H2O2 degradation is suppressed in kumquat leaves infected with Xanthomonas axonopodis pv. citri

We found in a previous study that after leaves of kumquat [Fortunella margarita (Lour.) Swingle] cv ‘Nagami’ were inoculated with Xanthomonas axonopodis pv. citri (Xac), total superoxide dismutase activity (SOD) increased to promote higher H₂O₂ concentrations that coincided with a 4-fold decline in Xac populations ( Kumar et al., 2011a). The objective of the current study was to determine how activities and isoforms of important enzymes that catabolize H₂O₂, specifically catalase (CAT), ascorbate peroxidase (APOD), and the Class III peroxidases (POD) that are located in the apoplast, change in infected kumquat leaves to affect concentration and compartmentalization of H₂O₂. DAB (3,3-diaminobenzidine) staining of the Xac-infected leaves confirmed higher overall concentration of H₂O₂ as in our earlier study. One day after inoculation (dai), APOD activity declined below the controls and declines steadily up to 10 dai when the experiment was terminated. CAT activity was similar to the controls until 4 dai then declined rapidly to about 60% the activity of the controls by 6 dai, after which it remained fairly constant until 10 dai. There were 4 CAT isoforms in control leaves and 5 isoforms in infected leaves. The CAT-1 isoform band was much smaller in infected plants than the control at all sampling times. The CAT-3 isoform band disappeared at 10 dai. The CAT-5 isoform band, which was not observed in control leaves, appeared only at 4 dai in infected leaves. POD activity of infected leaves increased above the controls starting 1 dai and reached a maximum of about 3-fold higher than the controls 8 dai after which it declined. Two POD isoforms were detected in control and infected plants. This study demonstrated that the higher accumulation of H₂O₂ in kumquat leaves infected with Xac was promoted during pathogenesis first by the suppression of APOD activity and later by suppression of CAT activity. We propose that the higher SOD and lower APOD and CAT activities in the symplast contributed H₂O₂ substrate for the higher POD activity in the apoplast, which is known to be involved in plant defense against pathogens.     

Induction of disease resistance and ROS metabolism in navel oranges by chitosan

The objective of this work was to evaluate how disease resistance and reactive oxygen species (ROS) metabolism in harvested navel oranges (Citrus sinensis L. Osbeck) may be affected by chitosan. Fresh navel oranges were treated with 2% chitosan or 0.5% glacial acetic acid (control) solution for 1 min, and some were inoculated with Penicillium italicum and Penicillium digitatum. Then, the fruit were stored at 20 °C and 85–95% RH. Treatment with 2% chitosan significantly reduced the disease incidence and the lesion diameter compared with control fruit. This treatment effectively enhanced the activities of peroxidase (POD) and superoxide dismutase (SOD), and levels of glutathione (GSH) and hydrogen peroxide (H₂O₂), inhibited the activities of catalase (CAT) and the decreases of ascorbate (AsA) content during navel orange fruits storage. Ascorbate peroxidase (APX) activity in the navel orange fruit was induced slightly by the chitosan treatment during 14–21 days storage. However, glutathione reductase (GR) activity in the fruit was not enhanced by the chitosan treatment. These results indicated that chitosan treatment could induce the navel orange fruit disease resistance by regulating the H₂O₂ levels, antioxidant enzyme and ascorbate–glutathione cycle.     

Stimulation of ROS-scavenging systems in squash (Cucurbita pepo L.) plants by compost supplementation under normal and low temperature conditions

The beneficial effect of compost, the final product of aerobic biodegradation of organic matter, on growth, lipid peroxidation [as malondialdehyde (MDA], hydrogen peroxide (H₂O₂) and superoxide anion (O₂^•−), activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), as well as reduced ascorbate (ASC) and glutathione (GSH) and their oxidized forms was investigated in squash (Cucurbita pepo L. cv. Eskandarany) plants exposed to normal and low temperature (LT) conditions. LT stress of 8 °C significantly reduced the plant growth of untreated plants, but compost alleviated the adverse effect of stress and significantly increased the fresh and dry weights under normal and stress conditions. LT also induced accumulation of H₂O₂ and O₂^•− and resulted in increased lipid peroxidation, pointing out to cellular oxidative stress. Under compost application, such reactive oxygen species (ROS) and peroxidized lipids were markedly reduced, but SOD, CAT, APX and GR activities, key enzymes of ROS-scavenging systems, were significantly increased. Data also indicated that there were general reductions in total ascorbate and glutathione pool in LT control plants, but compost-treated ones considerably have maintained higher levels of such redox metabolites. Significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulfide) were generally found in compost-treated plants than in untreated-ones. It is evident that compost induced enhancement of LT tolerance was related to up-regulation of enzymatic and non-enzymatic antioxidant systems. Such enhancement would eventually protect plant cells from LT-induced oxidative stress reactions via scavenging ROS.     

