Fura-2 Fluorescence Research Articles

Gabapentin-mediated inhibition of voltage-activated Ca 2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca 2+ entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG×neuroblastoma hybrid) cells. Gabapentin (2.5 μM) in contrast to GABA (10 μM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba 2+. Gabapentin attenuated high voltage-activated Ca 2+ channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca 2+ influx evoked by K + depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA B receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca 2+ channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca 2+ channels to gabapentin. We analysed the Ca 2+ channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca 2+ channel β 2 and α 2δ subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca 2+ current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca 2+ channel β 2 subunit type as critically important to drug sensitivity and interactions with α 1 and α 2δ subunits may be implicated in antihyperalgesic therapeutic action for this compound.

Regulation of intracellular calcium in dispersed fat body trophocytes of the cockroach, Periplaneta americana, by hypertrehalosemic hormone

Incubation of trophocytes from dissaggregated fat body of Periplaneta americana with either of the hypertrehalosemic hormones, HTH-I or HTH-II, leads to an increase in the cytosolic concentration of Ca 2+ from ∼80 to ∼310 nM with a rise time of approximately 110 s. The Ca 2+ concentration then declines to the resting level during the ensuing 5 min. In the absence of extracellular Ca 2+ the increase in [Ca 2+] i due to HTH is limited to ∼100 nM. The calmodulin inhibitors calmidazolium and W-7 also limit to a similar degree the ability of HTH to increase [Ca 2+] i. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, was shown to block Ca 2+ entry through the plasma membrane. Additional evidence to support the view that HTH enhances Ca 2+ influx has been obtained by measuring the quenching of fura-2 fluorescence when Ca 2+ is replaced with Mn 2+.

N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine reduces intracellular calcium response to noradrenaline in rat visual cortex

Using the fluorescent indicator Fura-2, we investigated the effects of N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4), a noradrenergic neurotoxin, on intracellular calcium responses to noradrenaline, N-methyl- D-aspartate, and carbamylcholine chloride in brain slices of the rat visual cortex. Noradrenergic depletion in the visual cortex of young rats was induced by DSP-4, and its selectivity was confirmed by two different methods, i.e., immunostaining with anti-dopamine-β-hydroxylase antibody and biochemical analysis by high-performance liquid chromatography. The treatment with DSP-4 (25 mg/kg i.p., ×2) caused disruption of noradrenergic fibers throughout all cortical layers, and reduced the content of noradrenaline to 6.4% of that in the normal control. In the normal cortex, bath-applied noradrenaline (100 μM) increased the intracellular calcium to 123% of the control in terms of the F 340/ F 380 ratio of Fura-2 fluorescence. Quantitative analysis of the F 340/ F 380 ratio was performed in layers II to IV, since the increase was mainly observed in these layers. The intracellular calcium response to noradrenaline was significantly ( P<0.0001) reduced in the DSP-4-treated animals to 63.2% of that in the normal control. The response to N-methyl- D-aspartate (100 μM) was also reduced, whereas the response to carbamylcholine chloride, a muscarinic cholinergic agonist (100 μM), was not affected by the DSP-4 treatment. From these findings we suggest that noradrenergic denervation by DSP-4 reduces the intracellular calcium response to noradrenaline through changes in the intracellular signal transduction.

Age-related differences in NMDA responses in cultured rat hippocampal neurons

The N-methyl- d-aspartate receptor (NMDAR) is expressed in the cerebral cortex and hippocampus and is important in learning and memory. NMDARs are influenced by aging and implicated in neurodegenerative disorders. We investigated age-related differences in NMDAR ionic currents and intracellular calcium in embryonic (E18), middle-age (9–10 month) and old (26 month) rat hippocampal neurons cultured in serum-free medium for 7–12 days. Responses to 200 μM NMDA with 50 μM glycine were measured using whole cell voltage clamp and fura-2 fluorescence. Embryonic neurons exhibited significantly larger and faster NMDA responses than adults. Old rats had 1.5 fold greater normalized NMDA peak current compared to middle-age rats, while intracellular calcium rose 1.3 fold higher. Differences in regression slopes generated from the integral of NMDA current versus normalized NMDA current indicate age-related differences are not exclusively due to changes in receptor density but likely influenced by changes in receptor function. Corresponding age-related measures of intracellular calcium by fura-2 fluorescence in response to NMDA showed a strong correlation with peak current ( r 2=0.996). Our data support the hypothesis that NMDAR responsiveness is altered during aging with an enhanced NMDA peak current in both old and embryonic neurons compared to middle-age neurons.

