Changes In Intracellular Free Ca2 Research Articles

P2Y2-receptor-mediated activation of a contralateral, lanthanide-sensitive calcium entry pathway in the human airway epithelium.

1. Receptor-mediated calcium entry (RMCE) was examined in well-differentiated cultures of normal human bronchial epithelial cells (HBECs). Changes in intracellular free Ca(2+) ([Ca(2+)](i)) were quantified using fluorescence ratio imaging of Fura-2-loaded cells during perfusion with Ca(2+) mobilizing agonists. 2. Initial studies revealed an agonist potency of ATP=uridine triphosphate (UTP) >ADP=uridine diphosphate, consistent with purinergic activation of an apical P2Y(2)-receptor mediating the increase in [Ca(2+)](i) in HBECs. 3. Apical UTP (30 microm) induced a sustained period of elevated [Ca(2+)](i) between 300 and 600 s following agonist stimulation that extracellular Ca(2+) free studies indicated was dominated by Ca(2+) influx. 4. RMCE was inhibited by 100 nm La(3+) (83+/-3%) or Gd(3+) (95+/-7%) (P<0.005, n=4-11) and was partially attenuated by Ni(2+) (1 mm) (58.7+/-5.0%, P<0.005, n=9). 5. RMCE was also partially sensitive (< 25% inhibition, P<0.01) to the cation channel blockers SKF96365 (30 microm) and econazole (30 microm), but was insensitive to both verapamil (1 microm) and ruthenium red (10 microm). 6. Using either a sided Ca(2+) readdition protocol or unilateral La(3+), established that the RMCE pathway was located exclusively on the basolateral membrane. 7. The pharmacological sensitivity of the P2Y(2)-receptor activated Ca(2+) entry pathway in the human airway epithelium is inconsistent with the established profile of TRP channel families and is therefore likely to be of an as-yet uncharacterized molecular identity.

NMDA-receptor regulation of muscarinic-receptor stimulated inositol 1,4,5-trisphosphate production and protein kinase C activation in single cerebellar granule neurons.

Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.

The Ca2+/calcineurin-regulated cup gene family in Dictyostelium discoideum and its possible involvement in development.

Changes in free intracellular Ca2+ are thought to regulate several major processes during Dictyostelium development, including cell aggregation and cell type-specific gene expression, but the mechanisms involved are unclear. To learn more about Ca2+ signaling and Ca2+ homeostasis in this organism, we used suppression subtractive hybridization to identify genes up-regulated by high extracellular Ca2+. Unexpectedly, many of the genes identified belong to a novel gene family (termed cup) with seven members. In vegetative cells, the cup genes were up-regulated by high Ca2+ but not by other ions or by heat, oxidative, or osmotic stress. cup induction by Ca2+ was blocked completely by inhibitors of calcineurin and protein synthesis. In developing cells, cup expression was high during aggregation and late development but low during the slug stage. This pattern correlates closely with reported levels of free intracellular Ca2+ during development. The cup gene products are highly homologous, acidic proteins possessing putative ricin domains. BLAST searches failed to reveal homologs in other organisms, but Western analyses suggested that Cup-like proteins might exist in certain other cellular slime mold species. Localization experiments indicated that Cup proteins are primarily cytoplasmic but become cell membrane-associated during Ca2+ stress and cell aggregation. When cup expression was down-regulated by antisense RNA, the cells failed to aggregate. However, this developmental block was overcome by partially up-regulating cup expression. Together, these results suggest that the Cup proteins in Dictyostelium might play an important role in stabilizing and/or regulating the cell membrane during Ca2+ stress and/or certain stages of development.

Prevention of NMDA‐induced death of cortical neurons by inhibition of protein kinase Cζ

Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.

Propofol increases pulmonary artery smooth muscle myofilament calcium sensitivity: role of protein kinase C.

