Fura-2AM Fluorescence Research Articles

Dexamethasone stimulates store-operated calcium entry and protein degradation in cultured L6 myotubes through a phospholipase A2-dependent mechanism

Muscle wasting in various catabolic conditions is at least in part regulated by glucocorticoids. Increased calcium levels have been reported in atrophying muscle. Mechanisms regulating calcium homeostasis in muscle wasting, in particular the role of glucocorticoids, are poorly understood. Here we tested the hypothesis that glucocorticoids increase intracellular calcium concentrations in skeletal muscle and stimulate store-operated calcium entry (SOCE) and that these effects of glucocorticoids may at least in part be responsible for glucocorticoid-induced protein degradation. Treatment of cultured myotubes with dexamethasone, a frequently used in vitro model of muscle wasting, resulted in increased intracellular calcium concentrations determined by fura-2 AM fluorescence measurements. When SOCE was measured by using calcium "add-back" to muscle cells after depletion of intracellular calcium stores, results showed that SOCE was increased 15-25% by dexamethasone and that this response to dexamethasone was inhibited by the store-operated calcium channel blocker BTP2. Dexamethasone treatment stimulated the activity of calcium-independent phospholipase A(2) (iPLA(2)), and dexamethasone-induced increase in SOCE was reduced by the iPLA(2) inhibitor bromoenol lactone (BEL). In additional experiments, treatment of myotubes with the store-operated calcium channel inhibitor gadolinium ion or BEL reduced dexamethasone-induced increase in protein degradation. Taken together, the results suggest that glucocorticoids increase calcium concentrations in myocytes and stimulate iPLA(2)-dependent SOCE and that glucocorticoid-induced muscle protein degradation may at least in part be regulated by increased iPLA(2) activity, SOCE, and cellular calcium levels.

PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation

Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (beta-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed >or=50% by the PDE5A inhibitor sildenafil (SIL, 1 microM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of beta3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in beta3-AR knockout (beta3-AR(-/-)) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not beta3-AR(-/-) myocytes. These data support a cascade involving beta3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial beta-adrenergic stimulation.

Spatiotemporal Effects of Sonoporation Measured by Real-Time Calcium Imaging

To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration ([Ca 2+] i) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison ™ microbubbles. At extracellular [Ca 2+] o of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of [Ca 2+] i in time) with durations of 100–180 s. Immediate [Ca 2+] i transients after ultrasound application were induced by ultrasound-mediated microbubble–cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium [Ca 2+] i levels, thereby indicating irreversible membrane damage. Spatial evolution of [Ca 2+] i in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7–20 μm/s over a distance >200 μm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca 2+ for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison ™ microbubbles can generate transient [Ca 2+] i changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane. (E-mail address: cxdeng@umich.edu)

GABAergic Mechanism of Propofol Toxicity in Immature Neurons

Certain anesthetics exhibit neurotoxicity in the brains of immature but not mature animals. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, is excitatory on immature neurons via its action at the GABAA receptor, due to a reversed transmembrane chloride gradient. GABAA receptor activation in immature neurons is sufficient to open L-type voltage-gated calcium channels. As propofol is a GABAA agonist, we hypothesized that it and more specific GABAA modulators would increase intracellular free calcium ([Ca2+]i), resulting in the death of neonatal rat hippocampal neurons. Neuronal [Ca2+]i was monitored using Fura2-AM fluorescence imaging. Cell death was assessed by double staining with propidium iodide and Hoechst 33258 at 1 hour (acute) and 48 hours (delayed) after 5 hours exposure of neurons to propofol or the GABAA receptor agonist, muscimol, in the presence and absence of the GABA receptor antagonist, bicuculline, or the L-type Ca2+ channel blocker, nifedipine. Fluorescent measurements of caspase-3,-7 activities were performed at 1 hour after exposure. Both muscimol and propofol induced a rapid increase in [Ca2+]i in days in vitro (DIV) 4, but not in DIV 8 neurons, that was inhibited by nifedipine and bicuculline. Caspase-3,-7 activities and cell death increased significantly in DIV 4 but not DIV 8 hippocampal neuronal cultures 1 hour after 5 hours exposure to propofol, but not muscimol, and were inhibited by the presence of bicuculline or nifedipine. We conclude that an increase in [Ca2+]i, due to activation of GABAA receptors and opening of L-type calcium channels, is necessary for propofol-induced death of immature rat hippocampal neurons but that additional mechanisms not elicited by GABAA activation alone also contribute to cell death.