Melatonin inhibits high voltage activated calcium currents in cultured rat dorsal root ganglion neurones

The actions of melatonin on high-voltage activated calcium channels (HVACC) and intracellular free Ca 2+ concentration in cultured dorsal root ganglion (DRG) neurones from neonatal rats were investigated using the whole-cell patch clamp and the fura-2 fluorescence ratio Ca 2+-imaging techniques. HVACC were pharmacologically and biophysically isolated and the effects of melatonin were investigated. Extracellular application of melatonin inhibited HVACC in a dose dependent manner. In calcium imaging experiments, application of extracellular recording medium containing 30 mM KCl evoked increases in intracellular free Ca 2+ that were dependent upon external Ca 2+ ions. This increase was prevented by both low (10 μM) and high dose (100 μM) of melatonin pre-treatment. The results of this study indicate that the pineal hormone melatonin has inhibitory actions on voltage dependent calcium entry in cultured rat DRG neurones.

Venom from Anemesia species of spider modulates high voltage-activated Ca2currents from rat cultured sensory neurones and excitatory post synaptic currents from rat hippocampal slices

The actions of crude venom from Anemesia species of spider were investigated in cultured dorsal root ganglion neurones from neonatal rats and hippocampal slices. Using mass spectrometry (MALDI-TOF MS), 10–12 distinct peptides with masses between about 3 and 10kDa were identified in the crude spider venom. At a concentration of 5μg/ml crudeAnemesia venom transiently enhanced the mean peak whole cell voltage-activated Ca2+current in avoltage-dependent manner and potentiated transient increases in intracellular Ca2+triggered by 30mM KCI as measured using Fura-2 fluorescence imaging. Additionally, 5–8μg/ml Anemesia venom increased the amplitude of glutamatergic excitatory postsynaptic currents evoked in hippocampal slices. Ω-Conotoxin GVIA (1μM) prevented the increase in voltage-activated Ca2+currents produced by Anemesia venom. This attenuation occurred when the cone shell toxin was applied before or after the spider venom. Anemesia venom (5μg/ml) created no significant change in evoked action potentials but produced modest but significant inhibition of voltage-activated K+currents. At a concentration of 50μg/ml Anemesia venom only produced reversible inhibitory effects, decreasing voltage-activated Ca2+currents. However, no significant effects on Ca2+currents were observed with a concentration of 0.5μg/ml. The toxin(s) in the venom that enhanced Ca2+influx into sensory neurones was heat-sensitive and was made inactive by boiling or repetitive freeze-thawing. Boiled venom (5μg/ml) produced significant inhibition of voltage-activated Ca2+currents and freeze-thawed venom inhibited Ca2+transients measured using Fura-2 fluorescence. Our data suggest that crude Anemesia venom contains components, which increased neuronal excitability and neurotransmission, at least in part this was mediated by enhancing Ca2+influx through N-type voltage-activated Ca2+channels.

Cadherins Mediate Intercellular Mechanical Signaling in Fibroblasts by Activation of Stretch-sensitive Calcium-permeable Channels

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.

Effect of tributyltin chloride on the release of calcium ion from intracellular calcium stores in rat hepatocytes

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca2+ from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca2+ was measured, using a fluorescent Ca2+ dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 μM TBT for 30 min, the extracellular Ca2+ concentration was high, but the inositol 1,4,5-trisphosphate (InsP3)-induced increase in Ca2+ concentration was suppressed, suggesting that the extracellular release of Ca2+ in response to TBT treatment was from intracellular stores. Images of the Ca2+ concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 μM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca2+ was released. When the hepatocytes were treated with 4.0 μM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 μM TBT and 2.0 μM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca2+ from intracellular stores. When the hepatocytes were preincubated with 0.1 μM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP3-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca2+ from the intracellular stores at high concentrations, and suppressed the InsP3-induced Ca2+ release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca2+ response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54–61).

Intracellular Calcium Signals Regulating Cytosolic Phospholipase A2 Translocation to Internal Membranes

Increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) promote cytosolic phospholipase A(2) (cPLA(2)) translocation to intracellular membranes. The specific membranes to which cPLA(2) translocates and the [Ca(2+)](i) signals required were investigated. Plasmids of EGFP fused to full-length cPLA(2) (EGFP-FL) or to the cPLA(2) C2 domain (EGFP-C2) were used in Ca(2+)/EGFP imaging experiments of cells treated with [Ca(2+)](i)-mobilizing agonists. EGFP-FL and -C2 translocated to Golgi in response to sustained [Ca(2+)](i) greater than approximately 100-125 nm and to Golgi, ER, and perinuclear membranes (PNM) at [Ca(2+)](i) greater than approximately 210-280 nm. In response to short duration [Ca(2+)](i) transients, EGFP-C2 translocated to Golgi, ER, and PNM, but EGFP-FL translocation was restricted to Golgi. However, EGFP-FL translocated to Golgi, ER, and PNM in response to long duration transients. In response to declining [Ca(2+)](i), EGFP-C2 readily dissociated from Golgi, but EGFP-FL dissociation was delayed. Agonist-induced arachidonic acid release was proportional to the [Ca(2+)](i) and to the extent of cPLA(2) translocation. In summary, we find that the differential translocation of cPLA(2) to Golgi or to ER and PNM is a function of [Ca(2+)](i) amplitude and duration. These results suggest that the cPLA(2) C2 domain regulates differential, Ca(2+)-dependent membrane targeting and that the catalytic domain regulates both the rate of translocation and enzyme residence.