Antioxidant and stomatal responses of grapevine (Vitis vinifera L.) to boron toxicity

Although of considerable agronomic importance, our understanding of B toxicity mechanism in plants is still not completely understood, and remains an open question. Therefore, we investigated the effect of increasing levels of B (0, 10, 20 and 30 mg kg⁻¹) on the growth, boron (B) concentrations, stomatal resistance, lipid peroxidation (MDA), membrane permeability (MP), lypoxygenase activity (LOX), proline (PRO) and H₂O₂ accumulation, and the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) of grapevine (Vitis vinifera L. cv. Kalecik Karasi) grafted on 5BB rootstock (V. berlandieri × V. riparia) was investigated. Applied toxic levels of B significantly reduced leaf and root growth and increased the B concentration of the leaf, and stem, bark and root of rootstock. In the all B levels leaf tissues of grapevine accumulated more B than that of the other plant parts. In order to restrict excessive uptake of B, stomatal resistance of the leaves increased especially at high B treatments (20 and 30 mg kg⁻¹). The concentrations of H₂O₂, MDA and membrane permeability were increased as the result of B toxicity while proline and the activity of lypoxygenase were decreased. Compared with control plants, the activities of SOD and CAT were increased by B treatments while the activity of APX was decreased. To our knowledge, this is the first report that B toxicity elevated the antioxidant enzymes to protect the membrane functions from reactive oxygen species (ROS) injury in grapevine and it was hoped that this study would provide a basis for developing strategies for reducing the risks associated with B toxicity.     

H2O2 metabolism during sweet orange (Citrus sinensis L. Osb.) ‘Hamlin’ Xanthomonas axonopodis pv. citri interaction

Sweet orange (Citrus sinensis L. Osb.) ‘Hamlin’ is a canker (Xanthomonas axonopodis pv. citri: Xac) susceptible citrus genotype grown commercially worldwide. Canker causes severe economic losses and restricts the marketability of crop for export. Little is known about the role of oxidative stress in canker development. In the present investigation, sweet orange ‘Hamlin’ leaves were artificially inoculated with Xac to determine the impact of Xac infection on hydrogen peroxide (H₂O₂) metabolism. Characteristic symptoms following artificial inoculation were water soaking of the infiltrated zone between 2 and 8 days after inoculation (dai); raised epidermis accompanying tiny yellow colored bacterial colonies at 8 dai; and yellowing and necrosis of the infected zone by 12–16 dai. In planta Xac population increased 1000 fold by 14 dai from an initial population of 7.3 × 10⁶ cfu cm⁻² (0 dai). Peak concentrations of H₂O₂ were observed at 24 h and between 8 and 10 dai and coincided with higher activity of total superoxide dismutase (SOD). Lower levels of H₂O₂ in infected leaves were maintained by Xac induced higher activities of catalase (CAT), ascorbate peroxidase (APOD), and guaiacol peroxidase (POD). It appears Xac altered H₂O₂ metabolism in C. sinensis L. Osb. ‘Hamlin’ to enhance survival and growth.     

Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2

Although proline is one of the major computable organic solutes that accumulate in many plant species in abiotic stresses, a hot debate continues about whether proline accumulation is a reaction to abiotic stress, or a plant's response is associated with stress tolerance. The effects of proline on antioxidative system in grape leaves of Vitis vinifera L. cv., ‘Öküzgözü’ exposed to oxidative stress by H₂O₂ was investigated. Endogenous proline, hydrogen peroxide (H₂O₂), malondialdehyde (MDA) concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) were measured spectrophotometrically. Inhibitory effect of H₂O₂ on antioxidant enzyme activities, MDA, and EL was found. In the presence of proline, SOD and CAT activities decreased, while POD and APX activities increased. Proline pre-treatment resulted in a decrease in cellular H₂O₂ content, MDA, and EL, while cellular concentration of proline increased. Based on the finding, it was suggested that proline and H₂O₂ could play an important role in oxidative stress injury of grapevine leaves grown in vitro culture. Also, proline might have a direct positive effect on antioxidant enzyme system, membrane phase change, MDA, and EL.     

