Selected contribution: PKC activation inhibits Ca(2+) signaling in tracheal epithelial cells kept in simulated microgravity.

Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca(2+) signaling and ATP-induced Ca(2+) oscillations, both of which are modulated by PKC. Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity. Mechanical stimulation of a single cell increased the cytosolic Ca(2+) concentration in 35-55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (smicroG). In outgrowth cultures, 12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca(2+) "waves" to about 10 cells in unit G and to significantly fewer cells in smicroG. TPA only slightly reduced the spread of Ca(2+) waves in epithelial sheets kept in smicroG but did not inhibit Ca(2+) waves of sheets kept in unit G. In both cell preparations from both conditions, TPA inhibited ATP-induced Ca(2+) oscillations; however, the effect was more pronounced in cells kept in smicroG. These results suggest that PKC activation is more robust in cells subjected to smicroG.

PKC role in mechanically induced Ca2+ waves and ATP-induced Ca2+ oscillations in airway epithelial cells.

Mechanical stimulation of airway epithelial cells generates the Ca2+ mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase (PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a "Ca2+ wave"); the role that diacylglycerol-activated PKC might play in the response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12-O-tetradecanoylphorbol 13-acetate- or 1, 2-dioctanyl-sn-glycerol-induced activation of PKC slows the Ca2+ wave, decreases the amplitude of induced intracellular free Ca2+ concentration ([Ca2+]i) increases, and decreases the number of affected cells. The PKC inhibitors bisindolylmaleimide and Gö 6976 slowed the spread of the wave but did not change the number of affected cells. We show that ATP-induced [Ca2+]i increases and oscillations, responses independent of intercellular communication, were inhibited by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2+]i increases and blocked oscillations, suggesting that PKC has an initial positive effect on Ca2+ mobilization and then mediates feedback inhibition. PKC activators also reduced the [Ca2+]i increase that followed thapsigargin treatment, indicating a PKC effect associated with the Ca2+ release mechanism.

Mechanical stimulation initiates intercellular Ca2+ signaling in intact tracheal epithelium maintained under normal gravity and simulated microgravity.

We investigated mechanically induced cell-to-cell Ca2+ signaling in a preparation of rabbit tracheal epithelium close to its in vivo condition. We used confocal microscopy to analyze changes in intracellular free calcium concentration ([Ca2+]i) in intact ciliated tracheal mucosal explants loaded with the Ca2+-indicator dye, fluo-3. When a single cell in the epithelium was transiently stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and then increased in surrounding cells. In the absence of extracellular Ca2+, the [Ca2+]i increases had a smaller amplitude and spread to fewer cells. Treatment of the cells with thapsigargin, in the presence of extracellular Ca2+, more markedly reduced the spread of elevated [Ca2+]i. These results suggest that the propagated [Ca2+]i increases are due to mobilization of Ca2+ from intracellular stores and, possibly, the influx of extracellular Ca2+. The mechanically stimulated [Ca2+]i increases were accompanied by propagated increases in ciliary beat frequency. Since microgravity has been shown to alter signal transduction, we investigated whether simulated microgravity affects the mechanically stimulated cell-to-cell Ca2+ signaling observed in tracheal epithelium. Tissues were maintained for 3-8 d in a rotating wall vessel which simulates microgravity conditions. Cells maintained in simulated microgravity exhibited mechanically induced [Ca2+]i increases not significantly different in magnitude, in speed of propagation, or in the number of cells involved, from tissue maintained at unit gravity. Our results suggest that intercellular Ca2+ signaling coordinates cellular activity, including ciliary beating, within the tracheal epithelium in vivo and that this function is not compromised in microgravity.

Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions.

