Stretch-activated Channel Blocker Research Articles

Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells.

Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.

Amiloride-sensitive pressure-induced myogenic contraction in rat cerebral artery.

The inhibitory action of amiloride on the pressure-induced contraction was assessed in isolated rat cerebral artery. The artery was mounted in an arteriograph, and the change in intracellular Ca2+ concentration ([Ca2+]i) and vessel diameter were simultaneously measured. The contractile response elicited by intraluminal pressurization was independent of endothelium, i.e. myogenic in nature, and abolished by nicardipine, a Ca2+ antagonist or by removal of extracellular Ca2+, and was potentiated by 25 mM KCl. Cyclopiazonic acid and thapsigargin, inhibitors of the Ca2+-ATPase pump of the sarcoplasmic reticulum, and a protein kinase C inhibitor calphostin C did not suppress the pressure-induced contraction. Amiloride, a putative stretch-activated cation channel blocker, attenuated with an IC50 (50% inhibitory concentration) of about 3 microM the increase in [Ca2+]i and contractile activity in response to pressure, whereas the drug showed no apparent effect on the contraction produced by high KCl or 9,11-dideoxy-11alpha,9alpha-epoxymethano prostaglandin F2alpha (U46619). Furthermore, amiloride (100 microM) did not significantly affect intracellular pH in the artery. In spite of its multiple pharmacological actions, it seems possible that amiloride is a useful alternative tool at the cellular or tissue level to study the mechanotransduction mechanisms involved in the pressure-induced contraction in rat cerebral artery.

Mechanoregulation of Chondrocyte Proliferation, Maturation, and Hypertrophy: Ion-Channel Dependent Transduction of Matrix Deformation Signals

Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on chondrocytes, we characterized primary chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled “Bio-Stretch” device. Cyclic matrix deformation greatly stimulated proliferation of immature chondrocytes, but not that of hypertrophic chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.

Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor.

We have studied whether activation of epidermal growth factor receptor (EGFR) is involved in stretch-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation and protein synthesis in cultured rat vascular smooth muscle cells (VSMC). Cyclic stretch (1 Hz) induced a rapid (within 5 min) phosphorylation of ERK1/2, an effect that was time and strength dependent and inhibited by an EGFR kinase inhibitor (AG-1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG-1296). The stretch rapidly (within 2 min) induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR as revealed by blockade with AG-1478 as well as immunoprecipitation with anti-EGFR antibody coupled with immunoblotting with anti-phosphotyrosine antibody. The stretch rapidly (within 2 min) induced association of tyrosine-phosphorylated EGFR with adaptor proteins (Shc/Grb2) as revealed by coprecipitation with glutathione-S-transferase-Grb2 fusion protein coupled with immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-Shc antibodies. Transfection of a dominant-negative mutant of H-Ras also inhibited stretch-induced ERK1/2 activation. Treatment with a stretch-activated ion channel blocker (Gd(3+)) and an intracellular Ca(2+) antagonist (TMB-8) inhibited stretch-induced phosphorylation of EGFR and ERK1/2. Treatment with AG-1478 and a mitogen-activated protein kinase kinase inhibitor (PD-98059), but not AG-1296, blocked [(3)H]leucine uptake stimulated by a high level of stretch. These data suggest that ERK1/2 activation by mechanical stretch requires Ca(2+)-sensitive EGFR activation mainly via stretch-activated ion channels, thereby leading to VSMC growth.

Evidence for the involvement of K+ channels and K(+)-Cl- cotransport in the regulatory volume decrease of newborn rat cardiomyocytes.

