Indolyl-3-butyric acid-induced Arabidopsis stomatal opening mediated by 3′,5′-cyclic guanosine-monophosphate

It has been pharmacologically suggested that 3′,5′-cyclic guanosine-monophosphate (cGMP) mediates indolyl-3-butyric acid (IBA)-induced stomatal opening. In Arabidopsis thaliana (L.) Heynh., such investigations compared the wild type (Columbia and Ws ecotypes) to mutants knockout for either GTP-binding protein (G protein) α subunit 1 (gpa1-4), putative G protein-coupled receptor 1 (gcr1-5), calcineurin B-like isoform 1 (cbl1) or 9 (cbl9), or the NADPH oxidases AtrbohD and AtrbohF (atrbohD/F). Stomatal opening to IBA or the permeant cGMP analogue, 8-bromo-cGMP (8-Br-cGMP) was abolished in the atrbohD/F mutant. The IBA response was fully or partially suppressed, respectively, in the gcr1-5 mutant, or the gpa1-4 and cbl1 mutants. In the cbl9 mutant, the response to IBA or 8-Br-cGMP, respectively, was partially or fully suppressed. Phenylarsine oxide (PAO) affected the IBA response, which the cbl1 mutant overlapped or the gpa1-4 and cbl9 mutants increased up to 100% inhibition. 6-anilino-5,8-quinolinedione, mas17, the (Rp)-diastereomer of 8-bromo-3′,5′-cyclic guanosine monophosphorothioate (Rp-8-Br-cGMPS), nicotinamide, ruthenium red (RRed), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), cyclosporine A (CsA) and FK506 converged to affect the IBA response, which the gpa1-4 and cbl9 mutants overlapped or the cbl1 mutant and PAO increased up to 100% inhibition. Rp-8-Br-cGMPS, nicotinamide, RRed, BAPTA, CsA or FK506 paralled the cbl9 and atrbohD/F mutants to abolish the 8-Br-cGMP response. Based on so far revealed features of these mutants and pharmacological compounds, these results confirmed cGMP as a Ca²⁺-mobilizing second messenger for apoplastic auxin whose perception and transduction would implicate a seven-transmembrane receptor – G protein – guanylyl cyclase unit at the guard cell plasma membrane.     

Pharmacological evidence for the implication of both cyclic GMP-dependent and -independent transduction pathways within auxin-induced stomatal opening in Commelina communis (L.)

It has been previously suggested that auxin-induced stomatal opening results from at least two transduction pathways, one of which involves cyclic GMP (cGMP) as the mediator within a Ca²⁺ signalling cascade. This hypothesis was investigated further in epidermal peels of Commelina communis by comparing the effects of potential inhibitors of plant Ca²⁺-dependent enzymes on the stomatal opening responses to the auxin indolyl-3-butyric acid (IBA) and to the cGMP membrane-permeable derivative 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP). In the 30–50 μM range, the potential plant calmodulin (CaM) antagonist N-(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) positively interacted with IBA but not with 8-Br-cGMP to open the stomata. The CaM antagonists W-7 (in the 10–20 μM range) and N-(aminohexyl)-1-naphthalenesulphonamide (40 μM), the potential inhibitors of plant protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (20 and 200 μM) and K-252a (0.6 μM), and cyclosporine A and FK506, potential inhibitors of plant homologs of Ca²⁺–CaM complex (Ca²⁺/CaM)-dependent protein phosphatase 2B, prevented the IBA and 8-Br-cGMP responses by about 70% and 100%, respectively. Together, these results provide indirect pharmacological evidence that, in addition to the cGMP-dependent pathway, the auxin signal is transduced through at least one cGMP-independent pathway.     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.     

Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 μM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1 mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 μM). The protein kinase A selective inhibitor Rp-cAMPS (50 μM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 μM dopamine increased cGMP levels two-fold in the presence of 1 mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 μM) impeded 10 mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca²⁺ channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca²⁺. Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca²⁺ influx.     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.     

