Mediator Of Ca2 Research Articles

Calcium-dependent regulation of voltage-gated sodium channels in cardiac myocytes: just the beginning?

The Ca2+-binding protein calmodulin (CaM) and the Ca2+/CaM-sensitive kinase (CaMKII) play an important role in the regulation of cardiac excitation–contraction coupling, regulating ryanodine receptor and phospholamban activity.1 CaM is also known to regulate a variety of ion channels; voltage-dependent Ca2+ channels are the classical example, and CaM has been identified as the mediator of Ca2+-dependent channel inactivation. The discovery that Ca2+-dependent inactivation is associated with a specific region in the C-terminus containing a consensus ‘IQ’ motif2 initiated the pursuance of calcium-dependent regulation of other ion channels. The recognition of the presence of an IQ-like motif within the C-terminus of voltage-gated sodium channels (VGSCs) in mammalian cells3 triggered interest in the possibility that VGSCs may also undergo regulation via this mechanism. In the heart, VGSCs are integral to conduction within the myocardium and maintenance of normal cardiac rhythm, underlying the action potential upstroke and velocity of impulse propagation. Impairment of VGSC function is frequently associated with clinical conditions, including Brugada syndrome, and conditions involving augmented late sodium VGSC current ( I NaL) such as long-QT syndrome (LQT3).4 At present, however, there is limited information regarding the effects of CaM/CaMKII modulation on activity of cardiac VGSCs. The results of a few individual studies that focused on the mechanisms … *Corresponding author. Tel: +44 1225 384471; fax: +44 1225 826 114. E-mail address : s.v.smirnov{at}bath.ac.uk

Characterization of promoter expression patterns of OsNrt2.1, a nitrate transporter gene of rice (Oryza sativa L.)

Nitrate is one form of available nitrogen nutrient, and its uptake and transport in plants was mediated by nitrate transporters. It is important to elucidate the transcription mechanism of nitrate genes at the molecular level. In this study, the promoter of OsNrt2.1, a rice nitrate transporter gene previously cloned in our group, has been characterized. Based on PLACE online analysis, some important cis-regulatory elements were identified. Under the control of the full length fragment of OsNrt2.1 promoter (P-2047), the expression of the reporter gene Gus was up-regulated when exposed to low nitrogen. Promoter deletion analysis indicated that the low nitrate responding elements located at position − 524 to − 1 and − 980 to − 525. Several cis-regulatory elements such as CIACADIANLELHC and − 10 promoter element, located at positions of − 524 to − 1, − 980 to − 525, − 1487 to − 981, and − 2047 to − 1488 in P-2047, were possibly involved in the circadian regulation of the OsNrt2.1 gene. Sugar signaling and sugar-responsive motif WBOXHVISO1 and TATCCAOSAMY were also identified in P-2047, suggesting that the exogenous sugar variations resulted from the photosynthesis changes were related to the up-regulated expression of Gus during the day time. It is guessed that the expression pattern of Gus with lownitrate inducible and diurnal rhythm was partly, at least, via the mediation of Ca2+ signal transduction pathway. Owing to its regulation pattern with low nitrate inducible and typical circadian pattern, the OsNrt2.1 promoter maybe has a potential role in the generation of transgenic crop varieties with high-N use efficiency in the future.

Functional analysis of C2H2 zinc finger transcription factor CrzA involved in calcium signaling in Aspergillus nidulans

Calcium signaling systems are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. Calcineurin is an important signaling component, which mediates ion homeostasis and virulence in several fungi. Based on intensive studies conducted on budding yeast, transcription factor Crz1p is thought to be a target of calcineurin. To provide insight into calcium signaling, a Crz1p homolog (CrzA) in a filamentous fungus Aspergillus nidulans was identified and its function with special reference to calcium response was characterized. A crzA gene disruption mutant exhibited sensitivity to high concentrations of Mn(2+) and Ca(2+), and mediated the expression of P-type calcium-ATPase homologous genes. Comprehensive transcriptional analysis with DNA microarrays indicated that CrzA regulates the expression of a vacuolar Ca(2+)/H(+) exchanger gene in response to external calcium stimuli. It is suggested that the calcineurin-CrzA pathway is the mediator of Ca(2+) homeostasis in A. nidulans. Moreover, a crzA/hogA double mutant showed hypersensitivity to osmotic stress, indicating the importance of calcium homeostasis for adaptation to osmotic stress, a universal stress in filamentous fungi.