Vascular smooth muscle tone is regulated by changes in intracellular free Ca2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity. These cellular mechanisms could serve as targets for anesthetic agents that alter vasomotor tone. This study tested the hypothesis that propofol increases myofilament Ca2+ sensitivity in pulmonary artery smooth muscle (PASM) via the protein kinase C (PKC) signaling pathway. Canine PASM strips were denuded of endothelium, loaded with fura-2/AM, and suspended in modified Krebs-Ringer's buffer at 37 degrees C for simultaneous measurement of isometric tension and [Ca2+]i. The KCl (30 mm) induced monotonic increases in [Ca2+]i and tension. Verapamil, an L-type Ca2+ channel blocker, attenuated KCl-induced increases in [Ca2+]i and tension to an equal extent. In contrast, propofol attenuated KCl-induced increases in [Ca2+]i to a greater extent than concomitant changes in tension and caused an upward shift in the peak tension-[Ca2+]i relation. Increasing extracellular Ca2+ in the presence of 30 mM KCl resulted in similar increases in [Ca2+]i in control and propofol-pretreated strips, whereas concomitant increases in tension were greater during propofol administration. The Ca2+ ionophore, ionomycin (0.1 microm), increased [Ca2+]i to approximately 50% of the value induced by 60 mm KCl. Under these conditions, propofol (10, 100 microm) caused increases in tension equivalent to 11 +/- 2 and 28 +/- 3% of the increases in tension in response to 60 mM KCl, whereas [Ca2+]i was slightly decreased. Similar effects were observed in response to the PKC activator, phorbol 12-myristate 13-acetate (PMA, 1 microm). Specific inhibition of PKC with bisindolylmaleimide I before ionomycin administration decreased the propofol- and PMA-induced increases in tension and abolished the propofol- and PMA-induced decreases in [Ca2+]i. Selective inhibition of Ca2+ -dependent PKC isoforms with Gö 6976 also attenuated propofol-induced increases in tension. These results suggest that propofol increases myofilament Ca2+ sensitivity in PASM, and this effect involves the PKC signaling pathway.

Multicolor Imaging of Ca 2+ and Protein Kinase C Signals Using Novel Epifluorescence Microscopy

Dynamic changes in intracellular free Ca 2+ concentrations ([Ca 2+] is) control many important cellular events, including binding of Ca 2+-calmodulin (Ca 2+-CaM) and phosphorylation by protein kinase C (PKC). The two signals compete for the same domains in certain substrates, such as myristoylated alanine-rich PKC-substrate (MARCKS). To observe the convergence and relative time of arrival of CaM and PKC signals at their shared domain of MARCKS, we need to image cells that are loaded with more than two fluorescent dyes at a reasonable speed. We have developed a simple and powerful multicolor imaging system using conventional fluorescence microscopy. The epifluorescence configuration uses a glass reflector and rotating filter wheels for excitation and emission paths. As it is free of dichroic (multichroic) mirrors, multiple fluorescence images can be acquired rapidly regardless of the colors of fluorophores. We visualized Ca 2+-CaM and PKC together with the dynamics of their common target, MARCKS, in single live cells. Receptor-activation resulted in translocation of MARCKS from the plasma membrane to cytosol through its phosphorylation by PKC. By observing fluorescence resonance energy transfer, we also obtained direct evidence that Ca 2+-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca 2+-mobilization predominates over PKC activation.

Glutamate induced modulation of free Ca 2+ in isolated inner hair cells of the guinea pig cochlea

To explore the possible involvement of glutamate (Glu) in modulation of inner hair cell (IHC) functions, the glutamate (Glu) induced changes in intracellular free Ca 2+ ([Ca 2+] i) concentration in isolated IHCs and outer hair cells (OHCs) of the guinea pig cochlea were investigated with fluo-3, a fluorescent probe for intracellular Ca 2+. Their unique flask shape identified the IHCs with a distinct neck and spherical base with a large spherical nucleus. Normal cell shapes could be maintained for about 2 h. Fluorescence of fluo-3 was distributed in the whole isolated IHC with brighter staining nuclei. Static [Ca 2+] i remained constant within the observation period in the absence of Glu. In the presence of a low concentration of Glu (3.85 μM), there was an increase of [Ca 2+] i in IHCs, whereas no obvious [Ca 2+] i change was found in OHCs. The increase of the fluorescence in IHCs reached peak level at 180 s and then gradually reduced at 400 s after the administration of Glu. The increases of [Ca 2+] i were observed in nine of 10 IHCs, but one IHC did not show any change. For 10 of the observed OHCs, seven showed no [Ca 2+] i change, and three showed minor reduction of [Ca 2+] i. The increase of the Glu concentration resulted in a corresponding change of [Ca 2+] i in the IHCs after three times administration of Glu. These results suggest that Glu acts on the IHCs presynaptic autoreceptor in a positive feedback manner.

Production of a novel calmodulin-binding DGK by alternative splicing

The importance of Ca2+ in the transduction of a multitude of signals in plant cells is now well established. A key mechanism by which changes in intracellular free Ca2+ are translated into cellular responses is via the activation of regulatory Ca2+-binding proteins such as calmodulin (CaM). Evidence for the importance of CaM in plant cell signalling has gained momentum in recent years with the identification of several CaM isoforms and CaM-like proteins, together with studies implicating CaM in cellular responses to pathogen invasion and a variety of environmental stimuli. However, unlike in animal cells, where numerous proteins under CaM control, including ion channels, transcription factors and kinases, have been reported, few effectors downstream of CaM have been unequivocally identified in plants. Therein lies the key to more fully elucidating Ca2+/CaM-mediated signalling pathways.