In order to delineate ion transport mechanisms involved in volume homeostasis of freshly isolated newborn rat ventricular myocytes, we investigated the effects of ion substitutions and pharmacological maneuvers upon (1) isotonic volume, (2) hypotonically induced initial swelling, and (3) the subsequent regulatory volume decrease (RVD), as determined by electronic cell sizing. Cardiomyocytes exposed to hypotonic medium (176 mosmol/l) swelled by 51+/-1% of isotonic volume, and they underwent a partial regulatory volume decrease (RVD), reaching a maximum regulation after 30 min (51+/-1% of initial swelling), with a half-time (t1/2) of 6+/-1 min (n=60). RVD was associated with significant cardiomyocyte K+ loss (12+/-4% at 5 min and 15+/-2% of isotonic control after 30 min: n=6, P<0.001), 71% of which was Cl- dependent (P<0.05). Within the 30-min experimental time frame, ouabain, a Na+/K+ pump inhibitor, had no significant effect on RVD (despite an inhibitory trend), cell swelling or on isotonic volume (n=6). Bumetanide (50 microM), a Na+-K+-Cl- co-transport blocker, induced a significant reduction of isotonic cell volume (3+/-2%, n=6. P<0.05), potentiated initial swelling by 16+/-1% (n=8, P<0.02), and it partially inhibited RVD (24+/-11% at 30 min, n=6), whereas Na+ omission had no significant effect on isotonic cell volume, cell swelling or RVD. The effects of bumetanide on initial swelling and RVD were prevented by gadolinium ion (10 microM), a stretch-activated cation channel blocker (n=5). Quinidine (500 microM), a non-selective Ca(2+)-activated potassium channel blocker with no side-effects on K(+)-Cl(-) cotransport, did not modify initial cell swelling, but inhibited RVD (50+/-3% at 5 min, n=9, P<0.01; 22+/-3% at 30 min), an effect which was cancelled by external Ca2+ chelation with EGTA (n=5), and reproduced by tetraethylammonium (TEA, 20 mM), another K+ channel blocker. 4,4'-Diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS, 100 microM), a non-selective swelling-activated Cl- channel blocker with marginal side-effects on K(+)-Cl(-)cotransport, did not modify initial swelling, but inhibited RVD to the same extent as quinidine (42+/-3% at 5 min, and 23+/-3% at 30 min, n=15, P<0.05), whereas hypotonic Cl(-)-free solution had no effect on isotonic volume, but potentiated initial swelling by 16+/-2% (P<0.05) and fully inhibited RVD (n=5, P<0.001). R(+)-[(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inde n-5yl)-oxy] acetic acid) (DIOA, 80 microM), a K(+)-Cl- cotransport blocker (with inhibitory potency toward Ca(2+)-activated K+ channels), inhibited 87+/-5% of the RVD process at 5 min (P<0.001) and 56+/-16% at 30 min (P<0.001), whereas it had a small effect on isotonic volume (+4%, P<0.01) and initial cell swelling (+2%, N.S.; n=9). In contrast to quinidine, DIOA was able to inhibit Ca(2+)-omission-resistant RVD (full inhibition at 5 min, and 56+/-9% at 30 min; P<0.01, n=5). In conclusion, our results suggest that at least three distinct ion transport mechanisms are involved in the RVD in newborn rat cardiomyocytes: (1) K+ and Cl-channels, (2) K(+)-Cl- cotransport, and (3) Na(+)-K(+)-Cl- co-transport.

Swelling-Induced, Cftr-Independent Atp Release from a Human Epithelial Cell Line

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 microM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 microM. Glibenclamide (500 microM) and arachidonic acid (100 microM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach approximately 13 microM by a biosensor technique using P2X(2) receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 microM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.

Mechanical responses and signal transduction pathways in stretched osteocytes.

Mechanotransduction in bone is complex in nature, being influenced by many modulators such as PTH, prostanoids, and extracellular Ca2+. It has been postulated that osteocytes, dendritic resident cells in bone, transduce signals of mechanical loading that result in anabolic responses such as the expression of c-fos, insulin-like growth factor-I (IGF-I), and osteocalcin. To date, however, neither the actual stimuli to which osteocytes respond nor the pathways of signal transduction are well understood. Cultured primary rat bone cells exhibit distinct responses to stretching depending on their developmental stages: young osteocytes that become progressively dendritic show striking responses to strain at physiological levels; these include an early response of cAMP secretion and the late responses such as the production of IGF-I and osteocalcin proteins. The upregulation of steady-state levels of their mRNA is biphasic, being preceded by two peaks of PGHS-2 (inducive prostaglandin G/H synthase; cox-2) gene expression. Compared to a typical transient immediate early expression of c-fos, PGHS-2 shows another distinct peak about 8 h after the initiation of stretching. Second peaks in IGF-I and osteocalcin expression are entirely dependent on the first wave of PGHS-2 expression judging from the inhibition by NS398. PGHS-2 is perhaps critically involved in the prolonged anabolic responses of bone "memory effect" to the osteogenic mechanical stimulation. In these cells, the extracellular Ca2+ is essential to their response to stretching. Furthermore, the blockers of stretch-activated channels, gadolinium (Gd3+), and of epithelial-like Na channels, benzamil, in combination abolish the effects of stretching such as elevated osteocalcin expression. Although voltage-operated or calcium-activated calcium channels or Na+-driven mechanisms, such as a Na-Ca exchanger, for example, are functioning, particulars of secondary Ca entry pathways are not certain at this point. It is conceivable, however, that the calcium influxes, both primary and secondary, trigger the anabolic reaction of bone to stretching via Ser/Thr kinase signaling pathways in osteocytic cells.