Transduction mechanism(s) of Na-saccharin in the blowfly <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>: evidence for potassium and calcium conductance involvement

The study on transduction mechanisms underlying bitter stimuli is a particularly intriguing challenge for taste researchers. The present study investigates, in the labellar chemosensilla of the blowfly Protophormia terraenovae, the transduction mechanism by which saccharin evokes the response of the “deterrent” cell, with particular attention to the contribution of K⁺ and Ca²⁺ current and the role of cyclic nucleotides, since second messengers modulate Ca²⁺, Cl⁻ and K⁺ currents to different extents. As assessed by extracellular single-sensillum recordings, our results show that the addition of a Ca²⁺ chelator such as EGTA or the Ca²⁺ current blockers SK&F-96365, Mibefradil, Nifedipine and W-7 decrease the response of the “deterrent” cell to saccharin. A similar decreasing effect was also obtained following the addition of 4-aminopyridine, a K⁺ current blocker. On the contrary, the membrane-permeable cyclic nucleotide 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) activates this cell and shows an additive effect when presented mixed with saccharin. Our results are consistent with the hypothesis that in the labellar chemosensilla of the blowfly both Ca²⁺ and K⁺ ions are involved in the transduction mechanism of the “deterrent” cell in response to saccharin. Our results also suggest a possible pathway common to saccharin and 8Br-cGMP.     

Putative primary involvement of <Emphasis Type="Italic">Arabidopsis</Emphasis> phosphoinositide-specific phospholipase C1 within abscisic acid-induced stomatal closing

Stomatal closing to abscisic acid (ABA) was studied in leaf epidermal peels of a dexamethasone (Dex)-inducible transgenic line expressing the phospholipase C AtPLC1 antisense in the Columbia genetic background. In the absence of Dex, the Ca²⁺ buffer, ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and the phopholipase C inhibitor, 1-[6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122) specifically inhibited the response to 20 μM ABA, whereas the Ca²⁺ buffer, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited the response to 20 or 30 μM ABA. Neither EGTA nor BAPTA increased the U73122 effect. Applying 30 μM Dex specifically affected 20 μM ABA-induced stomatal closing through reducing its magnitude as well as suppressing the EGTA, BAPTA and U73122 inhibitory effects. Neither Dex nor U73122 changed the specific inhibitory effects of both the antagonist of cyclic ADP-ribose synthesis, nicotinamide and the GTP-binding protein (G protein) modulators, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂ (GP Ant-2) and mas17 on 30 μM ABA-induced stomatal closing. When tested in combination, substituting nicotinamide for mas17, but not for GP Ant-2, enhanced their inhibitory effect to an extent that BAPTA did not increase. These results supported that AtPLC1 primarily mediates the Ca²⁺-dependent stomatal closing response to 20 μM ABA as much as 30 μM Dex did not affect 20 μM ABA-induced stomatal closing when tested on the wild type Columbia-4 ecotype. Furthermore, the present study suggested that Ca²⁺ mobilization did not involve any dependency between AtPLC1 and a putative G protein-coupled ADP-ribosyl cyclase at the tested ABA concentrations.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots

3′,5′-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na⁺/K⁺ ratio and a decrease in gene expression of the plasma membrane (PM) H⁺-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H₂O₂ or CaCl₂ alleviated the NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and increasing the PM H⁺-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H₂O₂ by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H₂O₂ on ionic homeostasis was abolished when Ca²⁺ was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, a Ca²⁺ chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H₂O₂ accumulation in salt stress, and Ca²⁺ was necessary in the cGMP-mediated signaling pathway. H₂O₂, as the downstream component of cGMP signaling pathway, stimulated PM H⁺-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.     

Inhibition of cyclic guanosine 5'-monophosphate-dependent protein kinase I (PKG-I) in lumbar spinal cord reduces formalin-induced hyperalgesia and PKG upregulation.

Nitric oxide-mediated nociception has been suggested to involve formation of cyclic guanosine 5'-monophosphate (cGMP) and activation of cGMP-dependent protein kinase (PKG). To further evaluate this pathway we assessed the effects of the PKG-inhibiting cGMP analog Rp-8-Br-cGMPS in the rat formalin assay and analyzed the regulation of PKG expression in rat lumbar spinal cord. Spinally delivered Rp-8-Br-cGMPS (0.1-0.5 micro mol i.t.) reduced the nociceptive behavior in a dose-dependent manner. Similar effects were achieved with Rp-8-Br-PET-cGMPS (0.5 micro mol i.t.), another PKG-inhibitory cGMP analog. In contrast, Rp-8-Br-cAMPS (0.5 micro mol i.t.), an inhibitor of protein kinase A, had no effect in this model. Formalin treatment resulted in a rapid (within 1h), long-lasting (up to 96h) upregulation of PKG-I protein expression. This increase was prevented in animals pretreated with Rp-8-Br-cGMPS (0.5 micro mol i.t.) or morphine (2.5-5mg/kg i.p.) 10min prior to formalin injection. Spinal delivery of 8-Br-cGMP, a PKG-activating cGMP analog, without subsequent formalin treatment also caused an increase of PKG-I protein expression. Hence, the upregulation of PKG-I might possibly be mediated by cGMP itself. Our data suggest that PKG-I activation is involved in the synaptic transmission of nociceptive stimuli in the spinal cord and that PKG-I inhibitors might be interesting novel drugs for pain treatment.     