Mechanical stimulation of a single cell in primary airway epithelial cell cultures induces an intercellular Ca2+ wave that has been proposed to be mediated via gap junctions. To investigate directly the role of gap junctions in this multicellular response, the effects of intracellularly-loaded sequence-specific connexin (gap junction) antibodies on the propagation of intercellular Ca2+ waves were evaluated. Electroporation of antibodies to the cytosolic loop (Des 1, generated to amino acids 102-112 + 116-124; and Des 5, amino acids 108-119), or to the carboxyl tail (Gap 9, amino acids 264-283) of connexin 32 inhibited the propagation of intercellular Ca2+ waves. The inhibitory effect of Des 1 antibody was competitively reversed by the co-loading of a peptide derived from a similar cytosolic loop sequence (Des 5 peptide). Conversely, the inhibitory effects on intercellular Ca2+ wave propagation of Gap 9 antibody was not altered by co-loading with the Des 5 peptide. Antibodies raised to peptide sequences within the extracellular loop (Gap 11, amino acids 151-187), or the cytoplasmically located amino terminus (Gap 10, amino acids 1-21) of connexin 32 did not inhibit mechanically-induced intercellular communication. Also ineffective in perturbing intercellular communication were antibodies raised to peptide sequences of the cytosolic loops of connexin 43 (Gap 15, amino acids 131-142) or connexin 26 (Des 3, amino acids 106-119). These data suggest that mechanically-induced Ca2+ waves in airway cell cultures are propagated through gap junctions made up of connexin 32 proteins.

Reduction of extracellular Na+ causes a release of Ca2+ from internal stores in airway epithelial cells.

Exchange of physiological salt solution with Na(+)-free solution caused an increase in intracellular Ca2+ concentration ([Ca2+]i) in 86.3% of cultured airway epithelial cells within 75 s. [Ca2+]i returned to near baseline levels within 45 s and frequently showed oscillatory increases thereafter. When extracellular Na+ concentration ([Na+]o) was reduced to 10 and 60 mM, 59.0 and 8.0% of the cells increased [Ca2+]i, respectively. Low [Na+]o-induced increase in [Ca2+]i was not blocked by amiloride, benzamil, La3+, or the absence of extracellular Ca2+. Low [Na+]o-induced [Ca2+]i increase did not occur after thapsigargin treatment. These results indicated that low [Na+]o-induced [Ca2+]i increase is due to release of Ca2+ from intracellular stores. Because mechanical stimulation of a single cell causes a Ca2+ increase among many cells (Sanderson, M. J., A. C. Charles, and E. R. Dirksen. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul. 1: 585-596, 1990.) we assayed the effect of low [Na+]o on this mechanically induced response. In low [Na+]o, mechanically induced [Ca2+]i increase in the stimulated cell was reduced; however, [Ca2+]i increase in adjacent cells was normal. We suggest that a mechanically induced Na+ conductance in the stimulated cell contributes to [Ca2+]i changes. These signaling pathways may be involved in the maintenance of periciliary ion concentrations.

Stretch increases inositol 1,4,5-trisphosphate concentration in airway epithelial cells.

Mechanical stimulation of airway epithelial cells with a microprobe leads to an increase in cytoplasmic [Ca2+] that appears to be due, in part, to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores (Boitano et al., Science 258:292[1992]). To investigate whether intracellular IP3 concentration ([IP3]i) increases in response to mechanical stimulation, we grew confluent monolayers from rabbit tracheal mucosal explants on flexible substrates and measured [IP3]i after stretching the substrate. The effect of stretch on [IP3]i was measured in the presence of Li+, an inhibitor of IP3 degradation. In unstretched cells, IP3 measured approximately 5.1 pmol/10(6) cells, from which we estimated [IP3]i to be 1.8 microM. Addition of Li+ had no effect on resting [IP3]i. When the flexible cell support was stretched to increase its surface area by 13%, mean [IP3]i increased about 3-fold with a half-time of approximately 1 s. The increased [IP3]i was maintained in a plateau phase for approximately 8 s and then decayed to near the unstretched level over the next 10 s, despite the sustained application of stretch. A transient stretch (0.5 s) induced a similar rate of increase and peak [IP3]i; however, [IP3]i subsided without a plateau phase. The magnitude of the [IP3]i increase was proportional to stimulus intensity between 0 and 13% increase in substrate surface area. In addition, dissociated airway epithelial cells were exposed to hypotonic solution to induce cell swelling. [IP3]i increased about 4-fold above control levels after 10 s of exposure to hypotonic solution. Basal [IP3]i of dissociated cells in isotonic solution was estimated to be 0.7 microM. These results are consistent with mechanical stimulation leading to phospholipase C synthesis of IP3, which mediates intracellular and intercellular Ca2+ signaling.

Evidence for voltage-sensitive, calcium-conducting channels in airway epithelial cells.