Leptin modulates the negative inotropic effect of interleukin-1β in cardiac myocytes

Interleukin-1beta (IL-1beta) is a potent negative inotrope implicated in the functional abnormalities of heart failure. Because the adipokine, leptin, protects against some of the cardiovascular effects of endotoxin, we hypothesized that leptin may modulate the cardiosuppressive effects of IL-1beta in isolated cardiomyocytes. Ventricular cardiac myocytes isolated from adult male Sprague Dawley rats were analyzed simultaneously for electrically stimulated contractility and calcium transients following 30 min exposure to IL-1beta (10 ng/ml) with or without 60 min pretreatment with leptin (25 ng/ml). IL-1beta decreased cell shortening, depressed maximal velocities of shortening and relengthening, and prolonged the time to 90% relaxation. The change in fura2-AM fluorescence ratio amplitude (Delta[Ca(2+)]) was significantly depressed and the time to return to baseline [Ca(2+)] was prolonged. The negative inotropic effects of IL-1beta were blocked by the neutral sphingomyelinase inhibitor Manumycin A (5 microM) or the ceramidase inhibitor N-oleoyl ethanolamine (1 microM). Prior exposure of myocytes to leptin blocked IL-1beta-induced cardiosuppression in conjunction with a blunting of IL-1beta stimulated ceramide accumulation. These data suggest that leptin may modulate IL-1beta signaling through the sphingolipid signaling pathway in cardiomyocytes.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour

Objectives The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E 2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca 2+] i ) during physiological pregnancy and labour. Methods Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. Results fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca 2+] i . Conclusions These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

Calcium‐Dependent Effects of Melatonin Inhibition of Glutamatergic Response in Rat Striatum

The effects of melatonin, amlodipine, diltiazem (L-type Ca2+ channel blockers) and omega-conotoxin (N-type Ca2+ channel blocker) on the glutamate-dependent excitatory response of striatal neurones to sensory-motor cortex stimulation was studied in a total of 111 neurones. Iontophoresis of melatonin produced a significant attenuation of the excitatory response in 85.2% of the neurones with a latency period of 2 min. Iontophoresis of either L- or N-type Ca2+ channel blocker also produced a significant attenuation of the excitatory response in more than 50% of the recorded neurones without significant latency. The simultaneous iontophoresis of melatonin + amlodipine or melatonin + diltiazem did not increase the attenuation produced by melatonin alone. However, the attenuation of the excitatory response was significantly higher after ejecting melatonin + omega-conotoxin than after ejecting melatonin alone. The melatonin-Ca2+ relationship was further supported by iontophoresis of the Ca2+ ionophore A-23187, which suppressed the inhibitory effect of either melatonin or Ca2+ antagonists. In addition, in synaptosomes prepared from rat striatum, melatonin produced a decrease in the Ca2+ influx measured by Fura-2AM fluorescence. Binding experiments with [3H]MK-801 in membrane preparations from rat striatum showed that melatonin did not compete with the MK-801 binding sites themselves although, in the presence of Mg2+, melatonin increased the affinity of MK-801. The results suggest that decreased Ca2+ influx is involved in the inhibitory effects of melatonin on the glutamatergic activity of rat striatum.

Clotrimazole inhibits the recombinant human cardiac L-type Ca2+ channel alpha 1C subunit.

1. Clotrimazole (CLT) is an antimycotic agent with a potential role in the treatment of cancer. Whole-cell patch clamp recordings and Fura-2 AM fluorescence measurements were used to investigate the inhibition by CLT of recombinant human cardiac L-type Ca2+ channel alpha 1C subunits, stably expressed in human embryonic kidney (HEK 293) cells. 2. CLT (100 nmol l-1 to 25 mumol l-1) reduced Ca2+ channel currents in a concentration-dependent manner. Inhibition was neither use- or voltage-dependent. The effects of CLT were rapid and maximal effects were attained within 3 min. Application of CLT also caused an acceleration of apparent Ca2+ channel current inactivation. 3. Basal current density and the degree of inhibition due to CLT were not significantly altered by pretreating cells with 3 mmol l-1 1-aminobenzotriazole for 1 h, or by dialysing cells for 10 min with 2 mmol l-1 alpha-napthoflavone via the patch pipette, suggesting that the inhibitory action of CLT was not due to inhibition of cytochrome P-450. 4. CLT (10 mumol l-1) did not influence [Ca2+]i, as determined by Fura-2 AM fluorescence measurements. 5. Dialysing cells for 10 min with the non-specific serine/threonine kinase inhibitor H-7 (10 mumol l-1) was without effect on basal current density or on the inhibitory response to 10 mumol l-1 CLT, indicating that CLT is not acting via an indirect effect on these kinases. 6. These data suggest that CLT exerts a direct blocking effect on the alpha 1C subunit at therapeutic concentrations. This effect may explain the abbreviation of the action potential duration by CLT observed in cardiac myocytes.

Effects of ethanol on mechanisms for secretory and aggregatory responses of human granulocytes

Since the mechanisms for inhibitory effects of ethanol on polymorphonuclear (PMN) leukocyte function are largely unknown we studied secretory and aggregatory responses after in vitro treatment of PMN with ethanol. Oxidative metabolism (assessed by chemiluminescence, Superoxide ion production and oxygen consumption) and aggregation were reduced dose-dependently in the presence of ethanol when elicited by stimuli binding to surface receptors or other structures (fMLP and ConA). Similar results were obtained with non-receptor-dependent stimuli, as NaF and the calcium ionophore A23187. In contrast, phorbol myristate acetate peak responses were not affected. FMLP induced changes in Fura-2AM fluorescence (as a probe for fast intracellular calcium concentration changes) were, however, not influenced by ethanol. Neither were membrane potential changes [assessed by di-O-C5(3) fluorescence]. Thus, it is suggested that ethanol may impair a step of the stimulus-response-coupling in PMNs distal to the initial intracellular calcium mobilization.