Mechanism of intracellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured endothelial CPAE cells

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Intercellular Ca(2+)-transient propagation in normal and high glucose solutions in rat retinal epithelial (RPE-J) cells during mechanical stimulation

We investigated the effect of high glucose and modulation of protein kinase C (PKC) on the intercellular propagation of Ca(2+)-waves in a rat retinal pigment epithelial cell line (RPE-J cells) in order to compare its properties with the properties previously investigated in primary LE-RPE cells. The intercellular propagation of the Ca(2+)-waves in RPE-J cells was analyzed by fluorescence imaging confocal microscopy and fluorescence recovery after photobleaching (FRAP). In control conditions the maximal normalized fluorescence in the mechanically stimulated (MS) cell and the propagation towards the neighboring RPE-J cells were similar to LE-RPE cells. As in LE-RPE cells, the propagation was reduced by the gap junction (GJ) blocker halothane, and FRAP experiments demonstrated the presence of functional GJ coupling. Similar to the effect in LE-RPE cells, the propagation of the Ca(2+)-transient was reduced by 25 mM glucose. However, unlike LE-RPE cells, the neighboring RPE-J cells presented a Ca(2+)-rise of amplitude similar to that in normal glucose levels. PKC activation with 1 microM PMA for 30 min resulted in inhibition of the Ca(2+)-wave propagation, which could be overcome by PKC downregulation as in LE-RPE cells. Cells grown for 72 h in a high glucose solution in which PKC activity was downregulated, did not develop the inhibitory effect on Ca(2+)-wave propagation that was induced by elevated glucose levels. However, the effects were not as pronounced as in LE-RPE cells. We concluded that despite marked similarities, the transduction and the modulation of intercellular propagation of the Ca(2+)-transients in RPE-J cells are not identical to the mechanisms in primary LE-RPE cells.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Properties of intra- and intercellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured RPE cells

Membrane deformation induced by a mechanical stimulus increases the [Ca2+]i in cultured retinal pigment epithelial (RPE) cells, and in many other cell types. In this study, confocal microscopy and Ca(2+)-measurements using the fluorescent dye fluo-3 were used to measure the spatiotemporal characteristics of the Ca(2+)-wave propagation during a mechanical stimulation in Long Evans (LE) RPE cells or dystrophic Royal College of Surgeons (RCS) RPE cells. Ca2+ signals were recorded in the mechanically stimulated cell and in the neighboring cells. A regenerative Ca(2+)-wave with a decreasing rate of propagation was found in the stimulated cells. The rate of propagation was significantly slower in RCS-RPE cells compared to LE-RPE cells. Incubation with thapsigargin significantly lowered the propagation rate in both LE- and RCS-RPE cells. The amplitude of the [Ca2+]i-rise in the nucleus and cytoplasm was differentially modulated by protein kinase C in RCS-RPE cells, but not in LE-RPE cells. It is concluded that RCS-RPE cells have intracellular Ca(2+)-regulating properties which are different from those of LE-RPE cells.     

Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is an essential regulator of T-lymphocyte Ca(2+)-signaling

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.     

Purinergic junctional transmission and propagation of calcium waves in spinal cord astrocyte networks

Micro-photolithographic methods have been employed to form discrete patterns of spinal cord astrocytes that allow quantitative measurements of Ca(2+) wave propagation. Astrocytes were confined to lanes 20-100 microm wide and Ca(2+) waves propagated from a point of mechanical stimulation or of application of adenosine triphosphate; all Ca(2+) wave propagation was blocked by simultaneous application of purinergic P2Y(1) and P2Y(2) antagonists. Stimulation of an astrocyte at one end of a lane, followed by further stimulation of this astrocyte, gave rise to Ca(2+) transients in the same astrocytes; however, if the second stimulation was applied to an astrocyte at the other end of the lane, then this gave rise to a different but overlapping set of astrocytes generating a Ca(2+) signal. Both the amplitude and velocity of the Ca(2+) wave decreased over 270 microm from the point of initiation, and thereafter remained, on average, constant with random variations for at least a further 350 microm. Also, the percentage of astrocytes that gave a Ca(2+) transient decreased with distance along lanes. All the above observations were quantitatively predicted by our recent theoretical model of purinergic junctional transmission, as was the Ca(2+) wave propagation along and between parallel lanes of astrocytes different distances apart. These observations show that a model in which the main determinants are the diffusion of adenosine triphosphates regeneratively released from a stimulated astrocyte, together with differences in the properties and density of the purinergic P2Y receptors on astrocytes, is adequate to predict a wide range of Ca(2+) wave transmission and propagation phenomena.     