In airways epithelial cultures, mechanical stimulation induces intracellular Ca2+ concentration ([Ca2+]i) changes by causing Ca2+ entry and intracellular Ca2+ release. Mechanically induced Ca2+ fluxes across the plasma membrane are blocked by Ni2+ (Boitano, S., M. J. Sanderson, and E. R. Dirksen. J. Cell. Sci. 107: 3037-3044, 1994). In this report we use fluorescence imaging microscopy with fura 2 and intracellular recording of the transmembrane potential to further characterize Ca2+ flux in the plasma membrane of these cells. Mechanically induced Ca2+ influx is blocked by nifedipine. Addition of the dihydropyridine agonist BAY K8644 (2 microM) leads to a delayed increase of [Ca2+]i that is dependent on extracellular Ca2+. Switching to high extracellular K+ concentration ([K+]o) causes depolarization of the plasma membrane and a transient increase in [Ca2+]i. The number of cells that respond to high [K+]o is significantly decreased by Ni2+ (1 mM) or nifedipine (10 microM). Mechanical stimulation causes a rapid depolarization of the stimulated cell that can be suppressed by the K+ ionophore valinomycin. Valinomycin treatment also blocks mechanically induced Ca2+ dux. These results suggest that voltage-sensitive Ca(2+)-conducting channels exist in airway epithelial cells, and these channels contribute to the [Ca2+]i changes observed after mechanical stimulation or depolarization of the plasma membrane.

A role for phospholipase C activity but not ryanodine receptors in the initiation and propagation of intercellular calcium waves.

Mechanical stimulation of a single cell in an airway epithelial culture initiates an increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates from cell to cell as an intercellular Ca2+ wave. These Ca2+ waves appear to require an increase in intracellular inositol 1,4,5-trisphosphate (IP3) concentration ([IP3]i) in the stimulated cell and are propagated between cells by the diffusion of IP3 through gap junctions. To test the hypothesis that the activation of phospholipase C (PLC) contributes to the elevation of [IP3]i and initiation of an intercellular Ca2+ wave, changes in [Ca2+]i induced by mechanical stimulation were measured by digital fluorescence microscopy in the presence of the PLC inhibitor, aminosteroid U73122. Following exposure to U73122 mechanical stimulation elevated [Ca2+]i of the stimulated cell, but did not initiate the propagation of an intercellular Ca2+ wave. By contrast, in the presence of U73343, a similar aminosteroid that does not inactivate PLC, mechanical stimulation increased the [Ca2+]i of the stimulated cell and initiated an intercellular Ca2+ wave. U73122 also blocked the elevation of [Ca2+]i of airway epithelial cells in response to ATP, a P2-receptor agonist that activates PLC to elevate [IP3]i and [Ca2+]i. In addition, the propagation of intercellular Ca2+ waves was not affected by the ryanodine-receptor agonists, caffeine or ryanodine. The hypotheses that: (1) an elevation of [IP3]i is required to initiate intercellular Ca2+ waves; (2) mechanical stimulation activates PLC; and (3) Ca2+ wave propagation in airway epithelial cells involves Ca2+ release from intracellular stores primarily via IP3 receptors are supported by these results.

A role for Ca(2+)-conducting ion channels in mechanically-induced signal transduction of airway epithelial cells.

Mechanical stimulation of a single cell in a cultured monolayer of airway epithelial cells initiates an intercellularly communicated increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates radically through adjacent cells via gap junctions, forming an intercellular Ca2+ wave. Mechanically-induced intercellular Ca2+ waves also occur in the absence of extracellular Ca2+. However, in Ca(2+)-free medium an increase in [Ca2+]i of the stimulated cell does not occur. Thus, mechanically-induced [Ca2+]i changes in the stimulated cell are influenced by the extracellular Ca2+ concentration. To investigate if a channel-mediated Ca2+ flux across the plasma membrane contributes to the elevation of [Ca2+]i in the stimulated cell we used digital image microscopy to measure mechanically-induced [Ca2+]i changes in the presence of Ca2+ channel blockers. In Ca(2+)-free medium containing Gd3+ (20 microM) mechanical stimulation resulted in an [Ca2+]i increase in the stimulated cell. The delay time between mechanical stimulation and increase in [Ca2+]i of the stimulated cell was dependent on extracellular [Gd3+], with a half-maximal effective concentration of approximately 40 microM. Mechanical stimulation in Ca(2+)-free medium containing La3+ (10 microM) or Ni2+ (100 microM) gave similar results. Mechanical stimulation in Ca(2+)-free medium containing the dihydropyridine Ca2+ channel blockers nifedipine (10 microM) and nimodipine (10 microM) also resulted in an increase of [Ca2+]i of the stimulated cell. Mechanical stimulation of cells treated with thapsigargin to deplete intracellular Ca2+ stores, in the presence of 1.3 mM extracellular Ca2+, results in an increase in [Ca2+]i of the stimulated cell without the propagation of an intercellular Ca2+ wave.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms and function of intercellular calcium signaling.