TRAAK Is a Mammalian Neuronal Mechano-gated K+Channel

The novel structural class of mammalian channels with four transmembrane segments and two pore regions comprise background K+ channels (TWIK-1, TREK-1, TRAAK, TASK, and TASK-2) with unique physiological functions (1-6). Unlike its counterparts, TRAAK is only expressed in neuronal tissues, including brain, spinal cord, and retina (1). This report shows that TRAAK, which was known to be activated by arachidonic acid (3), is also opened by membrane stretch. Mechanical activation of TRAAK is induced by a convex curvature of the plasma membrane and can be mimicked by the amphipathic membrane crenator trinitrophenol. Cytoskeletal elements are negative tonic regulators of TRAAK. Membrane depolarization and membrane crenation synergize with stretch-induced channel opening. Finally, TRAAK is reversibly blocked by micromolar concentrations of gadolinium, a well known blocker of stretch-activated channels. Mechanical activation of TRAAK in the central nervous system may play an important role during growth cone motility and neurite elongation.

Gadolinium suppresses stretch-induced increases in the differences in epicardial and endocardial monophasic action potential durations and ventricular arrhythmias in dogs.

We tested whether acute pressure overloading of the left ventricle (LV) had spatially different effects on repolarization, thereby causing arrhythmias. The effects of gadolinium (Gd3+), a nonspecific blocker of stretch-activated channels were also examined. In anesthetized dogs, 5 s clamping of the ascending aorta (AC), separated by 5-min intervals, was repeated while monophasic action potentials (MAPs) were recorded from the LV endocardium and epicardium. Gd3+ was injected into the left atrium before the second (500 micromol) and third AC (2500 micromol) (n=10). In a separate group (n=7), the effects of Gd3+ in the presence of verapamil were examined. Epicardial MAP durations at 50% and 90% repolarization (APD50; APD90) shortened in response to LV pressure rise and elongation of the segment length induced by the first AC, whereas endocardial MAP durations remained unchanged. Thus, the difference in APD50 and APD90 increased. Consistent with these changes, premature ventricular contractions (PVCs) developed. Gd3+ had no effect on baseline MAP durations, however it prevented an AC-induced increase in the difference by suppressing epicardial MAP shortening. Gd3+ also reduced PVCs in a dose-dependent manner at plasma concentrations of 1-4 micromol/L. The effects were also evident after administration of verapamil. Thus, gadolinium suppressed an increase in the spatial dispersion of repolarization and arrhythmias via a mechanism of action different from that of verapamil.

Sour transduction involves activation of NPPB-sensitive conductance in mouse taste cells.