Connexin mimetic peptides reversibly inhibit Ca(2+) signaling through gap junctions in airway cells

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.     

Intra- and intercellular Ca(2+)-transient propagation in normal and high glucose solutions in ROS cells during mechanical stimulation

Experiments using confocal laser microscopy on the rat osteosarcoma cell line (ROS 17/2.8) indicate that mechanical stimulation elicits pronounced [Ca2+](i)transients in the MS (mechanically stimulated) cell, which then propagate to the NB (neighbouring) cells. Experiments with Ca(2+)-free solutions or gadolinium suggest that Ca(2+)-influx through stretch-sensitive channels is required. When intracellular stores are depleted with thapsigargin, mechanical stimulation was able to evoke a Ca(2+)transient of reduced amplitude that disappeared entirely after subsequent blocking of Ca(2+)-influx. Heptanol inhibited intercellular propagation of the Ca(2+)transient, demonstrating the involvement of gap junctions in the propagation of the Ca(2+)transient in ROS cells. PKC activation has only a small inhibitory effect, while inhibition of PKC or tyrosine kinase was ineffective. PKA activation reduced the amplitude of the [Ca2+](i)-rise in NB cells, and decreased the percentage of responsive cells. Cells grown in 50mM glucose for 72h presented only a very limited decrease of the Ca(2+)-rise during mechanical stimulation in the MS and NB cells compared to control conditions. PKC downregulation in high glucose did not modulate this effect.The results of our experiments indicate that PKC or sustained high glucose concentrations do not affect gap junctional communication in ROS cells, while activation of PKA has an inhibitory effect. This might indicate that osteoblastic dysfunction in diabetes could be directly related to the high glucose concentrations and not to inhibition of the intercellular communication.     

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a Ca(2+) -influx" superimposed to Ca(2+) -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca(2+)](o) in the medium, from approximately 20-25% at [Ca(2+)](o) < or = 0.25 microM to 100% at [Ca(2+)](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [Ca(2+)] during membrane fusion. Next, we analyzed Ca(2+)-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca(2+) -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca(2+) -signal (due solely to Ca(2+) released from cortical stores) recovered with t(1/2) approximately 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca(2+)(o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45Ca(2+) -uptake by isolated ASs. In sum we find, (i) exogenous Ca(2+) increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) Ca(2+) -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.     

Mechanosensitivity and intercellular communication in HOBIT osteoblastic cells: a possible role for gap junction hemichannels

Mechanically induced intercellular Ca2+ signalling was investigated in differentiated HOBIT osteoblastic cells. HOBIT cells express connexin43 clustered at the cell-to-cell boundary and display functional intercellular coupling assessed by intercellular transfer of Lucifer yellow. Mechanical stimulation of single cells, besides leading to an intracellular Ca2+ rise, induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. Suramin and apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signalling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18alpha-glycyrrhetinic acid.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Modulation of CCK-8-evoked intracellular Ca2+ waves by hydrogen peroxide in mouse pancreatic acinar cells.

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Purinergic activation of Ca2+-permeable TRPV4 channels is essential for mechano-sensitivity in the aldosterone-sensitive distal nephron

Mechanical forces are known to induce increases of [Ca(2+)](i) in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca(2+)](i) in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca(2+) peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca(2+)](i). Furthermore, ATP-induced Ca(2+)-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca(2+)](i) similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca(2+)](i) response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca(2+) responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca(2+) appeared to contribute to the generation of ATP-induced Ca(2+) response in ASDN cells. Importantly, flow- and hypotonic-induced Ca(2+) elevations are markedly blunted in P2Y2 -/- mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca(2+)](i) elevation during purinergic stimulation. Consistent with this, ATP-induced Ca(2+) plateau are dramatically attenuated in TRV4 -/- mice. Inhibition of TRPC channels with 10 μM BTP2 also decreased ATP-induced Ca(2+) plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.     