Anti-oxidant enzyme responses during in vitro embryogenesis in Catharanthus roseus

Summary

The present study was carried out to analyse the activities of several anti-oxidant enzymes at various stages of somatic embryogenesis in Catharanthus roseus. The hypothesis was that anti-oxidant enzymes accumulated as part of a cellular defence mechanism in response to stress. We therefore measured superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities in various in vitro-grown tissues such as embryogenic and non-embryogenic calli, and in embryos at various stages. SOD activity increased gradually from the early embryogenic stage to heart-shaped stage embryos, but declined in the later stages (i.e., torpedo-shaped and cotyledonary embryos). In contrast, APX activity was high in non-embryogenic callus and decreased rapidly during the stage of embryo initiation. This pattern was the same for CAT. The maximum CAT activity was observed in non-embryogenic callus, then it declined almost linearly at the embryonic and post-embryonic developmental stages. The effect of exogenous hydrogen peroxide (H₂O₂) on in vitro embryogenesis was also evaluated. Lower H₂O₂ levels (0.025 mM) promoted embryo formation, whereas higher levels (0.10 mM) inhibited embryogenesis in C. roseus. Finally, higher soluble protein, free amino acid, and proline contents were found in embryogenic calli compared to non-embryogenic calli.     

外源草酸对冷藏绿竹笋的保鲜效果及其生理基础

 绿竹笋（Dendrocalamopsis oldhami）采后分别置于水和5 mmol · L^-1 草酸溶液中浸泡10 min, 晾干后在（4 ± 0.5）℃条件下贮藏。结果发现草酸处理抑制了竹笋切面的褐变,延缓了笋肉木质纤维化。 草酸处理降低了脂氧合酶（LOX）、多酚氧化酶（PPO）、过氧化物酶（POD）和苯丙氨酸解氨酶（PAL） 活性,提高了超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性,并降低了过氧化氢（H₂O₂）含量。     

Effect of CaCl2 treatment on the acclimatisation of mericlone seedlings of Cattleya and Phalaenopsis

Summary

To increase resistance to environmental stress during the acclimatisation of mericlone seedlings of Cattleya and Phalaenopsis, seedlings were treated with CaCl₂. The effectiveness of CaCl₂ treatment on acclimatisation was determined by investigating the levels of anti-oxidative enzyme activity and reactive oxygen species (ROS), the amounts of chlorophyll and malondialdehyde (MDA), the net rate of photosynthesis (A), and the area of leaf-burn caused by high temperature injury. The activities of four anti-oxidative enzymes [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)] in mericlone seedlings of Cattleya and Phalaenopsis increased with CaCl₂ treatment. The highest increase was in the treatment with 4 mM CaCl₂. Mericlone seedlings treated with 4 mM CaCl₂ had increased SOD, CAT, APX, and POD activities compared with the controls after 35 d of acclimatisation in a greenhouse. The levels of superoxide anions (O₂^?–) and hydrogen peroxide (H₂O₂) were also low. In line with this, any decrease in chlorophyll content, increase in MDA content, drop in A, or injury due to high temperature stress were mitigated by CaCl₂ treatment. These results show that CaCl₂ treatment is effective for the acclimatisation of mericlone seedlings of Cattleya and Phalaenopsis by increasing their resistance to environmental stress.     

Exogenous paraquat changes antioxidant enzyme activities and lipid peroxidation in drought-stressed cucumber leaves

In order to examine whether paraquat modifies the functioning of antioxidants and oxidative stress levels in drought-stressed plants, a cucumber cultivar (Cucumis sativus cv. Yuexiu no. 3) was grown hydroponically for 2 days. Drought stress, which was induced by polyethylene glycol (PEG), increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, while pretreatment of paraquat decreased them. Under drought stress induced by PEG, we observed the decreased contents of MDA, H₂O₂ and O₂⁻ in paraquat-pretreated plants in comparison to unpretreated stressed plants. Drought stress and paraquat both increased the activities of antioxidants such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reducatase (MDHAR, EC 1.6.5.4), glutathione reductase (GR, EC 1.6.4.2), reduced glutathione (GSH) and reduced ascorbate (AsA). But the combined effect of paraquat application and drought stress resulted in the highest activities of antioxidants. So paraquat is able to moderate the activities of scavenging system enzymes and to influence oxidative stress intensity under drought stress induced by PEG.     