Canonical Transient Receptor Potential 1 Channel Is Involved in Contractile Function of Glomerular Mesangial Cells

Contractility of mesangial cells (MC) is tightly controlled by [Ca(2+)](i). Ca(2+) influx across the plasma membrane constitutes a major component of mesangial responses to vasoconstrictors. Canonical transient receptor potential 1 (TRPC1) is a Ca(2+)-permeable cation channel in a variety of cell types. This study was performed to investigate whether TRPC1 takes part in vasoconstrictor-induced mesangial contraction by mediating Ca(2+) entry. It was found that angiotensin II (AngII) evoked remarkable contraction of the cultured MC. Downregulation of TRPC1 using RNA interference significantly attenuated the contractile response. Infusion of AngII or endothelin-1 in rats caused a decrease in GFR. The GFR decline was significantly reduced by infusion of TRPC1 antibody that targets an extracellular domain in the pore region of TRPC1 channel. However, the treatment of TRPC1 antibody did not affect the AngII-induced vasopressing effect. Electrophysiologic experiments revealed that functional or biologic inhibition of TRPC1 significantly depressed AngII-induced channel activation. Fura-2 fluorescence-indicated that Ca(2+) entry in response to AngII stimulation was also dramatically inhibited by TRPC1 antibody and TRPC1-specific RNA interference. These results suggest that TRPC1 plays an important role in controlling contractile function of MC. Mediation of Ca(2+) entry might be the underlying mechanism for the TRPC1-associated MC contraction.

Calcium/calmodulin-dependent protein kinase II-δ isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

Large Store-operated Calcium Selective Currents Due to Co-expression of Orai1 or Orai2 with the Intracellular Calcium Sensor, Stim1

The molecular nature of store-operated Ca(2+)-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca(2+)-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca(2+) entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca(2+) entry and Ca(2+)-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca(2+)-selective store-operated Ca(2+) entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca(2+) entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca(2+) sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.

Role of Endogenous TRPC6 Channels in Ca2+ Signal Generation in A7r5 Smooth Muscle Cells

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.

Inhibition of calpain blocks pancreatic β-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.

Annexin A2 is a novel RNA-binding protein.

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.

Differential Properties of Astrocyte Calcium Waves Mediated by P2Y1 and P2Y2 Receptors

Intercellular spread of Ca2+ waves is the primary manifestation of cell-to-cell communication among astrocytes. Ca2+ waves propagate via the release of a diffusible extracellular messenger that has been identified as ATP. In dorsal spinal astrocytes, Ca2+ waves are mediated by activation of two functionally distinct subtypes of metabotropic purinoceptor: the P2Y1 receptor and a receptor previously classified as P2U. Here, we show that the P2U receptor is molecularly and pharmacologically identical to the cloned P2Y2 receptor. Both P2Y1 and P2Y2 receptors are necessary for full Ca2+ wave propagation in spinal astrocytes. Conversely, heterologous expression of either P2Y1 or P2Y2 receptors is sufficient for Ca2+ waves, and expressing these receptor subtypes together recapitulates the characteristics of Ca2+ waves in spinal astrocytes. Thus, P2Y1 and P2Y2 receptors are both necessary and sufficient for propagation of Ca2+ waves. Furthermore, we demonstrate that there are dramatic differences in the characteristics of Ca2+ waves propagating through each receptor subtype: Ca2+ waves propagating via P2Y2 receptors travel faster and further than those propagating via P2Y1 receptors. We find that the nucleotidase apyrase selectively blocks Ca2+ wave propagation through P2Y2 receptors but accelerates Ca2+ waves propagating through P2Y1 receptors. Taking our results together with those from the literature, we suggest that mediation of Ca2+ waves by ATP leading to activation of two subtypes of receptor, P2Y1 and P2Y2, may be a general principle for gliotransmission in the CNS. Thus, processes that alter expression or function of these receptors may control the rate and extent of astrocyte Ca2+ waves.