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.     

Effects of calcium buffers and calbindin-D28k upon histamine-induced calcium oscillations and calcium waves in HeLa cells

The effects of the artificial Ca(2+) buffers EGTA and BAPTA upon histamine-induced Ca(2+) oscillations and calcium waves were studied in HeLa cells. These events were also examined in HeLa cell lines transfected with the intracellular calcium-binding protein calbindin-D28k (CaBP; HeLa-CaBP) or the pCINeo vector alone (HeLa-pCINeo). High concentrations of the Ca(2+) indicators fluo-3 and fura-2 significantly influenced the oscillatory pattern of intracellular Ca(2+) in HeLa-pCINeo cells exposed to 1 microM histamine. Loading cells with low concentrations of the cell-permeant esters of the artificial Ca(2+)-buffers EGTA or BAPTA, resulted in fewer cells with a distinct "baseline" oscillatory pattern, and loading with higher concentrations of BAPTA almost completely abolished them. In HeLa-CaBP cells, stimulation with 1 microM histamine resulted in individual Ca(2+) spikes that had a flattened profile when compared to control cells; peak [Ca(2+)](i) was lowered, the rate of increase in [Ca(2+)](i) was slower and transients were prolonged. When compared to HeLa-pCINeo cells, loading with EGTA or BAPTA, or transfection of CaBP, significantly reduced the propagation velocity (by up to 60%) of Ca(2+) waves induced by exposure to 100 microM histamine. We conclude that intracellular Ca(2+) buffering exerts a significant influence on global Ca(2+) responses in HeLa cells and the propagation of Ca(2+) waves that underlie them. The relative effectiveness of different Ca(2+) buffers, including CaBP, appears to be particularly dependent upon the rapidity of their binding kinetics, with BAPTA being the most effective.     

Hypertonicity triggers RhoA-dependent assembly of myosin-containing striated polygonal actin networks in endothelial cells

Electrical slow waves determine the timing and force of peristaltic contractions in the stomach. Slow waves originate from a dominant pacemaker in the orad corpus and propagate actively around and down the stomach to the pylorus. The mechanism of slow-wave propagation is controversial. We tested whether Ca(2+) entry via a voltage-dependent, dihydropyridine-resistant Ca(2+) conductance is necessary for active propagation in canine gastric antral muscles. Muscle strips cut parallel to the circular muscle were studied with intracellular electrophysiological techniques using a partitioned-chamber apparatus. Slow-wave upstroke velocity and plateau amplitude decreased from the greater to the lesser curvature, and this corresponded to a decrease in the density of interstitial cells of Cajal in the lesser curvature. Slow-wave propagation velocity between electrodes impaling cells in two regions of muscle and slow-wave upstroke and plateau were measured in response to experimental conditions that reduce the driving force for Ca(2+) entry or block voltage-dependent Ca(2+) currents. Nicardipine (0.1-1 microM) did not affect slow-wave upstroke or propagation velocities. Upstroke velocity, amplitude, and propagation velocity were reduced in a concentration-dependent manner by Ni(2+) (1-100 microM), mibefradil (10-30 microM), and reduced extracellular Ca(2+) (0.5-1.5 mM). Depolarization (by 10-15 mM K(+)) or hyperpolarization (10 microM pinacidil) also reduced upstroke and propagation velocities. The higher concentrations (or lowest Ca(2+)) of these drugs and ionic conditions tested blocked slow-wave propagation. Treatment with cyclopiazonic acid to empty Ca(2+) stores did not affect propagation. These experiments show that voltage-dependent Ca(2+) entry is obligatory for the upstroke phase of slow waves and active propagation.     

Intestinal calcium waves coordinate a behavioral motor program in C. elegans

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans, Genetics 124 (1990) 855-872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C. elegans, J. Neurosci. 14 (1994) 1953-1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA 92 (1995), 10317-10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50s behavioral rhythm in C. elegans, Cell 98 (1999) 757-767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca(2+) dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca(2+)-wave propagation in the intestine. Lack of the Ca(2+)-wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca(2+)-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca(2+)-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans.