Calcium Chelator and Channel Blockers Suppress the IAA-Induced Membrane Hyperpolarization without Inhibiting the Following Growth Promotion in Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

The effects of the Ca²⁺ chelator EGTA and the Ca²⁺ channel blockersLa³⁺, Gd³⁺, nifedipine, and verapamil on the IAA-induced earlyresponses of hypocotyl sections of Vigna unguiculata were studiedto assess the involvement of Ca²⁺ in the IAA-induced hyperpolarizationfollowed by growth promotion using the xylem perfusion method.The IAA-induced hyperpolarization was distinctly inhibited bypretreatment with EGTA, La³⁺, and/or Gd³⁺ (5 mM each). EGTAinhibited by about 80–90% while La³⁺ and Gd³⁺ inhibitedby about 60–75%. The sustained hyperpolarization afterIAA application was also inhibited by these reagents. Nifedipineand verapamil (50 µM each) had little effect on the IAA-inducedhyperpolarization and growth promotion. The inhibitory effectsof EGTA, La³⁺ and Gd³⁺ on the membrane potentials were not dueto changes in a passive component, but mainly to decreases inan elec-trogenic component. The effect of EGTA was reversible,while those of La³⁺ and Gd³⁺ were not. Present results suggestthat the influx of Ca²⁺ through the plasma membrane might berequired not only for the initiation of hyperpolarization byIAA but also for the following sustained hyperpolarization andmay therefore play an essential role in the IAA-induced activationof the electrogenic proton pumps at the plasma membrane in Vignahypocotyls. Inhibition of the hyperpolarization by EGTA, La³⁺,and Gd³⁺, however, did not involve inhibition of the promotionof growth by IAA. It seems that activation of the electrogenicproton pumps at the plasma membrane is not always the indispensableprimary step of the IAA-induced promotion of elongation growth.Some alternative pathway of proton extrusion might take part. ³Present address: Laboratory of Biochemistry, Graduate SchoolofBioagricultural Sciences, Nagoya University, Nagoya, 464-8601Japan.     

Mechanically Activated Currents in Chick Heart Cells

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na⁺ and K⁺ but not Cl⁻, and were independent of external Ca²⁺. The currents had linear I/V curves reversing at −16 mV and were completely blocked by Gd³⁺≥ 30 μm and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations—the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at −2 mV and a 90 pS K⁺ selective reversing at −70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd³⁺ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd³⁺. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels. Received: 8 April 1996/Revised: 8 August 1996     

TRPM7 is a stretch- and swelling-activated cation channel involved in volume regulation in human epithelial cells

Stretch- and swelling-activated cation (SSAC) channels play essential roles not only in sensing and transducing external mechanical stresses but also in regulating cell volume in living cells. However, the molecular nature of the SSAC channel has not been clarified. In human epithelial HeLa cells, single-channel recordings in cell-attached and inside-out patches revealed expression of a Mg²⁺- and Gd³⁺-sensitive nonselective cation channel that is exquisitely sensitive to membrane stretch. Whole cell recordings revealed that the macroscopic cationic currents exhibit transient receptor potential (TRP) melastatin (TRPM)7-like properties such as outward rectification and sensitivity to Mg²⁺ and Gd³⁺. The whole cell cation current was augmented by osmotic cell swelling. RT-PCR and Western blotting demonstrated molecular expression of TRPM7 in HeLa cells. Treatment with small interfering RNA (siRNA) targeted against TRPM7 led to abolition of single stretch-activated cation channel currents and of swelling-activated, whole cell cation currents in HeLa cells. The silencing of TRPM7 by siRNA reduced the rate of cell volume recovery after osmotic swelling. A similar inhibition of regulatory volume decrease was also observed when extracellular Ca²⁺ was removed or Gd³⁺ was applied. It is thus concluded that TRPM7 represents the SSAC channel endogenously expressed in HeLa cells and that, by serving as a swelling-induced Ca²⁺ influx pathway, it plays an important role in cell volume regulation. regulatory volume decrease     

Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry

The aim of the present study was to investigate the properties and role of capacitative Ca²⁺ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca²⁺ entry in IC was larger in cells with depleted intracellular Ca²⁺ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca²⁺ entry blockers Gd³⁺ (10 µM), La³⁺ (10 µM), and Ni²⁺ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca²⁺ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at –60 mV. These events were not significantly affected by Gd³⁺ (10 µM) or La³⁺ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni²⁺. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents     

