Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate.

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.     

Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor.

Receptor activation may result in distinct subcellular patterns of Ca2+ release. To define the subcellular distribution of Ca2+i signals induced by stimulation of the vasopressin V1a receptor, we expressed the cloned receptor in Xenopus oocytes. Oocytes were then loaded with fluo-3 and observed using confocal microscopy. Vasopressin induced a single concentric wave of increased Ca2+ that radiated inward from the plasma membrane. With submaximal stimulation, however, regions of the Ca2+ wave spontaneously reorganized into repetitive (oscillatory) waves. Focal stimulation of a small part of the plasma membrane resulted in a Ca2+ wave which began at the point of stimulation, radiated toward the center of the cell, then reorganized into multiple foci of repetitive, colliding waves and spirals of increased Ca2+i. The pattern of Ca2+ signaling induced by focal or global stimulation was not altered in Ca(2+)-free medium, although signals did not propagate as fast. Finally, subcellular Ca2+ signaling patterns induced by vasopressin were inhibited by caffeine, while neither vasopressin nor microinjection of inositol trisphosphate blocked caffeine-induced increases in cytosolic Ca2+. Thus, stimulation of the V1a receptor in this cell system induces a complex pattern of Ca2+ signaling which is influenced by (1) the magnitude of the stimulus, (2) the distribution of the surface receptors that are stimulated, and (3) mobilization of Ca2+ from the extracellular space as well as from two distinct endogenous Ca2+ pools. The manner in which a single type of receptor is activated may represent an important potential mechanism for subcellular Ca2+i signaling.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

Control of astrocyte Ca(2+) oscillations and waves by oscillating translocation and activation of protein kinase C

BACKGROUND: Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves. RESULTS: Glutamate stimulation of astrocytes triggered highly localized GFP-PKCgamma plasma membrane translocation events, induced rapid oscillations in GFP-PKCgamma translocation, and generated GFP-PKCgamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca(2+) waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca(2+) and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca(2+) spikes, suggesting that PKC activation is involved in terminating individual Ca(2+) spikes and waves and in defining the time period between Ca(2+) spikes. CONCLUSIONS: Our study suggests that cPKCs have a negative feedback role on Ca(2+) oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca(2+) concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca(2+) spikes and waves and can define the Ca(2+) spike and wave frequencies.     

G protein-dependent Ca2+ signaling complexes in polarized cells

Polarized cells signal in a polarized manner. This is exemplified in the patterns of [Ca2+]i waves and [Ca2+]i oscillations evoked by stimulation of G protein-coupled receptors in these cells. Organization of Ca(2+)-signaling complexes in cellular microdomains, with the aid of scaffolding proteins, is likely to have a major role in shaping G protein-coupled [Ca2+]i signal pathways. In epithelial cells, these domains coincide with sites of [Ca2+]i-wave initiation and local [Ca2+]i oscillations. Cellular microdomains enriched with Ca(2+)-signaling proteins have been found in several cell types. Microdomains organize communication between Ca(2+)-signaling proteins in the plasma membrane and internal Ca2+ stores in the endoplasmic reticulum through the interaction between the IP3 receptors in the endoplasmic reticulum and Ca(2+)-influx channels in the plasma membrane. Ca2+ signaling appears to be controlled within the receptor complex by the regulators of G protein-signaling (RGS) proteins. Three domains in RGS4 and related RGS proteins contribute important regulatory features. The RGS domain accelerates GTP hydrolysis on the G alpha subunit to uncouple receptor stimulation from IP3 production; the C-terminus may mediate interaction with accessory proteins in the complex; and the N-terminus acts in a receptor-selective manner to confer regulatory specificity. Hence, RGS proteins have both catalytic and scaffolding function in Ca2+ signaling. Organization of Ca(2+)-signaling proteins into complexes within microdomains is likely to play a prominent role in the localized control of [Ca2+]i and in [Ca2+]i oscillations.     

Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.

We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals.     

Propagation of calcium waves along endothelium of hamster feed arteries

An increase in tissue blood flow requires relaxation of smooth muscle cells along entire branches of the resistance vasculature. Whereas the spread of hyperpolarization along the endothelium can coordinate smooth muscle cell relaxation, complementary signaling events have been implicated in the conduction of vasodilation. We tested the hypothesis that Ca(2+) waves propagate from cell to cell along the endothelium of feed arteries exhibiting spontaneous vasomotor tone. Feed arteries of the hamster retractor muscle were isolated, pressurized to 75 mmHg at 37 degrees C, and developed myogenic tone spontaneously. Smooth muscle cells and endothelial cells were loaded with the Ca(2+) indicator Fluo-4. An acetylcholine stimulus was delivered locally using microiontophoresis (1-microm pipette tip, 1 microA, 1 s), and Ca(2+) signaling within and along respective cell layers was determined using laser-scanning confocal microscopy. Acetylcholine triggered an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) of endothelial cells at the site of stimulation that preceded two distinct events: 1) a rapid synchronous decrease in smooth muscle [Ca(2+)](i) along the entire vessel and 2) an ensuing Ca(2+) wave that propagated bidirectionally along the endothelium at approximately 111 microm/s for distances exceeding 1 mm. Maximal dilation of vessels with either nifedipine (1 microM) or sodium nitroprusside (SNP, 100 microM) reduced the distance that Ca(2+) waves traveled to approximately 300 microm (P < 0.05). Thus Ca(2+) waves propagate along the endothelium of resistance vessels with vasomotor tone, and this signaling pathway is compromised during maximal dilation with nifedipine or SNP.     

UV-A induces two calcium waves in Physcomitrella patens

Our understanding of the role of Ca2+ in blue/UV-A photoreceptor signaling in a single cell is limited. Insight into calcium signaling has now been attained in Physcomitrella patens and its cryptochrome and phototropin knock-outs. Physcomitrella patens caulonemal filaments grow in the dark by apical extension and their apical cells are highly polarized. Fura-2-dextran ratio images of the apical cell from wild type (WT), Ppcry1a/1b and PpphotA2/B1/B2 were obtained immediately following UV-A exposure (30 microW cm(-2) at 340 nm for 1,000 ms plus 30 microW cm(-2) at 380 nm for 1,000 ms) [abbreviated as 1,000 ms (340/380 nm)] and demonstrated two intracellular waves: a Ca2+ wave from the growing apical tip through the apical cap, and a wave from the junction of the neighboring cell through the vacuolar, nuclear and plastid regions. In WT, the UV-A-induced tip wave increase had a magnitude of 454.0 +/- 40 nM, traveled at a rate of 3.4 +/- 0.7 microm s(-1) and was complete within 26.6 +/- 2.3 s, while the basal vacuolar wave had a magnitude of 596.8 +/- 110 nM, a rate of 8.4 +/- 0.8 microm s(-1) and duration of 25.3 +/- 4.9 s. Subsequent Ca2+ spikes of similar magnitude followed these waves. The amplitude of the Ca2+ waves in the apical cap and basal vacuolar regions of Ppcry1a/1b were higher than those in the WT, while the duration of those in PpphotA2/B1/B2 was longer. Subsequent Ca2+ spikes occurred in WT and Ppcry1a/1b but not in PpphotA2/B1/B2. When Mn2+ was added to the culture medium, the [Ca2+](cyt) increase was delayed, did not move as a wave and lasted longer. The results indicate that plants respond to blue light and UV-A radiation by generating a wave of changes in the [Ca2+](cyt). The characteristics of these Ca2+ waves were dependent upon cryptochrome and phototropin. Blue/UV-A signaling in P. patens appears to differ from that in Arabidopsis.     

Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells.

Intercellular communication of epithelial cells was examined by measuring changes in intracellular calcium concentration ([Ca2+]i). Mechanical stimulation of respiratory tract ciliated cells in culture induced a wave of increasing Ca2+ that spread, cell by cell, from the stimulated cell to neighboring cells. The communication of these Ca2+ waves between cells was restricted or blocked by halothane, an anesthetic known to uncouple cells. In the absence of extracellular Ca2+, the mechanically stimulated cell showed no change or a decrease in [Ca2+]i, whereas [Ca2+]i increased in neighboring cells. Iontophoretic injection of inositol 1,4,5-trisphosphate (IP3) evoked a communicated Ca2+ response that was similar to that produced by mechanical stimulation. These results support the hypothesis that IP3 acts as a cellular messenger that mediates communication through gap junctions between ciliated epithelial cells.     