草酸对冷藏期间桃果实抗氧化系统和PPO活性的影响

 桃( Prunus persica L. ) 栽培品种‘八月脆’果实采后经5 mmol·L ^{- 1}草酸溶液浸泡10 min后在低温条件下贮藏, 与对照相比, 果实的CAT和PPO活性提高, SOD和POD活性在贮藏10 d后较高; 还原型抗坏血酸(AsA) 含量下降减缓; 超氧阴离子(O₂· ) 生成速率随贮藏增加, 过氧化氢(H₂O₂ ) 含量先升后降, 但经草酸处理后O₂ ·生成速率和H₂O₂ 含量分别在5 d和5、10 d时显著低于对照。说明草酸处理通过提高果实抗氧化防御系统的能力和PPO活性来延缓果实成熟和增强果实抗病能力。     

Effect of silicon on antioxidant and stomatal response of two grapevine (Vitis vinifera L.) rootstocks grown in boron toxic,saline and boron toxic-saline soil

We investigated individual and combined effects of B toxicity and salinity in the presence or absence of silicon on the shoot growth, concentrations of sodium (Na), chloride (Cl), boron (B) and silicon (Si), and stomatal resistance (SR), lipid peroxidation (MDA), proline accumulation, H₂O₂ accumulation and the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) activity grapevine rootstocks of 41B (V. Vinifera × V. Berlandieri) and 1103P (V. Berlandieri × V. Rupestris). Applied Si counteracted the deleterious effects of salinity and boron toxicity on shoot growth by lowering the accumulation of Na in 1103P, and B and Cl in the both rootstocks. Stomatal resistance, MDA, and the concentrations of H₂O₂ and proline were higher in the plants grown in conditions of B toxicity, salinity and their combination while applied Si lowered these parameters. Lowering SOD and CAT but increasing APX, Si treatment significantly affected the enzyme activities of both rootstocks. Based on the present work, it can be concluded that Si alleviates the adverse effects of salinity, B toxicity and combined salinity-B toxicity on grapevine rootstocks by preventing both oxidative membrane damage and translocation of Na and B from root to shoots and/or soil to plant, and also lowering the phytotoxic effects of Na and B within plant tissues. When considering the antioxidative response and membrane systems, it was concluded that the rootstock 1103P was responsive to Si under stress conditions. To our knowledge, this is the first report that Si improves the combined salt and B tolerance of grapevine grown under saline, B toxic, and B toxic and saline conditions which describes membrane related parameters and antioxidant responses.     

Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation

Bitter gourd (Momordica charantia L.) seedlings treated with elevated concentrations of dimethoate (100 and 200 ppm) and fixed ultraviolet-B (0.4 Wm⁻²/30 min) irradiation showed stunted growth and less photosynthetic pigments chlorophylls (Chl) content. The synergistic effects of both the stresses were more pronounced than the individual effect. However, dimethoate at low concentration (50 ppm) stimulated growth and pigmentation but with UV-B it showed slight inhibition. Reactive oxygen species (ROS) accumulated considerably in leaves due to UV-B and high concentrations of dimethoate. Combined exposure further increased the ROS leading to lipid peroxidation and electrolyte leakage. Both the stresses alone and together also caused the increase activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). High concentration of dimethoate and UV-B accelerated the accumulation of ROS particularly H₂O₂ in leaves, causing heavy damage to photosynthetic pigments and growth of bitter gourd seedlings. Simultaneous exposure of UV-B and dimethoate inhibit the growth, photosynthetic pigment and enhanced the accumulation of ROS more severely than the individual exposure. Interestingly, low concentration (50 ppm) of dimethoate significantly reduced the effects of UV-B. The results suggested synergistic effect of dimethoate and UV-B on plant growth as a function of decreased photosynthetic pigments despite increase in the activities of the antioxidant enzyme.     

低温对仁用杏雌蕊抗坏血酸-谷胱甘肽循环的影响低温对仁用杏雌蕊抗坏血酸-谷胱甘肽循环的影响

 利用人工智能模拟霜箱,设8 个温度（18、–1、–2、–3、–4、–5、–6、–7 ℃）,对 抗寒性不同的两个仁用杏（Prunus armeniaca Linn.）品种雌蕊的过氧化氢（H₂O₂）含量和抗坏血酸-谷胱 甘肽（AsA-GSH）循环进行了研究。结果表明,低温胁迫增加了仁用杏雌蕊H₂O₂ 含量,–7 ℃时抗晚霜 品种‘围选1 号’和晚霜敏感品种‘龙王帽’分别为对照（18 ℃）的 5.49 倍和7.22 倍。低温期间,‘围 选1 号’雌蕊AsA 等酶含量及APX 等酶活性均高于‘龙王帽’,–7 ℃时,‘围选1 号’雌蕊AsA、MDHA、 DHA、GSH 和GSSG 含量比对照分别下降了66%、69%、48%、52%、53%,‘龙王帽’下降了78%、79%、 59%、66%、65%,–7 ℃时,‘围选1 号’雌蕊APX、MDHAR、DHAR、GPX、GR 活性、AsA/DHA、 GSH/GSSG 分别是对照的2.87 倍、1.07 倍、1.42 倍、1.60 倍、1.20 倍、65%和1.03 倍,‘龙王帽’是对照 2.41 倍、96%、1.13 倍、1.08 倍、98%、54%、97%。结果表明‘围选1 号’雌蕊在低温期间有较高的AsA-GSH 循环效率,可有效抑制H₂O₂ 的积累,这可能是其抗晚霜能力较强的重要原因之一。     