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells

In isolated rat pancreatic -cells, hypotonic stimulation elicited an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at 2.8 mM glucose. The hypotonically induced [Ca²⁺]_c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca²⁺ channel blocker, and by Gd³⁺, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca²⁺]_c elevation was not inhibited by suramin, a P₂ purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd³⁺ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd³⁺ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium     

Aluminium Effects on Pollen Germination and Tube Growth of Chamelaucium uncinatum. A Comparison with Other Ca2+Antagonists

An interaction between aluminium (Al) and calcium (Ca) may bea cause of Al toxicity in plants. The pollen tube is a suitablesystem to test the interaction between Al and Ca since Ca ionsplay a pivotal role in pollen germination and tube growth. Weinvestigated how Al and other known blockers of Ca²⁺-permeablechannels (trivalent cations, ruthenium red, verapamil and nifedipine)influence pollen of an Australian native species Geraldton waxflower(Chamelaucium uncinatum). Pollen germination was inhibited bymicromolar concentrations of trivalent cations (La³⁺>Al³⁺>Gd³⁺)and ruthenium red, but it was relatively insensitive to a micromolarconcentration of verapamil. Exposure of the growing pollen tubesto micromolar concentrations of Al³⁺and La³⁺, and a millimolarconcentration of Ca²⁺chelator ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA) led to rapid tip bursting.In contrast, exposure to Gd³⁺, nifedipine, ruthenium red, verapamiland the organic trivalent cation tris (ethylenediamine)cobalt(TEC³⁺) caused only inhibition of pollen tube growth. The Al³⁺-relatedpollen tube bursting was reduced significantly by increasingeither solution pH from 4.5 to 6 or activity of Ca²⁺from 0.25to 5 m M. In contrast, La³⁺-related pollen tube bursting wasinsensitive to changes in Ca²⁺activity. The results are discussedin terms of Al interactions with cell wall Ca²⁺and the plasmamembrane Ca²⁺-permeable channels. Copyright 1999 Annals of BotanyCompany Aluminium toxicity, Ca²⁺-channel blockers, cell wall, Chamelaucium uncinatum, pollen germination, pollen tube growth.     

Stimulation of Fibroblast Proliferation by Insoluble Gadolinium Salts

The purpose of this study was to assess insoluble salts containing gadolinium (Gd³⁺) for effects on human dermal fibroblasts. Responses to insoluble Gd³⁺ salts were compared to responses seen with Gd³⁺ solubilized with organic chelators, as in the Gd³⁺-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd³⁺ phosphate or Gd³⁺ carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd³⁺ concentrations between 12.5 and 125 μM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd³⁺ salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd³⁺) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd³⁺). Finally, high concentrations of the insoluble Gd³⁺ salts failed to prevent fibroblast lysis under low-Ca²⁺ conditions, while similar concentrations of chelated Gd³⁺ were effective. In conclusion, while insoluble Gd³⁺ salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.     

Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia

The nature of the pathway forK⁺ release activated duringregulatory volume decrease (RVD) in A6 epithelia was investigated bymeasuring cell thickness (T_c) asan index of cell volume and by probingK⁺ efflux with⁸⁶Rb as tracer forK⁺(R_Rb). Cell swelling was inducedby sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH₂O). Experiments wereperformed in the absence of Na⁺transport. Apical R_Rb wasnegligible in iso- and hyposmotic conditions. On the other hand,osmotic shock increased basolateralR_Rb(R^bl_Rb) rapidly, reaching a maximum 7 minafter the peak in T_c. Quinine (0.5 mM) completely inhibited RVD and R^bl_Rb.Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine(0.2 mM) did not exert a noticeable effect. TheK⁺ channel blockerBa²⁺ (30 mM) delayed RVD but couldnot prevent complete volume recovery. Cs⁺ inhibited RVD noticeably atconcentrations <40 mM. With large Cs⁺ concentrations (>40 mM), theinitial osmometric swelling was followed by a gradual increase ofT_c, suggesting activation of Cs⁺ influx. Chronic exposure ofthe basolateral surface to 0.5 mM La³⁺ orGd³⁺ completely abolished RVD andR^bl_Rb. Acute administration oflanthanides at the time of osmolality decrease did not affect theinitial phase of RVD and reduced R^bl_Rbonly slightly. Apical Gd³⁺ exertedan inhibitory effect on RVD and R^bl_Rb.The effect of Gd³⁺ shouldtherefore be localized at an intracellular site. The role ofCa²⁺ entry could be excluded byfailure of extracellular Ca²⁺removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administeredMg²⁺ (0.5 mM) inhibited RVD andR^bl_Rb by ~50%. These data suggest thatK⁺ excretion during RVD occursthrough a rather poorly selective pathway that does not seem to bedirectly activated by membrane stretch.

     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

Calcium-Dependent Voltage Transients Evoked by Illumination in the Liverwort Conocephalum conicum

The liverwort Conocephalum conicum with anion channels blockedby anthracene-9-carboxylic acid (A-9-C) and potassium channelsblocked by tetraethylammonium (TEA) generates dose-dependentresponses to illumination further called voltage transients(VTs). Unlike the action potentials in untreated Conocephalumthalli, VTs do not propagate and cannot be evoked by electricalstimuli. Except A-9-C, two other anion channel inhibitors: ethacrinicand niflumic acids were effective in inducing VTs. These responseswere blocked by DCMU, diethylstilbestrol and vanadate, whichindicates that the photosynthetic electron transfer chain andthe proton pump mediate in their generation. Light-induced VTswere considerably suppressed by calcium channel inhibitors:Mn²⁺, Gd³⁺, verapamil and nifedipine, and to a less extent byLa³⁺ and diltiazem, provided that the incubation lasted morethan 2 h. The participation of voltage-independent Ca²⁺ permeablechannels in ionic mechanism of VTs is postulated. (Received July 14, 1998; Accepted October 16, 1998)     

Evidence that signal transduction for osmoreception is mediated by stretch-activated ion channels in tilapia

Prolactin (PRL) playsa central role in the freshwater osmoregulation of teleost fish,including the tilapia (Oreochromis mossambicus). Consistentwith this action, PRL release from the tilapia pituitary increases asextracellular osmolality is reduced both in vitro and in vivo.Dispersed tilapia PRL cells were incubated in a perfusion chamber thatallowed simultaneous measurements of cell volume and PRL release.Intracellular Ca²⁺ concentrations were measured from fura2-loaded PRL cells treated in a similar way. Gadolinium(Gd³⁺), known to block stretch-activated cation channels,inhibited hyposmotically induced PRL release in a dose-related mannerwithout preventing cell swelling. Nifedipine, an L-typeCa²⁺ channel blocker, did not prevent the increase in PRLrelease during hyposmotic stimulation. A high, depolarizingconcentration of KCl induced a transient and marked increase ofintracellular Ca²⁺ and release of PRL but did not preventthe rise in intracellular Ca²⁺ and PRL release evoked byexposure to hyposmotic medium. These findings suggest that a decreasein extracellular osmolality stimulates PRL release through the openingof stretch-activated ion channels, which allow extracellularCa²⁺ to enter the cell when it swells.

     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Direct Effects of Ca2+-Channel Blockers on Plasma Membrane Cation Channels of Amaranthus tricolor Protoplasts

Ca²⁺-channel blockers at concentrations greater than 1 mmolm^–3, directly affect the activity of K ⁺selective channelsin the plasma membrane of Amaranthus tricolor protoplasts. Theseeffects are not mediated by the blockade of Ca²⁺ channels. Blockers tested included 1, 4-dihydropyridines (nifedipine,nicardipine), verapamil, bepridil, Gd³⁺ and La³⁺, applied towhole-cell and detached outside-out patches of plasma membraneat concentrations from 50µmol m^–3 to 100 mmol m^–3.For certain experiments the concentration of Ca²⁺ on the cytoplasmicside of the plasma membrane ([Ca²⁺]cyt) was buffered at either50ftmol m^–3 or 500 µmol m^–3. The principal currents observed in whole-cells flowed throughcation outward rectifier (OR) channels. Each blocker causedan immediate reduction of time-dependent outward currents atdoses down to 1 mmol m^–3 and produced a different, reversible,kinetic block of the outward current, independent of the levelof [Ca²⁺]cyt. Verapamil also activated a sustained inward cationcurrent at negative p.d. The same effects were found with individualchannels in detached outside-out patches. Conductance and selectivityof the cation OR channels were unchanged by the drugs. [Ca²⁺]ex, was varied over a range from 0 to 10 mol m^–3.Progressively lower [Ca²⁺]eI, increasingly enhanced the maximumamplitude of the time-dependent currents. Time-constants fordecay of inward tail currents were increased at low [Ca²⁺]eit.These effects were rapidly reversible. Although there was noevidence that the cation ORs in plasma membrane of Amaranthustricolor were dependent on [Ca²⁺]cyl for their activation, theywere sensitive to the concentration of free Ca²⁺ in the extracellularmedium. Key words: Verapamil, blocker, cation channels, Amaranthus, protoplasts     