Influence of Ovariectomy on Ventricular Myocyte Contraction in Simulated Diabetes

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a ‘diabetes-simulated high-glucose’ environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR₉₀), maximal velocity of shortening and relengthening (± dL/dt), intracellular Ca²⁺ fura-2 fluorescence intensity and decay rate (τ). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR₉₀, slower ± dL/dt, lower fluorescence intensity and higher τ (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR₉₀, reduced fluorescence intensity, higher τ and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR₉₀ and τ), reversed (PS and NOS) or lost (± dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.

Effects of doxorubicinol on excitation–contraction coupling in guinea pig ventricular myocytes

The cardiotoxicity of the anticancer drug doxorubicin may be related to its main metabolite doxorubicinol. In this study, the acute effects of doxorubicinol on excitation–contraction coupling in isolated guinea pig ventricular myocytes were investigated and compared with doxorubicin using the whole-cell patch-clamp-, fura-2 fluorescence- and cell-edge tracking techniques. Both drugs were applied intracellularly by diffusion from the patch electrode for 15–20 min. Doxorubicin (100 μM) prolonged the action potential duration (APD) by 31% and enhanced cell shortening by 26%. Contrary to doxorubicin, doxorubicinol (10 μM) shortened APD by 25% and decreased cell shortening by 31%. APD shortening by doxorubicinol was due to an increase of the delayed rectifier K + current. Neither the inward rectifier K + current nor the L-type Ca 2+ current was influenced by doxorubicinol. The decline in cell shortening induced by doxorubicinol was not exclusively due to APD shortening because doxorubicinol reduced the peak Ca 2+ transient by 23% in cells clamped with an action potential of constant duration. Despite opposite effects on APD and contractility, both doxorubicin and doxorubicinol produced a considerable delay in the activation and inactivation of contraction and Ca 2+ transient, compatible with an impaired function of the sarcoplasmic reticulum. It is suggested that doxorubicinol-induced APD shortening may amplify the detrimental effects of both doxorubicin and doxorubicinol on sarcoplasmic reticulum Ca 2+ load and hence on contractile function. The accumulation of doxorubicinol in the cardiac myocytes may play an important role in the time-dependent development of doxorubicin-induced ventricular dysfunction.

Protein Kinase C Activates Store-operated Ca2+Channels in Human Glomerular Mesangial Cells

Store-operated Ca(2+) channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin. The measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](i). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca(2+)](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N',N'tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP(O) of SOC, whereas calphostin C did not affect base-line channel activity. In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 microm ATP. Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC. Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.

Influence of ovariectomy on ventricular myocyte contraction in simulated diabetes.

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a 'diabetes-simulated high-glucose' environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR(90), slower +/- dL/dt, lower fluorescence intensity and higher tau (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR90, reduced fluorescence intensity, higher tau and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR90 and tau), reversed (PS and NOS) or lost (+/- dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.

Molecular Cloning of a Third Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family,NCKX3

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.

Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells. Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence. Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim. Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.

Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit gamma1.

1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca(2+) entry (L-type current) and internal Ca(2+) release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit gamma1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca(2+) release and force production in muscle preparations of a mouse deficient in the gamma1 subunit (gamma-/-). 2. The rate of internal Ca(2+) release at large depolarization (+20 mV) was determined in voltage-clamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca(2+). On average, gamma-/- cells showed an increase in release of about one-third of the control value and no alterations in the time course. 3. Voltage of half-maximal activation (V(1/2)) and voltage sensitivity (k) were not significantly different in gamma-/- myotubes, either for internal Ca(2+) release activation or for the simultaneously measured L-type Ca(2+) conductance. The same was true for maximal Ca(2+) inward current and conductance. 4. Contractions evoked by electrical stimuli were recorded in isolated extensor digitorum longus (EDL; fast, glycolytic) and soleus (slow, oxidative) muscles under normal conditions and during fatigue induced by repetitive tetanic stimulation. Neither time course nor amplitudes of twitches and tetani nor force-frequency relations showed significant alterations in the gamma1-deficient muscles. 5. In conclusion, the overall results show that the gamma1 subunit is not essential for voltage-controlled Ca(2+) release and force production.