Involvement of phospholipase C-independent calcium-mediated abscisic acid signalling during <Emphasis Type="Italic">Arabidopsis</Emphasis> response to drought

The present study investigated whether Ca²⁺ mobilization independent of phosphoinositide-specific phospholipase C (PI-PLC) would delay wilting in Arabidopsis thaliana (L.) Heynh. cv. Columbia through mediating stomatal closure at abscisic acid (ABA) concentrations rising beyond a drought-specific threshold value. In wild type (WT) epidermis, the PI-PLC inhibitor (U73122) affected the stomatal response to 20 μM ABA but not to 30 μM ABA. Disruption in GTP-binding protein ά subunit 1 (GPA1) affected the stomatal response to 30 μM ABA, but not to 20 μM ABA. In the gpa1-4 mutant, the inhibitory effects of the Ca²⁺ buffer, 1,2-bis(0-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), the inactive mastoparan analogue, mas17 and the antagonist of cyclic ADP-ribose synthesis, nicotinamide, were differentially attenuated on 30 μM ABA-induced stomatal closure. By contrast, the NADPH oxidase atrbohD/F double mutation fully suppressed inhibition of 20 μM ABA-induced stomatal closure by BAPTA or U73122 as well as inhibition of 30 μM ABA-induced stomatal closure by BAPTA, mas17 or nicotinamide. On the contrary, The Al resistant alr-104 mutation modulated ABA-induced stomatal closure by a stimulatory effect of U73122 and an increased sensitivity to mas17, nicotinamide and BAPTA. Compared to WT, the atrbohD/F double mutant was more hypersensitive than the gpa1-4 mutant to wilting under the tested water stress conditions, whereas wilting was delayed in the alr-104 mutant. Since the atrbohD/F mutation breaks down ABA-induced Ca²⁺ signalling through fully preventing apoplastic Ca²⁺ to enter into the guard cells, these results showed that a putative guard cell GPA1-dependent ADP-ribosyl cyclase activity should contribute to drought tolerance within PI-PLC-independent-Ca²⁺-mediated ABA signalling.     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Two calcium mobilizing pathways implicated within abscisic acid-induced stomatal closing in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The present study investigated whether, depending on the abscisic acid (ABA) concentration, phospholipase C (PLC) would be implicated within a Ca²⁺ mobilizing pathway that would regulate stomatal aperture under standard watering conditions. Among Al sensitive mutants the als1-1 mutant of Arabidopsis thaliana (L.) Heynh. (Columbia-4 ecotype) was selected for a pharmacological approach of stomatal closing in leaf epidermal peels induced by 3, 20 or 30 μM ABA. Comparison with the wild type (WT) revealed that, exclusively in the als1-1 mutant, the stomatal response to 3 or 20 μM ABA was inhibited by about 40 %, whereas the stomatal response to 30 μM ABA and the wilting response to drought were unaffected. In WT, the Ca²⁺ buffer EGTA and the PLC inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), specifically inhibited by about 70 and 40 %, respectively, the response to 3 or 20 μM ABA, while the Ca²⁺ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited by about 70 % the response to 3, 20 or 30 μM ABA. EGTA, BAPTA and U73122 did not inhibit the part of the response to 3 or 20 μM ABA that was unaffected by the als1-1 mutation. Together, these results showed that ABA closes the stomata through two different Ca²⁺ mobilizing pathways. Since PLC could be indirectly deactivated in the als1-1 mutant, these results might suggest that, under sufficient water supply, PLC-mediated Ca²⁺ mobilization is needed for the regulation of stomatal aperture by endogenous ABA resting at concentrations below a drought-specific threshold value.     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca²⁺ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca²⁺ concentration, which peaked within 2–2.5 min of treatment. The Ca²⁺ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca²⁺ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca²⁺ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca²⁺ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca²⁺ signals were inhibited by both the Ca²⁺ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca²⁺-channel inhibitor La³⁺. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca²⁺ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca²⁺ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca²⁺ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses. Received: 23 July 1998 / Accepted: 16 October 1998     

Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

The stimulation of IP₃ production by muscarinic agonists causes both intracellular Ca²⁺ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na⁺ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na⁺ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na⁺ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent K _D of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca²⁺ influx and electrical excitability. In N1E-115 cells, Ca²⁺ entry by this pathway is necessary to refill the IP₃-sensitive intracellular Ca²⁺ pool during repeated stimulation and CNG channels may play a similar role in other neurons.