The protein kinase inhibitor, staurosporine, inhibits L-type Ca2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.     

Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca²⁺ current (I_Ca), followed by a Ca²⁺-activated Cl^- current, and, on stepping back to -80 mV, large Cl^- tail currents were observed. Both currents were abolished when the cells were superfused with Ca²⁺-free bath solution, suggesting that Ca²⁺ influx was necessary for activation of the Cl^- current. The Cl^- current was also abolished when Ba²⁺ was substituted for Ca²⁺ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl^- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca²⁺-induced Ca²⁺ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation. interstitial cells; urethra; calcium-activated chloride current; calcium-induced calcium release; inositol 1,4,5-trisphosphate; ryanodine     

Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

Ca2+-activated Cl- current from human bestrophin-4 in excised membrane patches

Bestrophins are a newly discovered family of Cl(-) channels, some members of which are activated by intracellular Ca(2+). So far, all studies were carried out with whole-cell recordings from plasmid-transfected cultured cells, so it is unclear whether Ca(2+) activates bestrophin through a metabolic mechanism or in a more direct way. We report here experiments that addressed this question with excised, inside-out membrane patches. We chose human bestrophin-4 (hBest4) for heterologous expression because it gave particularly large Cl(-) currents when expressed, thus allowing detection even in excised membrane patches. hBest4 gave a negligible Cl(-) current in a Ca(2+)-free solution on the cytoplasmic (bath) side, but produced a Cl(-) current that was activated by Ca(2+) in a dose-dependent manner, with a K(1/2) of 230 nM. Thus, Ca(2+) appears to activate the bestrophin Cl(-) channel without going through a freely diffusible messenger or through protein phosphorylation. Because the activation and deactivation kinetics were very slow, however, we cannot exclude the involvement of a membrane-associated messenger.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Mechanical stress-induced Ca2+ entry and Cl- current in cultured human aortic endothelial cells

A fluid streamthrough a microtube was applied to cultured human aortic endothelialcells to investigate the endothelial responses of both the ioniccurrents and intracellular Ca²⁺concentration([Ca²⁺]_i)to mechanical stimulation. The fluid stream induced an increase in[Ca²⁺]_ithat was dependent on both the flow rate and the extracellular Ca²⁺ concentration.Gd³⁺ and niflumic acid inhibitedthe fluid stream-induced increase in[Ca²⁺]_i,whereas Ba²⁺ andtetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca²⁺]_iincrease was accompanied by the activation of an inward current at52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the externalCl concentration wasreduced but was not affected by variation of the externalNa⁺ concentration. During theexposure to the fluid stream,[Ca²⁺]_iwas voltage dependent, i.e., depolarization decreased[Ca²⁺]_i.We therefore conclude that the fluid stream-induced current is largelycarried by Cl and that theCl current may thus play arole in modulating the Ca²⁺ influxby altering the membrane potential of endothelial cells.

     

Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Ca2+-sensitive permeability changes caused by influenza virus

Influenza virus added to Lettré cells at pH 5.3 induces a permeability change similar to that elicited by Sendal virus at pH 7.4: K⁺ and Na⁺ equilibrate across the plasma membrane and low-molecular-weight phosphorylated compounds leak out of cells, which remain impermeable to trypan blue.     

Mg2+-sensitive alterations in Ca2+ regulation associated with cell transformation

The intracellular Ca²⁺ content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg²⁺ causes a 2- to 3-fold increase in their Ca²⁺ content over a 24-hr period. Restoring Mg²⁺ to the medium decreases the Ca²⁺ content of the cells to their original values in about the same time. The increase in Ca²⁺ content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg²⁺ deprivation also decreases the initial rate of Ca²⁺ efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca²⁺ to the levels found in the nontransformed cells. Since Mg²⁺ deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca²⁺ in this normalization was studied. Large changes in extracellular Ca²⁺ produced large changes in the Ca²⁺ content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca²⁺ deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca²⁺ content of these cells. The possible role of Mg²⁺ in regulating cellular Ca²⁺ content and distribution is discussed, as is the relation of Ca²⁺ content and distribution to the development of the transformed state.     

Transient expression of a Ca2+-activated Cl- current during development of quail sensory neurons

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

EDTA-binding and acylation of the Ca2+-sensitive photoprotein aequorin

The rate of phosphorylation and concomitant inactivation of purified pig heart muscle pyruvate dehydrogenase complex by intrinsic kinase (EC 2.7.1.99) is markedly accelerated by the addition of coenzyme A to the incubation medium, showing a half-maximum effect at 1.8 μM. The pantetheine moiety is the effective part of the coenzyme A molecule. The free thiol group is prerequisite for the stimulatory action, acetyl-CoA, benzoyl-CoA or CoAS-SCoA being ineffectual. The thiol's specificity is evidenced by showing that dithiothreitol, 2-mercaptoethanol or glutathione up to 5 mM failed to replace coenzyme A. The possibility is considered that coenzyme A might act as a physiological modifier of pyruvate dehydrogenase kinase activity.     

Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins

The recent improvement in the design and use of genetically encoded fluorescent Ca2+ indicators should foster major progress in three aspects of Ca2+ signaling. At the subcellular level, ratiometric probes with expanded dynamics are now available to measure accurately the local Ca2+ changes occurring within specific cell compartments. These tools will allow to determine precisely the role of organelles and of cellular microdomains in Ca2+ handling. At the cellular level, the permanent labeling offered by the genetic probes enables large-scale, long-term Ca2+ measurements with robotic multiplexing technologies such as fluorescence plate readers or automated microscopes. This opens the way to large-scale pharmacological or genetic screens based on organelle-specific functional assays. At the whole animal level, probes with improved dynamics and reduced interference with endogenous proteins will allow to generate transgenic animals bearing Ca2+ sensitive indicators in specific cells and tissues. With this approach, Ca2+ signals can be recorded in neurons, heart, and pancreas of live animals during physiological and pathological stimulations. In this chapter, I will review the progress made in the design and use of genetic Ca2+ indicators, and discuss how these new tools provide an opportunity to challenge several unsolved questions in Ca2+ signaling.     

Pentoxifylline inhibits Ca2+-dependent and ATP proteasome-dependent proteolysis in skeletal muscle from acutely diabetic rats

Previous studies from this laboratory have shown that catecholamines exert an inhibitory effect on muscle protein degradation through a pathway involving the cAMP cascade. The present work investigated the systemic effect of pentoxifylline (PTX; cAMP-phosphodiesterase inhibitor) treatment on the rate of overall proteolysis, the activity of proteolytic systems, and the process of protein synthesis in extensor digitorum longus muscles from normal and acutely diabetic rats. The direct in vitro effect of this drug on the rates of muscle protein degradation was also investigated. Muscles from diabetic rats treated with PTX showed an increase (22%) in the cAMP content and reduction in total rates of protein breakdown and in activity of Ca2+-dependent (47%) and ATP proteasome-dependent (23%) proteolytic pathways. The high content of m-calpain observed in muscles from diabetic rats was abolished by PTX treatment. The addition of PTX (10(-3) M) to the incubation medium increased the cAMP content in muscles from normal (22%) and diabetic (51%) rats and induced a reduction in the rates of overall proteolysis that was accompanied by decreased activity of the Ca2+-dependent and ATP proteasome-dependent proteolytic systems, in both groups. The in vitro addition of H-89, an inhibitor of protein kinase A (PKA), completely blocked the effect of PTX on the reduction of proteolysis in muscles from normal and diabetic rats. The present data suggest that PTX exerts a direct inhibitory effect on protein degradative systems in muscles from acutely diabetic rats, probably involving the participation of cAMP intracellular pathways and activation of PKA, independently of tumor necrosis factor-alpha inhibition.