Acamprosate inhibits Ca2+ influx mediated by NMDA receptors and voltage-sensitive Ca2+ channels in cultured rat mesencephalic neurones.

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-D-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to omega-conotoxin (omega-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 microM and 300 microM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of omega-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as omega-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.

Growth hormone secretagogue actions on the pituitary gland: multiple receptors for multiple ligands?

1. Growth hormone (GH) secretion is thought to occur under the reciprocal regulation of two hypothalamic hormones, namely GH-releasing hormone (GHRH) and somatostatin (SRIF), through their engagement with specific cell-surface receptors on the anterior pituitary somatotropes. 2. In addition to GHRH and SRIF, synthetic GH-releasing peptides (GHRP) or GH secretagogue(s) (GHS) regulate GH release through the activation of a novel receptor, the GHS receptor (GHS-R). 3. The cloning of the GHS-R from human, swine and rat identifies a novel G-protein-coupled receptor involved in the control of GH secretion and supports the existence of an undiscovered hormone that may activate this receptor. 4. Varieties of intracellular signalling systems are suggested to mediate the action of GHS, which include changes in intracellular free Ca2+ ([Ca2+]i), cAMP, protein kinases A and C, phospholipase C etc. 5. With regard to the use of signalling systems by GHS, especially a new form of GHRP or GHRP-2, a clear species difference has been demonstrated, supporting the possibility of more than one type of GHS-R.

In vitro effects of beta-carotene for the motility, ATP, and intracellular free Ca(2+) concentrations of fowl spermatozoa.

The motility of intact fowl spermatozoa was vigorous at 25 degrees C, but decreased gradually following the addition of 0-100 microM beta-carotene in a dose-dependent manner. Even in the presence of stimulators of fowl sperm motility, such as Ca(2+) or calyculin A, the motility of intact spermatozoa at both 25 and 40 degrees C remained inhibited following the addition of beta-carotene. Under all of these circumstances, sperm ATP concentrations were not reduced by the addition of beta-carotene. Moreover, the motility of demembranated spermatozoa was not inhibited by the addition of the same concentrations of beta-carotene. No changes in intracellular free Ca(2+) concentrations, measured by means of a fluorescent Ca(2+) indicator, fura-2, were observed in intact beta-carotene -treated spermatozoa. These results suggest that beta-carotene is involved in the inhibition of the flagellar movement of fowl spermatozoa without change in energy production, and that the target of beta-carotene might be present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers--an in vivo study.

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.

Nitric oxide induces dose-dependent CA(2+) transients and causes temporal morphological hyperpolarization in human neutrophils.

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.

Ischemia/reperfusion-induced changes in intracellular free Ca

Ischemia/reperfusion-induced changes in intracellular free Ca

Contributions of the C-terminal Domain to the Control of P2X Receptor Desensitization

The P2X purinergic receptor channels (P2XRs) differ among themselves with respect to the rates of desensitization during prolonged agonist stimulation. Here we studied the desensitization of recombinant channels by monitoring the changes in intracellular free Ca(2+) concentration in cells stimulated with ATP, the native and common agonist for all P2XRs. The focus in our investigations was on the relevance of the P2XR C terminus in controlling receptor desensitization. When expressed in GT1 cells, the P2XRs desensitized with rates characteristic to each receptor subtype: P2X(1)R = P2X(3)R > P2X(2b)R > P2X(4)R > P2X(2a)R > P2X(7)R. A slow desensitizing pattern of P2X(2a)R was mimicked partially by P2X(3)R and fully by P2X(4)R when the six-amino acid sequences of these channels located in the cytoplasmic C terminus were substituted with the corresponding arginine 371 to proline 376 sequence of P2X(2a)R. Changing the total net charge in the six amino acids of P2X(4)R to a more positive direction also slowed the receptor desensitization. On the other hand, substitution of arginine 371-proline 376 sequence of P2X(2a)R with the corresponding sequences of P2X(1)R, P2X(3)R, and P2X(4)R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X(2a)R and mutant P2X(3)R having the C-terminal six amino acids of P2X(2a)R at its analogous position resulted in a functional channel whose desensitization was significantly delayed. These results suggest that composition of the C-terminal six-amino acid sequence and its electrostatic force influence the rate of receptor desensitization.