We examined the sour taste transduction mechanism in the mouse by applying whole cell patch-clamp technique to nondissociated taste cells from the fungiform papillae. Localized stimulation with 0.5 M NaCl and 25 mM citric acid (pH 3.0) of the apical membrane enabled us to obtain responses from single taste cells under a quasi-natural condition. Of 28 taste cells examined, 11 cells (39%) responded to 0. 5 M NaCl alone and 2 cells (7%) responded to 25 mM citric acid alone, indicating the presence of salty- and sour-specific taste cells. Ten cells (36%) responded to both NaCl and citric acid and 5 cells (18%) responded to neither salt nor citric acid. Amiloride reversibly suppressed NaCl-induced responses in mouse taste cells but not citric acid-induced responses. On the other hand, a Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), reversibly suppressed all the citric-acid-induced responses. Most of the NaCl-induced current responses displayed an inwardly rectifying property, whereas all the citric-acid-induced responses displayed an outwardly rectifying property. The reversal potential for NPPB-sensitive component in citric-acid-induced current responses was -2 +/- 7 mV (mean +/- SE, n = 4), which was close to the equilibrium potential of Cl- (ECl), whereas the reversal potential for NPPB-insensitive component was 34 +/- 8 mV (n = 4). The reversal potential of citric-acid-induced current responses (19 +/- 8 mV, n = 4) was mostly present at the middle point between reversal potentials of NPPB-sensitive and -insensitive current components. In some taste cells, an inorganic cation channel blocker, Cd2+, suppressed citric-acid-induced responses, but an inorganic stretch-activated cation channel blocker, Gd3+, did not affect these responses. These results suggest that salt- and acid-induced responses were mediated by differential transduction mechanisms in mouse taste cells and that NPPB-sensitive Cl- channels play a more important role to sour taste transduction rather than amiloride-sensitive Na+ channels. However, the fact that the reversal potentials of citric-acid-induced responses had more positive than ECl suggests that Ca2+ or H+ permeable and poorly selective cation channels, which should be amiloride insensitive, may be activated by citric acid.

Gene expression in the electrically stimulated differentiation of PC12 cells

Cell differentiation of PC12 cells was electrically induced to grow neurites in the absence of nerve growth factor (NGF) on the electrode surface, of which potential was modulated by a rectangular wave of potential. The electric stimulation induced the c-fos expression which is essential for cell differentiation. Non-specific calcium channel blocker, lanthanum ion, inhibited the electrically induced differentiation, while NGF-induced differentiation was not suppressed. An L-type calcium channel blocker, nifedipine, also inhibited the electrically induced calcium influx and c-fos expression. Moreover, a stretch-activated (SA) channel blocker, gadolinium ion, inhibited the electrically stimulated differentiation by blocking the calcium influx, but gave no prominent effects on the potassium ion-induced differentiation. Chelerythrine, a specific protein kinase C (PKC) inhibitor, almost inhibited the cell differentiation by the electric stimulation but not by the NGF treatment. These results indicate that the alternative potential may stimulate cell differentiation through a PKC cascade.

Effects of Mechanical Forces on Pulmonary Epithelial Gene Expression and Differentiation • 1738

Fetal breathing movements (FBM) are necessary for normal fetal lung growth and maturation. Absence of FBM or lung constriction results in lung hypoplasia. The molecular mechanisms by which FBM stimulate lung growth are not known. We analyzed fetal rat lungs (E19) cultured ± lung distension and tracheal ligation. We also examined the effects of mechanical stretch on rat fetal lung type II cells, human pulmonary adenocarcinoma A549 cells, and a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins SP-A and SP-B. Cells were grown to confluence on matrix-coated silastic membranes and mounted in a FX-3000 Strain Unit (Flexcell). Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5-15% radial deformation) at 50 cycles/min for 2-24h. Unstretched cells were used as controls. We separately evaluated the effects of fluid shear forces (without cellular radial elongation) and cell deformation. SP-A and SP-B mRNA abundance was assayed by northern blot. Parathyroid hormone-related protein (PTHrP, a stretch-dependent lung developmental regulator), and cyclin-dependent kinase (cdk) inhibitors were analyzed by RNase protection assay (RPA). Our results indicate that static distension for as brief as 4h decreased steady-state SP-A and SP-B mRNA levels in whole lung to 51% and 31%, respectively, of control levels (n=5-6; p SP-A expression 2-4 fold over controls and PTHrP expression by 30-60%. These effects were not mediated by fluid shear but were partially inhibited by lanthanum (a stretch-activated channel blocker). Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine in the different epithelial culture models. Cyclic stretching for 48h altered cell cycle phase distribution, apparently by inducing a block in SG2 progression, and did not activate apoptosis(assayed by DNA flow cytometry). The cell cycle effects do not appear to be due to transcriptional regulation of cdk inhibitor proteins. Our results in lung organ culture are consistent with observations made in vivo in tracheal ligation models, and suggest that continuous lung distension may interfere with airway epithelial cytodifferentiation. In contrast, dynamic mechanodeformation (distension-relaxation) may be a critical stimulus for fetal lung maturation. The molecular mechanisms by which mechanotransduction alters gene expression and cytodifferentiation are under investigation.