Ryanodine receptors and the mediation of Ca2+-dependent anion secretion across rat colon.

The properties of the Na(+)-Ca(2+) exchanger in isolated crypts from rat colon were studied using the Fura-2 imaging technique. The transport mode of the exchanger was reversed by replacing extracellular Na(+) by the impermeable cation, N-methyl-D-glucamine (NMDG(+)), so that the transporter mediated a Ca(2+) influx into the cells. Depletion of intracellular Ca(2+) stores by inhibitors of sarcoplasmatic endoplasmatic calcium ATPases (SERCA), i.e., cyclopiazonic acid (10(-5) mol l(-1)) or thapsigargin (10(-6) mol l(-1)), reduced the increase in [Ca(2+)](i) evoked by superfusion with NMDG(+), suggesting a cross-talk between the exchanger and the Ca(2+) stores. However, measurement of Ca(2+) influx with the Mn(2+) quench technique revealed that the activity of the exchanger was independent of the filling state of the stores. Instead, the obvious inhibition of the [Ca(2+)](i)response by SERCA blockers was due to a reduction of Ca(2+)-induced Ca(2+) release after inhibition of store-refilling. The functional presence of ryanodine receptors was demonstrated by the increase in [Ca(2+)](i)evoked by ryanodine (10(-7) to 3x10(-4) mol l(-1)) in a concentration-dependent manner. This effect was mimicked by cADP ribose (10(-5) mol l(-1)) in crypts permeabilized with saponin (10 mg l(-1)). Ruthenium red (5x10(-5) mol l(-1)) or high concentrations of ryanodine (3x10(-4) mol l(-1)) inhibited this response. In Ussing chamber experiments ruthenium red (5x10(-4) mol l(-1)) or a high concentration of ryanodine (10(-3) mol l(-1)) inhibited the increase in short-circuit current evoked by the cholinergic agonist, carbachol (5x10(-5) mol l(-1)). Consequently, Ca(2+)-induced Ca(2+) release may act as kind of amplifier during Ca(2+)-dependent Cl(-) secretion in order to maintain a long-lasting increase in the intracellular Ca(2+) concentration.

A model of the L‐type Ca2+ channel in rat ventricular myocytes: ion selectivity and inactivation mechanisms

1. We have developed a mathematical model of the L-type Ca2+ current, which is based on data from whole-cell voltage clamp experiments on rat ventricular myocytes. Ion substitution methods were employed to investigate the ionic selectivity of the channel. Experiments were configured with Na+, Ca2+ or Ba2+ as the majority current carrier. 2. The amplitude of current through the channel is attenuated in the presence of extracellular Ca2+ or Ba2+. Our model accounts for channel selectivity by using a modified Goldman-Hodgkin-Katz (GHK) configuration that employs voltage-dependent channel binding functions for external divalent ions. Stronger binding functions were used for Ca2+ than for Ba2+. 3. Decay of the ionic current during maintained depolarization was characterized by means of voltage- and Ca2+-dependent inactivation pathways embedded in a five-state dynamic channel model. Particularly, Ca2+ first binds to calmodulin and the Ca2+-calmodulin complex is the mediator of Ca2+ inactivation. Ba2+-dependent inactivation was characterized using the ttau same scheme, but with a decreased binding to calmodulin. 4. A reduced amount of steady-state inactivation, as evidenced by a U-shaped curve at higher depolarization levels (>40 mV) in the presence of [Ca2+]o, was observed in double-pulse protocols used to study channel inactivation. To characterize this phenomenon, a mechanism was incorporated into the model whereby Ca2+ or Ba2+ also inhibits the voltage-dependent inactivation pathway. 5. The five-state dynamic channel model was also used to simulate single channel activity. Calculations of the open probability of the channel model are generally consistent with experimental data. A sixth state can be used to simulate modal activity by way of introducing long silent intervals. 6. Our model has been tested extensively using experimental data from a wide variety of voltage clamp protocols and bathing solution manipulations. It provides: (a) biophysically based explanations of putative mechanisms underlying Ca2+- and voltage-dependent channel inactivation, and (b) close fits to voltage clamp data. We conclude that the model can serve as a predictive tool in generating testable hypotheses for further investigation of this complex ion channel.

Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin.

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+ inhibition. We propose that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition.

Cytoplasmic calcium gradients and calmodulin in the early development of the fucoid alga Pelvetia compressa.

The predicted existence of cytoplasmic Ca2+ gradients during the photopolarization of the zygotes of the brown algae, Pelvetia and Fucus, has proved to be difficult to establish, and the downstream targets of the putative gradients are not known. We have used quantitative microinjection of the long excitation wavelength Ca2+ indicator, Calcium Crimson, and of antibodies against calmodulin to investigate these matters in the zygotes and early embryos of Pelvetia. We found that there is a window of cytoplasmic Calcium Crimson concentration that gives an adequate signal above autofluorescence yet allows normal development of the zygotes. As Calcium Crimson is not a ratiometric indicator, we injected other zygotes with a Ca2+-insensitive dye, rhodamine B, and imaged the cells at the same time that Calcium Crimson-injected cells were imaged. Ratios were calculated by dividing the averaged pixel values of Calcium Crimson images by the averaged pixel values of corresponding rhodamine B images. By this method, we observed the formation of a cytoplasmic Ca2+ gradient within one hour of the exposure of the cells to unilateral blue light during the photosensitive period. The region of high Ca2+ was localized to and predictive of the site of future rhizoid formation. We validated this somewhat indirect method by applying it to the growing rhizoid, where the existence of a tip-localized Ca2+ gradient is well established. The method clearly revealed the known gradient. The injection of ungerminated zygotes with antibodies made against Dictyostelium calmodulin inhibited germination, and this inhibition was abolished if the calmodulin antibodies were coinjected with an excess of purified maize calmodulin. Likewise, the growth of the rhizoids was inhibited by calmodulin antibody injections. The fungus-derived calmodulin antagonist, ophiobolin A, which has previously been shown to be a potent inhibitor of germination, also inhibited rhizoidal growth. Our results provide evidence that a cytoplasmic Ca2+ gradient is present during photopolarization and that calmodulin acts as a mediator of Ca2+ gradients throughout the early developmental processes of germination and rhizoidal growth in Pelvetia compressa.

Inhibition of calmodulin-activated smooth-muscle myosin light-chain kinase by calmodulin-binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives.

Aspects of the biochemistry of calmodulin have been addressed that bear on its cell biological role as a mediator of Ca2+ regulation. Calmodulin-binding peptides derived from the amino acid sequence of smooth-muscle myosin light-chain kinase (MLCK) were characterized as inhibitors of calmodulin activation of MLCK-catalyzed phosphorylation of the smooth-muscle regulatory light chain (MLC). MLCK activity was determined by measuring the rate of formation of one of the reaction products, ADP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0 and 21 degreesC. The Km value of calmodulin was 3.5 nM, a value 16-35-fold greater than the Kd value of calmodulin for MLCK [Török, K., and Trentham D. R. (1994) Biochemistry 33, 12807-12820]. The different Km and Kd values are most likely associated with the rate-limiting step in MLC phosphorylation being associated with product release from MLCK. The values of the inhibition constants, Ki, were the following: Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide), 8.6 (+/-1. 4 sd) pM; Y4-analogue of Trp peptide (Tyr peptide), 7.3 (+/-0.1) nM; and A-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-S-S (RS20-like peptide), 0. 11-0.39 nM. The Ki values were consistent with kinetically determined Kd values of the peptides to calmodulin. Kinetic determination of Kd values required the use of a fluorescently labeled calmodulin, 2-chloro-(epsilon-amino-Lys75)-[6-(4-N, N-diethylamino-phenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin).1 Since, as here, Lys75 is a convenient labeling site on calmodulin for the introduction of fluorescent probes, the biological activity of the Lys-modified calmodulins was evaluated. TA-calmodulin and calmodulin selectively modified by 1-N, N-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-C1) at Lys75 (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK. The Km value for dansyl-calmodulin was equal to that of calmodulin, and that of TA-calmodulin was 3.5-fold greater. TA-calmodulin and Lys75-labeled dansyl-calmodulin thus distinguish between PDE and MLCK being agonists to the former and antagonists to the latter.