The UVA Light Used during the Fluorescence Microscopy Assay Affects the Level of Intracellular Calcium Being Measured in Experiments with Electric-Field Exposure

In the present paper, the induction of calcium signals in neuroblastoma cells, cells of T-cell leukemia, and osteogenic sarcoma cells were investigated in relation to the UVA irradiation used in fluorescence microscopy. Methods were developed to measure both the mean UVA irradiance and the intensity profile in the UVA-illuminated area of the microscope. This allowed us to calculate the applied UVA radiant exposure of the cells during each experiment. This investigation was undertaken because of the conflicting results in the literature on the effects of electromagnetic fields on the signals of the calcium-sensitive fluorescence probe FURA-2 in lymphocytes. Taking into account that each group used a different system with different optics and lamps, these conflicting results are now at least partially understandable. Our measurements indicate that in a typical experiment with FURA-2 the cells were irradiated with up to 776 kJ m(-2) during 25 min of exposure to UVA light. This causes changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Designating cells in which the [Ca(2+)](i) was distinctly increased during the experiment as "responding", we found Hill-type dependences on the irradiance. Jurkat cells showed a 50% response even at 10 kJ m(-2) and osteosarcoma cells at about 60 kJ m(-2), whereas neuroblastoma cells even at the maximum possible dose responded only minimally. In the case of neuroblastoma cells, we found a dependence of this effect on the CO(2) partial pressure during the preincubation. An electrical treatment with an a.c. field (5 kHz sinusoidal, amplitude modulation 16 Hz 100%, 800 V m(-1), 5 min) had a significant effect on intracellular calcium in neuroblastoma cells only in the case of cells that were not pretreated with CO(2) with high fluences of UVA irradiation. In conclusion, these results indicate that the possibility of UVA artifacts must be considered in all experiments using fluorescence microscopy. Furthermore, our results lead to the hypothesis that oxidative stress could be the link between UVA and electric-field effects.

ATP-Induced Ca2+ Release in Cochlear Outer Hair Cells: Localization of an Inositol Triphosphate-Gated Ca2+ Store to the Base of the Sensory Hair Bundle

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Regulation of p21rac Activation in Human Neutrophils

The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca(2+) ([Ca(2+)](i)) stimulated by fMLP; however, changes in [Ca(2+)](i) were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF(-), a potent activator of heterotrimeric G-protein alpha-subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca(2+)](i), tyrosine phosphorylation, or PI3K.

Involvement of K+ Channels in the Inhibitory Effects of Adenosine on Anoxia-Induced [Ca2+ ]i Increase in Cultured Rat Hippocampal CA1 Neurons

Changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cultured hippocampal CA1 neurons isolated from newborn rats were measured by a confocal laser scanning microscope, using the Ca²⁺ indicator Fluo-3. The results showed that exogenous adenosine (100 μM) significantly attenuated the increase of neuronal [Ca²⁺]_i induced by acute anoxia. This effect of adenosine could be suppressed by the adenosine A1 receptor antagonist 8-cyclopentyltheophylline. Moreover, potassium channel blockers, aminopyridine, and glipizide could also block the inhibitory role of adenosine, but tetraethylammonium had no effect. These results suggest that adenosine may activate 4-AP or ATP-sensitive potassium channels via an A1-receptor-mediated mechanism and consequently inhibit anoxia-induced [Ca²⁺]_i elevation in hippocampal neurons.

Comparison of the Effects of Endothelin-1, -2 and -3 (1-31) on Changes in [Ca2+]i in Human Coronary Artery Smooth Muscle Cells

We have previously found that human chymase selectively cleaves big endothelins (ETs) at the Tyr31-Gly32 bond to produce 31-amino-acid endothelins, ETs (1-31). In the present study, we investigated the effects of ETs (1-31) on changes in intracellular free Ca2+ ([Ca2+]i) in cultured human coronary artery smooth muscle cells (HCASMCs) using confocal laser microscopy. ETs (1-31) increased [Ca2+]i in a concentration-dependent manner. Phosphoramidon did not inhibit the increases in [Ca2+]i caused by ETs (1-31). The [Ca2+]i increases induced by ETs (1-31) were compared to those of ETs (1-21) and big ETs. ET-1 (1-21) was about 10-times more potent than big ET-1 or ET-1 (1-31), whereas big ET-2 was 10-times less potent than ET-2 (1-21) or ET-2 (1-31). ETs (1-31) may induce [Ca2+]i increase through ETA-type or ETA-type-like receptors. The 10-12 M ET (1-31)-induced increases in [Ca2+]i were not affected by removal of extracellular Ca2+, but were inhibited by thapsigargin. These results suggested that ET-1, -2 and -3 (1-31) showed similar potencies in increasing [Ca2+]i and mechanisms of ET (l-31)-induced increases in [Ca2+]i may be similar among the three ETs (1-31).