Intercellular Ca2+ waves initiated by mechanical or chemical stimuli propagate between cells via gap junctions. The ability of a wide diversity of cells to display intercellular Ca2+ waves suggests that these Ca2+ waves may represent a general mechanism by which cells communicate. Although Ca2+ may permeate gap junctions, the intercellular movement of Ca2+ is not essential for the propagation of Ca2+ waves. The messenger that moves from one cell to the next through gap junctions appears to be IP3 and a regenerative mechanism for IP3 may be required to effect multicellular communication. Extracellularly mediated Ca2+ signaling also exists and this could be employed to supplement or replace gap junctional communication. The function of intercellular Ca2+ waves may be the coordination of cooperative cellular responses to local stimuli.

Intercellular calcium signaling induced by extracellular adenosine 5'-triphosphate and mechanical stimulation in airway epithelial cells.

Airway epithelial cells in culture respond to extracellular adenosine 5'-triphosphate (ATP) by increasing their intracellular Ca2+ concentration ([Ca2+]i). The effective concentration of ATP that elicited a Ca2+ response equal to 50% of the maximal response (EC50) was 0.5 microM. Release of ATP from a pipette to form a local gradient of ATP increased [Ca2+]i of individual cells in a sequential manner. Cells closest to the pipette showed an immediate increase in [Ca2+]i while more distal cells displayed a delayed increase in [Ca2+]i. This response to the local release of ATP appeared as a wave of increasing [Ca2+]i that spread to several cells and, in this respect, was similar to the intercellularly communicated Ca2+ waves initiated by mechanical stimulation in airway epithelial cells (Sanderson et al., Cell Regul. 1, 585-596, 1990). In the presence of a unidirectional fluid flow, the Ca2+ response to a local release of ATP was biased such that virtually all the cells responding with an increase in [Ca2+]i were downstream of the release site. By contrast, an identical fluid flow did not bias the radial propagation of intercellular Ca2+ waves induced by mechanical stimulation. Suramin, a P2-purinergic receptor antagonist, did attenuate the Ca2+ response induced by ATP but did not block the propagation of mechanically induced Ca2+ waves. Cells from young cultures (3-5 days) or those at the leading edge of an outgrowth elevated their [Ca2+]i in response to ATP. However, these cells do not respond to mechanical stimulation by the propagation of a Ca2+ wave. From these results we conclude that the intercellular Ca2+ waves elicited by mechanical stimulation are not the result of ATP or another compound released from the stimulated cell, diffusing through the extracellular fluid. This conclusion is consistent with previous experimental evidence suggesting that intercellular Ca2+ signaling in epithelial cells is mediated by the movement of inositol trisphosphate through gap junctions.

Stretch-activated channels in airway epithelial cells.

Two type of stretch-activated (SA) ion channels were identified in the basolateral membrane of isolated rabbit airway epithelial cells by patch-clamp techniques. Pressure activation and deactivation of one channel, which had a conductance of 29 pS, occurred after a delay of approximately 20-30 s. The open probability of this delayed stretch-activated (DSA) channel was increased from < 0.01 to 0.45 at 50 mmHg of suction. The reversal potential of the DSA channel, calculated from the pipette potential at which membrane currents reversed [-31.3 +/- 3.6 (SD) mV] and the resting membrane potential (-27.8 +/- 3.3 mV) was +3.5 +/- 3.3 mV. None of the equilibrium potentials of the ions used were similar to the calculated reversal potential of the DSA channel, suggesting that this channel is nonselective for cations. The DSA channel gating behavior was characterized by bursts of rapid transitions between open and closed states. The distribution of the open and closed times revealed that this gating behavior could be fitted with two open states and two closed states. Only the slow time constant of the closed state was decreased by suction. The second SA channel was selective for K+ and had a conductance of 65 pS but a long delay was not associated with the pressure sensitivity of this channel. The open probability of the K(+)-selective SA channel was increased from < 0.01 to 0.30 by 50 mmHg of suction. The K(+)-selective SA channel was distinct from the well-characterized basolateral K+ channel.