Calcium-stimulated Phosphorylation of MAP-2 in Pancreatic ॆTC3-cells Is Mediated by Ca2+/Calmodulin-dependent Kinase II

An understanding of the role of CaM kinase II in the pancreatic beta-cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta-cell in situ, and that this event may provide an important link in the mediation of Ca2+-dependent insulin secretion.

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas.

Nitric oxide (NO) production reportedly regulates guanosine 3', 5'-cyclic monophosphate (cGMP) formation and Ca2+ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca2+]([Ca2+]i), NO synthase, and cGMP were measured. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 0.01-100 microM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 microM did not affect the changes in Ca2+ spiking evoked by these secretagogues; nor was Ca2+ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 microM) altered neither amylase secretion nor Ca2+ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1-1 mM stimulated amylase secretion and Ca2+ transients to a level equal to 10-15% and 13-24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1-10 microM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca2+ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca2+]i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca2+ signaling and amylase secretion stimulated by carbachol and CCK-8.

CD45 Regulates Apoptosis Induced by Extracellular Adenosine Triphosphate and Cytotoxic T Lymphocytes

Several lines of evidence implicate protein tyrosine phosphatases (PTP) in the regulation of apoptotic cell death. We have evaluated the role of CD45, the major PTP of hematopoietic cells, in apoptosis induced by extracellular ATP (ATPe) and cytotoxic T lymphocytes (CTL). We observed that two CD45−clones obtained by mutagenesis of the Fas−cell line L1210, exhibit a higher susceptibility to apoptosis induced by ATPe, which was also evident in Ca2+-free conditions, when compared to the parental cell line or CD45+variants. The CD45−cells were also more susceptible to death mediated by an alloreactive CTL clone. When the cytotoxic assay was performed in the presence of EGTA, a Ca2+chelator, which prevents cytotoxic granule exocytosis and perforin polymerization on target cell membranes, only the CD45−target cells were killed by the CTL clone. These results suggest that a cytotoxic pathway other than the secretory or Fas-dependent pathways was responsible for the enhanced susceptibility of CD45−cells to death, and therefore provide further evidence for the role of ATPeas a possible mediator of Ca2+-independent target cell destruction by CTL.

Protein kinase C mediation of Ca(2+)-independent contractions of vascular smooth muscle.

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca(2+)-independent isoenzyme of PKC. Of the two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, identified in ferret aorta, PKC epsilon is more likely to mediate the contractile response because (i) PKC epsilon, but not PKC zeta, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKC epsilon translocates from the sarcoplasm to the sarcolemma, whereas PKC zeta, translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKC epsilon, but not PKC zeta, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKC epsilon is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of alpha, beta, and gamma isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKC epsilon but is a very poor substrate of PKC zeta. A signal transduction pathway is proposed to explain Ca(2+)-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKC epsilon would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (approximately 0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKC epsilon is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to alpha 1-adrenergic agonists than to K+.

Ca2+ regulates calmodulin binding to IQ motifs in IRS-1.

IRS-proteins couple the receptors for insulin and various cytokines to signalling proteins containing Src homology 2 (SH2) domains. Here we demonstrate that calmodulin, a mediator of Ca(2+)-dependent physiological processes, associates with IRS-1 in a phosphotyrosine-independent manner. IRS-1 coimmunoprecipitated with calmodulin from lysates of Chinese hamster ovary cells expressing IRS-1. The interaction was modulated by Ca2+, and calmodulin binding to IRS-1 was enhanced by increasing intracellular Ca2+ with A23187. In contrast, trifluoperazine, a cell-permeable calmodulin antagonist, decreased binding of calmodulin to IRS-1. Insulin stimulated tyrosine phosphorylation of IRS-1, but did not significantly alter the interaction between calmodulin and IRS-1. IQ-like motifs occur between residues 106-126 and 839-859 of IRS-1. Synthetic peptides based on the these sequences inhibited the association between IRS-1 and calmodulin. These data demonstrate that calmodulin binds to IRS-1 in intact cells in a Ca(2+)-regulated manner, providing a molecular link between the signalling pathways.