Effects of Anion Channel Inhibitors on Light-Induced Potential Changes in the Liverwort Conocephalum conicum

The effect of three different anion channel inhibitors, namely(5-nitro-2-3-phenylpropyl-amino)benzoic acid (NPPB), Zn²⁺ andanthracene-9-carboxylic acid (A-9-C) on the action potentialin the liverwort Conocephalum conicum were tested. All threecaused an increase of the excitability threshold and a decreaseof action potential amplitudes. This confirms the involvementof anion channels in the action potentials in Conocephalum.In plants treated with 1 or 2 mM A-9-C but not with NPPB (50or 100 µM) and Zn²⁺ (100 or 500 µM), a light-inducedtransient depolarization occurred. In contrast to action potentials,the amplitude of this voltage transient depended on the lightintensity and on the duration of preceding dark period. Alsoin contrast to action potentials, which are blocked by TEA,when applied together with A-9-C, TEA even increased the amplitudesof the light-induced voltage transients to up to 170 mV. Thedepolarization was obviously limited by the voltage-dependentopening of K⁺ channels in the absence of TEA. The amplitudeof the light-induced voltage transients (in the presence ofTEA) increased in elevated CaCl₂ concentrations pointing toa Ca²⁺ permeability giving rise to the depolarization. However,none of the Ca²⁺ channel blockers tested, La³⁺, Gd³⁺, nifedipine,verapamil or diltiazem, had an effect. The light-induced voltagetransients in A-9-C treated plants are quite different fromlight- and electrically triggered action potentials but sharesome similarities with light-induced generator potentials. (Received July 9, 1996; Accepted February 20, 1997)     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd³⁺. Isolated fiber cells were viable in physiological [Ca²⁺] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 µm) contained variable combinations of Ba²⁺-and tetraethylammonium-sensitive K⁺ currents. Longer fiber cells (150–650 µm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na⁺ with N-methyl-D-glucamine, indicating that the lens contains both Gd³⁺-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K⁺ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate. electrophysiology; potassium channel; anion channel; nonselective cation channel     

Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes

Localized Ca²⁺ transients inisolated murine colonic myocytes depend on Ca²⁺ releasefrom inositol 1,4,5-trisphosphate (IP₃) receptors.Localized Ca²⁺ transients couple to spontaneous transientoutward currents (STOCs) and mediate hyperpolarization responses inthese cells. We used confocal microscopy and whole cell patch-clamprecording to investigate how muscarinic stimulation, which causesformation of IP₃, can suppress Ca²⁺ transientsand STOCs that might override the excitatory nature of cholinergicresponses. ACh (10 µM) reduced localized Ca²⁺ transientsand STOCs, and these effects were associated with a rise in basalcytosolic Ca²⁺. These effects of ACh were mimicked bygeneralized rises in basal Ca²⁺ caused by ionomycin(250-500 nM) or elevated external Ca²⁺ (6 mM).Atropine (10 µM) abolished the effects of ACh. Pretreatment of cellswith nicardipine (1 µM), or Cd²⁺ (200 µM) had no effecton responses to ACh. An inhibitor of phospholipase C, U-73122, blockedCa²⁺ transients and STOCs but did not affect the increasein basal Ca²⁺ after ACh stimulation. Xestospongin C (Xe-C;5 µM), a membrane-permeable antagonist of IP₃ receptors,blocked spontaneous Ca²⁺ transients but did not prevent theincrease of basal Ca²⁺ in response to ACh. Gd³⁺(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca²⁺ after ACh and increased thefrequency and amplitude of Ca²⁺ transients and waves.Another inhibitor of receptor-mediated Ca²⁺ influxchannels, SKF-96365, also prevented the rise in basal Ca²⁺after ACh and increased Ca²⁺ transients and development ofCa²⁺ waves. FK-506, an inhibitor ofFKBP12/IP₃ receptor interactions, had no effect onthe rise in basal Ca²⁺ but blocked the inhibitory effectsof increased basal Ca²⁺ and ACh on Ca²⁺transients. These results suggest that the rise in basalCa²⁺ that accompanies muscarinic stimulation of colonicmuscles inhibits localized Ca²⁺ transients that couldcouple to activation of Ca²⁺-activated K⁺channels and reduce the excitatory effects of ACh.

