and contraction. In electrically stimulated myocytes, activation of purinergic receptors triggers oscillatory contractions and potentiates the amplitude of electrically triggered contractions.

Arachidonic acid-dependent phosphorylation of troponin I and myosin light chain 2 in cardiac myocytes.

Recent evidence has suggested that arachidonic acid (AA) may be an important signaling molecule in cardiac excitation-contraction coupling. We previously showed that AA and endothelin-1 (ET) inhibit distinct K+ channels via protein kinase C-dependent pathways in rat ventricular myocytes. In addition, we demonstrated that Ca2+ transients in populations of fura 2-loaded myocytes were potentiated by AA and ET via activation of protein kinase C. In this study, we have used suspensions of [32P]orthophosphate (32Pi)-labeled rat ventricular myocytes to study the effects of AA and ET at the level of the myofilaments. After a 10-minute incubation of the labeled cells with phorbol 12-myristate 13-acetate (PMA), AA, or ET in the presence or absence of the protein kinase C inhibitor calphostin C, the myofibrillar proteins were separated by PAGE. Measurement of unloaded cell shortening using video edge detection in single electrically stimulated myocytes was also used to assess the effects of AA and ET on myocyte contractility. Incubation with either PMA, AA, or ET resulted in similar increases in 32Pi incorporation into troponin I (TnI) and myosin light chain 2 (MLC2), which was inhibited by preincubation with the protein kinase C antagonist calphostin C. In addition, the ability of these agonists to stimulate phosphorylation of TnI or MLC2 did not require extracellular Ca2+ or intact intracellular Ca2+ stores. The effects of AA and ET together on phosphorylation of TnI or MLC2 were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)