Stretch-induced protection shares a common mechanism with ischemic preconditioning in rabbit heart.

We sought to determine whether stretch-induced preconditioning may be related to activation of adenosine receptors, ATP-sensitive K+ (K+ATP) channels, and/or protein kinase C (PKC) in the rabbit heart. Anesthetized rabbits underwent 30 min of coronary artery occlusion followed by 3 h of reperfusion. Ischemic preconditioning was induced by one episode of 5 min of ischemia followed by 5 min of reperfusion, and stretch preconditioning was induced by a transient volume overload. The abilities of gadolinium (Gd3+), a blocker of stretch-activated channels, glibenclamide (Glib), a blocker of K+ATP channels, 8-(p-sulfophenyl)-theophylline (8-SPT), a blocker of adenosine receptors, and polymyxin B (PMXB), an antagonist of PKC, to prevent the infarct size-limiting effect of stretch-induced preconditioning were evaluated. Because the infarct size-reducing effect of stretch occurred in the absence of ischemia and was prevented by previous administration of Gd3+, Glib, 8-SPT, and PMXB, we propose that activation of mechanosensitive ion channels protects the rabbit heart from subsequent sustained ischemic insult, likely through a mechanism that involves downstream activation of PKC, adenosine receptors, and/or K+ATP channels.

Thigmotropism and stretch-activated channels in the pathogenic fungus Candida albicans.

The direction of growth of hyphae of the pathogenic fungus Candida albicans responds thigmotropically to surface contours by following scratches, ridges and grooves and by penetrating pores. Here it is shown that the thigmotropic response to ridges is attenuated by GdCl3 and verapamil [blockers of stretch-activated (SA) ion channels and L-type calcium channels, respectively]. At low concentrations, both compounds reduced the percentage of hyphae reorienting on contact with a ridge without markedly affecting hyphal extension rate, suggesting a possible role for SA or other calcium channels in the transduction of the thigmotropic response. In addition, patch-clamp recordings demonstrated SA channel activity in the plasma membrane of both yeast and hyphal cells of C. albicans. Two distinct SA channels with conductances of 54 pS and 20-25 pS in 200 mM KCl were observed in protoplasts from yeast cells and one channel of 51 pS was found in protoplasts from hyphal cells.

Transmural compression-induced proliferation and DNA synthesis through activation of a tyrosine kinase pathway in rat astrocytoma RCR-1 cells

Gliosis results from abnormal proliferation of glial cells and often occurs in response to brain or spinal cord injury. There are many factors that trigger gliosis associated with such injuries, including ischemia, humoral factors produced by the injured tissue, and possibly mechanical compression itself. In the present study, the effects of mechanical compression on cell proliferation and DNA synthesis were examined in vitro with the rat astrocyte cell line RCR-1. Pressure was applied to cells by instilling compressed helium into sealed plates or flasks in which the partial pressure of oxygen were maintained constant. Compression resulted in time- and intensity-dependent increases in cell number and [ 3H]thymidine incorporation, with maximum effects apparent at 10 min and 120 mmHg. Compression-induced cell proliferation and DNA synthesis were not inhibited by gadolinium (Gd 3+), a blocker of stretch-activated ion channels, or by inhibitors of protein kinase A, protein kinase C, or Ca 2+/calmodulin-dependent protein kinases. However, the tyrosine kinase inhibitor genistein inhibited these effects of compression in a concentration-dependent manner. Conditioned medium from compressed cells also induced cell proliferation and DNA synthesis at atmospheric pressure in a genistein-sensitive manner. These results suggest that transmural compression triggers the release of a factor (or factors) that induces cell proliferation and DNA synthesis through a tyrosine kinase pathway in RCR-1 cells.

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca(2+)-independent K+ channel.

We used single-channel recording techniques to identify and characterize a large-conductance, Ca(2+)-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability (Po) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel Po was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3<-->C2<-->C1<-->O. Arachidonic acid (AA) increased the Po of the channel, with an apparent stimulatory constant (Ks) of 1.39 microM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased Po also, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect Po. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 microM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl- secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

Guidance of CNS growth cones by substratum grooves and ridges: effects of inhibitors of the cytoskeleton, calcium channels and signal transduction pathways.