Functional mapping of Ca2+ signaling complexes in plasma membrane microdomains of polarized cells

Many cells cluster signaling complexes in plasma membrane microdomains. Polarized secretory cells cluster all Ca2+ signaling proteins, including GPCRs, at the apical pole. The functional significance of such an arrangement is not known because of a lack of techniques for functional mapping of signaling complexes at plasma membrane patches. In the present work, we developed such a technique based on the use of two patch pipettes, a recording and a stimulating pipette (SP). Including 20% glycerol in the SP solution increased the viscosity and the hydrophobicity to prevent leakage and formation of tight seals on the plasma membrane. This allowed moving the SP between sites to stimulate multiple patches of the same cell and with the same agonist concentrations. Functional mapping of Ca2+ signaling in pancreatic acinar cells revealed that the M3, cholecystokinin, and bombesin signaling complexes at the apical pole are much more sensitive to stimulation than those at the basal pole. Furthermore, at physiological agonist concentrations, Ca2+ signals could be evoked only by stimulation of membrane patches at the apical pole. [Ca2+](i) imaging revealed that Ca2+ waves were invariably initiated at the site of apical membrane patch stimulation, suggesting that long range diffusion of second messengers is not obligatory to initiate and propagate apical-to-basal Ca2+ waves. The present studies reveal a remarkable heterogeneity in responsiveness of Ca2+ signaling complexes at membrane microdomains, with the most responsive complexes confined to the apical pole, probably to restrict the Ca2+ signals to the site of exocytosis and allow the polarized functions of secretory cells.     

Arachidonic acid modulates the spatiotemporal characteristics of agonist-evoked Ca2+ waves in mouse pancreatic acinar cells

In pancreatic acinar cells analysis of the propagation speed of secretagogue-evoked Ca2+ waves can be used to examine coupling of hormone receptors to intracellular signal cascades that cause activation of protein kinase C or production of arachidonic acid (AA). In the present study we have investigated the role of cytosolic phospholipase A2 (cPLA2) and AA in acetylcholine (ACh)- and bombesin-induced Ca2+ signaling. Inhibition of cPLA2 caused acceleration of ACh-induced Ca2+ waves, whereas bombesin-evoked Ca2+ waves were unaffected. When enzymatic metabolization of AA was prevented with the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid, ACh-induced Ca2+ waves were slowed down. Agonist-induced activation of cPLA2 involves mitogen-activated protein kinase (MAPK) activation. An increase in phosphorylation of p38(MAPK) and p42/44(MAPK) within 10 s after stimulation could be demonstrated for ACh but was absent for bombesin. Rapid phosphorylation of p38(MAPK) and p42/44(MAPK) could also be observed in the presence of cholecystokinin (CCK), which also causes activation of cPLA2. ACh-and CCK-induced Ca2+ waves were slowed down when p38(MAPK) was inhibited with SB 203580, whereas inhibition of p42/44(MAPK) with PD 98059 caused acceleration of ACh- and CCK-induced Ca2+ waves. The spreading of bombesin-evoked Ca2+ waves was affected neither by PD 98059 nor by SB 203580. Our data indicate that in mouse pancreatic acinar cells both ACh and CCK receptors couple to the cPLA2 pathway. cPLA2 activation occurs within 1-2 s after hormone application and is promoted by p42/44(MAPK) and inhibited by p38(MAPK). Furthermore, the data demonstrate that secondary (Ca2+-induced) Ca2+ release, which supports Ca2+ wave spreading, is inhibited by AA itself and not by a metabolite of AA.     

Properties of intracellular Ca2+ waves generated by a model based on Ca(2+)-induced Ca2+ release.

Cytosolic Ca2+ waves occur in a number of cell types either spontaneously or after stimulation by hormones, neurotransmitters, or treatments promoting Ca2+ influx into the cells. These waves can be broadly classified into two types. Waves of type 1, observed in cardiac myocytes or Xenopus oocytes, correspond to the propagation of sharp bands of Ca2+ throughout the cell at a rate that is high enough to permit the simultaneous propagation of several fronts in a given cells. Waves of type 2, observed in hepatocytes, endothelial cells, or various kinds of eggs, correspond to the progressive elevation of cytosolic Ca2+ throughout the cell, followed by its quasi-homogeneous return down to basal levels. Here we analyze the propagation of these different types of intracellular Ca2+ waves in a model based on Ca(2+)-induced Ca2+ release (CICR). The model accounts for transient or sustained waves of type 1 or 2, depending on the size of the cell and on the values of the kinetic parameters that measure Ca2+ exchange between the cytosol, the extracellular medium, and intracellular stores. Two versions of the model based on CICR are considered. The first version involves two distinct Ca2+ pools sensitive to inositol 1,4,5-trisphosphate (IP3) and Ca2+, respectively, whereas the second version involves a single pool sensitive both to Ca2+ and IP3 behaving as co-agonists for Ca2+ release. Intracellular Ca2+ waves occur in the two versions of the model based on CICR, but fail to propagate in the one-pool model at subthreshold levels of IP3. For waves of type 1, we investigate the effect of the spatial distribution of Ca(2+)-sensitive Ca2+ stores within the cytosol, and show that the wave fails to propagate when the distance between the stores exceeds a critical value on the order of a few microns. We also determine how the period and velocity of the waves are affected by changes in parameters measuring stimulation, Ca2+ influx into the cell, or Ca2+ pumping into the stores. For waves of type 2, the numerical analysis indicates that the best qualitative agreement with experimental observations is obtained for phase waves. Finally, conditions are obtained for the occurrence of "echo" waves that are sometimes observed in the experiments.     

Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering.

Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns.     

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos

Intact zebrafish embryos were used as an in vivo animal model to investigate the role of Ca2+ signaling during the differentiation of slow muscle cells (SMCs) within forming skeletal muscle. Transgenic zebrafish were generated using an a-actin promoter that targeted apoaequorin expression specifically to muscle cells. Two distinct Ca2+ signaling periods (CSPs) were visualized in the developing SMCs: between ~17.5-19.5 hours post-fertilization (hpf) and after ~23 hpf, separated by a ~3.5 h Ca2+ signaling quiet period. Further spatial characterization of these Ca2+ signals using confocal fluorescent microscopy and calcium green-1 dextran as a reporter, indicated that the earlier CSP displayed distinct nuclear and cytoplasmic components, whereas the later CSP was predominantly cytoplasmic. Both CSPs consisted of a series of oscillating Ca2+ waves generated at distinct frequencies, while the earlier CSP also displayed a slow rise then fall in the Ca2+ baseline-level. Imaging of cyclopamine- and forskolin-treated wild-type, or smo-/- mutant embryos, where SMCs do not form, confirmed the specific cell population generating the signals. Treating embryos with antagonists indicated that both IP3Rs and RyRs are responsible for generating the temporal characteristics of the Ca2+ signaling signature, and that the latter plays a necessary role in SMC differentiation and subsequent myotome patterning. Together, these data support and extend the proposition that specific spatiotemporal patterns of spontaneous Ca2+ signals might be used for different as well as combinatorial regulation of both nuclear and cytosolic signal transduction cascades, resulting in myofibrillogenesis in SMCs as well as myotome patterning.     

Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle

Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling.     

The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.     

Intercellular Ca2+ waves in mechanically stimulated articular chondrocytes

Articular cartilage is a tissue designed to withstand compression during joint movement and, in vivo, is subjected to a wide range of mechanical loading forces. Mechanosensitivity has been demonstrated to influence chondrocyte metabolism and cartilage homeostasis, but the mechanisms underlying mechanotransduction in these cells are poorly understood. In many cell types mechanical stimulation induces increases of the cytosolic Ca2+ concentration that propagates from cell to cell as an intercellular Ca2+ wave. Cell-to-cell communication through gap junctions underlies tissue co-ordination of metabolism and sensitivity to extracellular stimuli: gap junctional permeability to intracellular second messengers allows signal transduction pathways to be shared among several cells, ultimately resulting in co-ordinated tissue responses. Mechanically-induced Ca2+ signalling was investigated with digital fluorescence video imaging in primary cultures of rabbit articular chondrocytes. Mechanical stimulation of a single cell, obtained by briefly distorting the plasmamembrane with a micropipette, induced a wave of increased Ca2+ that was communicated to surrounding cells. Intercellular Ca2+ spreading was inhibited by 18 alpha-glycyrrhetinic acid, suggesting the involvement of gap junctions in signal propagation. The functional expression of gap junctions was assessed, in confluent chondrocyte cultures, by the intercellular transfer of Lucifer yellow dye in microinjection experiments while the expression of connexin 43 could be detected in Western blots. A series of pharmacological tools known to interfere with the cell calcium handling capacity were employed to investigate the mechanism of mechanically-induced Ca2+ signalling. In the absence of extracellular Ca2+ mechanical stimulation induced communicated Ca2+ waves similar to controls. Mechanical stress induced Ca2+ influx both in the stimulated chondrocyte but not in the adjacent cells, as assessed by the Mn2+ quenching technique. Cells treatment with thapsigargin and with the phospholipase C inhibitor U73122 blocked mechanically-induced signal propagation. These results provide evidence that in chondrocytes mechanical stimulation activates phospholipase C, thus leading to an increase of intracellular inositol 1,4,5-trisphosphate. The second messenger, by permeating gap junctions, stimulates intracellular Ca2+ release in neighbouring cells. Intercellular Ca2+ waves may provide a mechanism to co-ordinate tissue responses in cartilage physiology.