     

Essential role for extracellular Ca(2+) in JNK activation by mechanical stretch in bladder smooth muscle cells

Mechanicalstretch has been implicated in phenotypic changes as an adaptiveresponse to stretch stress physically loaded in bladder smooth musclecells (BSMCs). To investigate stretch-induced signaling, we examinedthe mitogen-activated protein kinase (MAPK) family using rat primaryBSMCs. When BSMCs were subjected to sustained mechanical stretch usingcollagen-coated silicon membranes, activation of c-JunNH₂-terminal kinase (JNK) was most relevant among three subsets of MAPK family members: the activity was elevated from 5 minafter stretch and peaked at 10 min with an 11-fold increase. Activationof p38 was weak compared with that of JNK, and ERK was notactivated at all. JNK activation by mechanical stretch was totallydependent on extracellular Ca²⁺ and inhibited byGd³⁺, a blocker of stretch-activated (SA) ion channels.Nifedipine and verapamil, inhibitors for voltage-dependentCa²⁺ channels, had no effect on this JNK activation.Moreover, none of the inhibitors pertussis toxin, genistein,wortmannin, or calphostin C affected stretch-induced JNK activation,indicating that G protein-coupled and tyrosine kinase receptors areunlikely to be involved in this JNK activation. On the other hand, W-7,a calmodulin inhibitor, and cyclosporin A, a calcineurin inhibitor,prevented JNK activation by stretch. These results suggest a novelpathway for stretch-induced activation of JNK in BSMCs: mechanicalstretch evokes Ca²⁺ influx via Gd³⁺-sensitiveSA Ca²⁺ channels, resulting in JNK activation underregulation in part by calmodulin and calcineurin.

     

The Role of Light-Induced Membrane Potential Changes in Guttation in Gametophytes of Asplenium trichomanes

Gametophytes of the fern Asplenium trichomanes exhibit guttationwhen illuminated. Membrane potential changes evoked by lightwere measured in the presence of ion channel and proton pumpinhibitors to elucidate the nature of the response and a possiblelink to guttation. Light-induced depolarization was suppressedby the anion channel inhibitors: anthracene-9-carboxylic acidand niflumic acid. Potassium channel blockers: TEA and Ba²⁺caused an increase of the amplitude of light-induced membranepotential changes. Calcium channel inhibitors, La³⁺ Gd³⁺ diltiazem,nifedipine and verapamil had no significant effect on the membranepotential changes. Similarly, proton pump inhibitors, diethylstilbestroland vanadate, had only minor effects on the response. A possiblerole of Cl^– and K⁺ fluxes in light-induced guttation isdiscussed. (Received July 8, 1999; Accepted October 13, 1999)     

The Involvement of Calcium Ions in Maintenance of Apple Fruit Tissue 9

The hypothesis that the cell walls of apple fruit tissue arebound together by Ca²⁺ ions was tested by infiltration withother cations of similar size. Sr²⁺ and Ba²⁺ were as effectiveas Ca²⁺ in increasing the resistance of apple tissue to failureunder tension while Mg²⁺, Sm³⁺, La³⁺ and Ce³⁺ were less effective.Infiltration with Ca²⁺ increased the tensile strength of tissuefrom air-stored apples to 85% of that of untreated CA-storedfruit. It was concluded that both movement of Ca²⁺ from themiddle lamella and loss of its binding sites occurred duringapple softening, with the movement of Ca²⁺ predominating andthat these processes contribute to changes in tissue structure. Substitution of D₂O for H₂O in infiltration solutions did notaffect the strength of tissue. Key words: Calcium ions, apple fruit, cell walls