We exploited our observation that embryonic Xenopus spinal neurites align parallel to grooves in a quartz surface and that embryonic rat hippocampal neurites align perpendicular to shallow, narrow grooves (see companion paper: A. M. Rajnicek, S. Britland and C. D. McCaig, 1997) (J. Cell Sci. 110, 2905-2913) to investigate the mechanism of growth cone contact guidance. Substratum topography affected the pattern of growth cone filopodia and microtubules but parallel orientation of Xenopus neurites and perpendicular orientation of hippocampal neurites were unperturbed by cytochalasin B, which virtually eliminated filopodia. Hippocampal growth cone orientation and turning in response to grooves was unaffected by disruption of microtubules using taxol or nocodazole. Gross cytoskeletal reorganization on grooved substrata was therefore not required for growth cone steering. Inhibitors were used to identify the signal transduction pathway for perpendicular alignment of hippocampal neurites. Alignment persisted in the presence of gadolinium chloride, a blocker of stretch-activated calcium channels, the G protein inhibitor pertussis toxin, the protein tyrosine kinase inhibitor genistein, the protein kinase A and G inhibitor HA1004, the protein kinase A inhibitor KT5720and the protein kinase G inhibitor KT5823. Low concentrations of the protein kinase C inhibitors stauro-sporine, bisindolylmaleimide or H-7 did not affect perpendicular orientation but higher concentrations inhibited it. The calcium channel blockers flunarizine, nifedipine and diltiazem also inhibited perpendicular orientation. Influx of calcium and protein kinase C activity therefore appear to be involved in perpendicular contact guidance.

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes.

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.

Resting Tension Participates in the Modulation of Active Tension in Isolated Guinea Pig Ventricular Myocytes

We studied active and passive properties of intact isolated guinea-pig ventricular myocytes in auxotonic conditions. Cells were attached using carbon fibres. The passive properties of the myocytes, in the presence of the stretch-activated channel blocker streptomycin sulphate, could be separated into two groups: stiff cells (stiffness slope=2.88±0.93 nN/μm3,n=63 cells) and compliant cells (stiffness slope=0.91±0.35 nN/μm3,n=52 cells). The study and the localization of the different kind of cells indicated that endocardium is mainly constituted of stiff cells (80%) while the epicardium contained more compliant cells (60%). When a longitudinal strain was applied to compliant cells, an increase in resting tension, diastolic sarcomere length and active tension were observed. On the other hand, in stiff cells, it induced an increase in resting tension and active tension with little change of diastolic sarcomere length. In both kinds of cells, strain had no effect on Ca2+transient amplitude and shape. Plotting active tensionvdiastolic sarcomere length also clearly showed two separated populations of cells, corresponding to stiff and compliant cells. The results of the two groups of cells when plotting active tensionvresting tension could not be distinguished. We conclude that resting tension is an important factor in the modulation of active tension by stretch in addition to interfilament lattice spacing or sarcomere length.

An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes.

Hyposmotically induced changes in membrane conductance were measured in embryonic chick cardiac myocytes using conventional and perforated patch-clamp recording techniques; simultaneous measurements of cell volume were made from the video image of the voltage-clamped cell. Hyposmotic challenge was associated with a rapid, transient current coincident with the onset of cell swelling; cell volume subsequently recovered towards control values (regulatory volume decrease; RVD). The transient swelling-induced current (I(swell)) reversed at +15 mV, and was not found to be carried exclusively by any single ion in the physiological solutions. I(swell) was abolished by gadolinium (Gd3+), a blocker of stretch-activated ion channels, and was absent when the cytoskeleton was disrupted by treatment with cytochalasin B. I(swell) was also prevented when intracellular [Ca2+] was buffered with BAPTA AM. Under those experimental conditions which prevented the generation of I(swell), cell volume regulation failed so that the cells remained swollen in hyposmotic solution. Our data reveal a functional relationship between I(swell) and RVD, whereby I(swell) is a necessary prerequisite, although not exclusively sufficient, for volume recovery following cell swelling. We propose that I(swell) is an important early signalling event which activates subsequent mechanisms to regulate cell volume.