Ca2+-dependent exocytosis of L-glutamate by alphaTC6, clonal mouse pancreatic alpha-cells.

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by alphaTC6 cells (clonal mouse pancreatic alpha-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited approximately 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 micromol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that alphaTC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by alpha-cells through Ca2+-dependent regulated exocytosis.

The anti-inflammatory interactions of epinephrine with human neutrophils in vitro are achieved by cyclic AMP-mediated accelerated resequestration of cytosolic calcium

This study was designed to evaluate the effects of epinephrine (0.01–1 μM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) (1 μM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5′ cyclic monophosphate (cAMP) and cytosolic free Ca 2+. Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca 2+ fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective β 1-, α 1- or α 2-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca 2+ from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca 2+. These effects of epinephrine on the clearance of Ca 2+ from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca 2+ sequestering/resequestering endo-membrane Ca 2+-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca 2+.

Inhibition of ATP-induced surfactant exocytosis by dihydropyridine (DHP) derivatives: a non-stereospecific, photoactivated effect and independent of L-type Ca 2+ channels

Purinergic stimulation of surfactant secretion via exocytosis of lamellar bodies is mediated by an elevation of the intracellular Ca 2+ concentration ([Ca 2+] i). We tested the dihydropyridine (DHP) analogues isradipine (+/-enantiomers), nifedipine and Bay K 8644 (racemic forms) on ATP-induced surfactant secretion and [Ca 2+] i in single type II cells, using FM1-43 and fura-2 fluorescence. None of the DHPs (2 μM) had an effect on ATP-induced surfactant secretion in the dark. They did, however, inhibit secretion in a concentration-dependent manner during illumination, particularly with UV light. This effect was not stereospecific, because it was mimicked by (−)-isradipine. In addition, (+)- or (−)-isradipine, but not nifedipine or Bay K 8644, elicited a slow increase of [Ca 2+] i during illumination with UV light, which was reversible by exposure to dark. None of the DHPs inhibited the ATP-induced Ca 2+ signal. In perforated patch clamp experiments, depolarizing voltage steps did not induce L-type Ca 2+ (Sr 2+) currents, even in the presence of the agonist Bay K 8644 (1 μM). We conclude that impairment of ATP-induced surfactant secretion by all tested DHPs and alterations of Ca 2+ homeostasis by isradipine are photoactivated effects, independent of L-type Ca 2+ channels.

Characterization of cardiac myocyte and tissue beta-adrenergic signal transduction in rats with heart failure.

The cellular basis of alterations in beta-adrenergic signal transduction in rats with chronic heart failure (CHF) remains unclear. The aim of the present study was to examine this signal transduction system in isolated ventricular cardiomyocytes of rats with CHF. We focused on changes in the levels of stimulatory (Gs) and inhibitory G-proteins (Gi). CHF was induced in male Wistar rats by coronary artery ligation (CAL). Hemodynamic and biochemical parameters were measured 8 weeks after CAL. Alterations in contractile function and Ca(2+) transients via beta-adrenergic receptor signaling of cardiomyocytes isolated from rats with CHF were characterized by simultaneous measurements of cell shortening and fura-2 fluorescence intensity. Coronary artery-ligated rats showed symptoms of CHF, such as decreased contractile function, increased left ventricular volume, decreased chamber stiffness, and about 40% infarct formation of the left ventricle, by 8 weeks after surgery. The contractile function and Ca(2+) dynamics of cardiomyocytes from the rats with CHF remained normal under basal conditions. Only cardiac cell length was increased. The responses of peak shortening, fura-2 fluorescence ratio amplitude, and cAMP content to beta-adrenoceptor stimulation were reduced in cardiomyocytes of the rats with CHF, whereas direct stimulation of adenylate cyclase did not affect the response of these variables. Cardiomyocyte Gsalpha protein was decreased, whereas no changes in Gialpha proteins were seen in these cells. Increases in tissue Gsalpha and Gialpha proteins in the scar zone were detected. The results on tissue levels of collagen and G-proteins in the viable left ventricle appeared to depend on the presence of nonmyocytes. The results suggest that impaired contractile function of cardiomyocytes is unlikely to account for global LV contractile dysfunction, and that down-regulation of beta-adrenoceptors occurs in cardiomyocytes per se. The difference in changes of G-protein between the cardiomyocyte and myocardial tissue suggests an appreciable contribution of nonmyocytes to myocardial G-protein levels.