Mechanical stimulation induces intercellular calcium signaling in bovine aortic endothelial cells.

To study the mechanism by which endothelial cells respond to mechanical forces, we used digital fluorescence microscopy to measure changes in intracellular Ca2+ concentration ([Ca2+]i) in primary cultures of bovine aortic endothelial cells in response to mechanical stimulation. Before stimulation, [Ca2+]i was stable (approximately 50-75 nM). When an individual cell within the monolayer was mechanically stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and spread in the form of a wave from the site of contact to the cell edges. After a delay of approximately 1 s, nonstimulated adjacent cells showed a similar spreading rise in [Ca2+]i. This outwardly radiating [Ca2+]i wave involved progressively more distal cells to a radius of 4-6 cells. The time delay before the wave appeared in adjacent cells increased, and peak [Ca2+]i in each cell decreased with distance from the stimulated cell. In the absence of extracellular Ca2+, there was no increase in [Ca2+]i in the stimulated cell, yet a wave of increased [Ca2+]i occurred in neighboring cells as if communicated from the stimulated cell. These results indicate that endothelial cell mechanosensitivity results in increases in [Ca2+]i and that the temporospatial dynamics of intercellular Ca2+ signaling are mediated by a diffusible substance other than Ca2+.

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin.

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca2+]i) that was communicated to surrounding cells. Following propagation of the Ca2+ wave, many cells showed asynchronous oscillations in [Ca2+]i. Dantrolene sodium (10 microM) inhibited the increase in [Ca2+]i associated with this Ca2+ wave by 60-80%, and prevented subsequent Ca2+ oscillations. Despite the markedly decreased magnitude of the increase in [Ca2+]i, the rate of propagation and the extent of communication of the Ca2+ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 microM) induced an initial increase in [Ca2+]i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca2+]i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of Ca2+ wave from the stimulated cell to adjacent cells. Glutamate (50 microM) induced an initial increase in [Ca2+]i in most cells that was followed by sustained oscillations in [Ca2+]i in some cells. Dantrolene (10 microM) inhibited this initial [Ca2+]i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 micron) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca2+ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca2+ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP3) mediates the propagation of Ca2+ waves whereas Ca(2+)-induced Ca2+ release amplifies Ca2+ waves and generates subsequent Ca2+ oscillations.

Intercellular propagation of calcium waves mediated by inositol trisphosphate.

Two types of calcium (Ca2+) signaling-propagating intercellular Ca2+ waves of increasing intracellular Ca2+ concentration ([Ca2+]i) and nonpropagating oscillations in [Ca2+]i-co-exist in a variety of cell types. To investigate this difference in Ca2+ signaling, airway epithelial cells were loaded with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist, by pulsed, high-frequency electroporation. Heparin inhibited propagation of intercellular Ca2+ waves but not oscillations of [Ca2+]i. In heparin-free cells, Ca2+ waves propagated through cells displaying [Ca2+]i oscillations. Depletion of intracellular Ca2+ pools with the Ca2+-pump inhibitor thapsigargin also inhibited the propagation of Ca2+ waves. These studies demonstrate that the release of Ca2+ by IP3 is necessary for the propagation of intercellular Ca2+ waves and suggest that IP3 moves through gap junctions to communicate intercellular Ca2+ waves.

Control of the beat cycle of respiratory tract cilia by Ca2+ and cAMP.

Beat frequency and the duration of the constituent recovery, effective, and rest phases of the beat cycle of respiratory tract cilia were measured photoelectronically before and after manipulation with ionomycin or isoproterenol. Both ionomycin, acting by increasing intracellular Ca2+, and isoproterenol, acting by elevating intracellular adenosine 3',5'-cyclic monophosphate (cAMP), increased beat frequency by reducing the duration of the three phases of the ciliary beat cycle in a similar manner. The addition of increasing concentrations of ATP to ciliated cells permeabilized by exposure to saponin caused a pattern of phase reduction indistinguishable from that observed in whole cells. The beat frequency of permeabilized cells was slower than that of whole cells and insensitive to changes in Ca2+ and cAMP. Ca2+ and cAMP may regulate ciliary beat frequency by acting at a common site within intact cells, possibly regulating the rate at which the axoneme can use ATP or the availability of ATP to the axoneme.

Intercellular calcium signaling via gap junctions in glioma cells.

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.