Silicon mediates changes to some physiological and enzymatic parameters symptomatic for oxidative stress in spinach (Spinacia oleracea L.) grown under B toxicity

The effect of exogenous silicon (Si) on the growth, boron (B) uptake, stomatal conductance, lipid peroxidation (MDA), membrane permeability, lipoxygenase activity (LOX), proline and H₂O₂ accumulation, non-enzymatic antioxidant activity (AA) and the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) of spinach were investigated under greenhouse conditions. Spinach plants were grown with 0 or 30 mg kg⁻¹ B combined with 0 and 150 mg kg⁻¹ Si. The severity of leaf symptoms of B toxicity was lower when the plants were grown with 150 mg kg⁻¹ Si. Silicon supplied to the soil with high B counteracted the deleterious effects of B on root and shoot growth. Application of B significantly increased B concentration in shoot and in root tissues. However, Si decreased B concentration in the shoots but increased it in the roots. Shoot tissues of spinach contained higher B than the roots in all treatments. Applied Si increased the Si concentration of the root and shoot. Stomatal conductance of the plants was decreased by B, but was increased by Si. The concentrations of H₂O₂ and proline were increased by B toxicity but were decreased by Si applied to plants. Boron toxicity increased the membrane permeability, MDA content and LOX activity of excised leaves of spinach. Applied Si ameliorated the membrane deterioration significantly. Compared with control plants, the activities of AA, SOD, CAT and APX in B-stressed plants without Si applied increased, and application of Si decreased their activities under toxic B conditions. Based on the present work, it can be concluded that Si alleviates B toxicity by preventing oxidative membrane damage and also translocation of B from root to shoots. To our knowledge, this is the first report on the effect of Si in improving B tolerance in spinach.     

Photosynthetic characteristics and protective mechanisms against photooxidation during high temperature stress in two citrus species

Exposure of Satsuma mandarin (Citrus unshiu Marc.) and Navel orange (Citrus sinensis Osbeck) plants to high temperature (38 °C) led to reductions of the net photosynthetic rate (Pn), the photorespiration rate (Pr), the quantum efficiency CO₂ assimilation (ΦCO₂${\Phi }_{\text{C}{\text{O}}_2}$), the maximal photochemical efficiency of PS2 (Fv/Fm), the photochemical quenching (qP) and the quantum efficiency of PS2 photochemistry (Φ_PS2), whereas the minimal fluorescence yield (Fo) and the non-photochemical quenching (qN) increased. Increase in the value of Pr/Pn and ΦPS₂/ΦCO₂${\Phi }_{\text{PS}2}/{\Phi }_{\text{C}{\text{O}}_2}$ was attributed to the greater decrease in Pn and ΦCO₂${\Phi }_{\text{C}{\text{O}}_2}$ than Pr and Φ_PS2. In addition, the superoxide radical (O₂⁻) production, the H₂O₂ concentration and the activities of antioxidant enzymes such as the superoxide dismutase (SOD, EC 1.15.1.1), the ascorbate peroxidase (APX, EC 1.11.1.11), the dehydroascorbate reductase (DHAR, EC 1.8.5.1) and the catalase (CAT, EC 1.11.1.6) were raised. On the other hand, the chlorophyll concentration in leaves decreased during high temperature stress. These results suggest that decline in Pn related to inactivation of PS2 reaction centers may be due to the enhanced number of active oxygen species in the citrus leaves. The water–water cycle may play a role in limiting the degree of photodamage caused by high temperature. Lower O₂⁻ production rate, the H₂O₂ concentration and the antioxidant enzymes activity were observed in high temperature tolerant species of citrus. The exogenous active oxygen scavenger ascorbic acid (Asc) enhanced the ability to clear the O₂⁻ in citrus plants, and quicken the recovery of photosynthetic apparatus.     

The role of calcium in regulating photosynthesis and related physiological indexes of cucumber seedlings under low light intensity and suboptimal temperature stress

The response of photosynthesis, antioxidant enzyme activity, and proline content to low light intensity and suboptimal temperature in Cucumis sativa L. seedlings pretreated with either distilled water, 10 mM CaCl₂, 1 mM LaCl₃, 3 mM ethyleneglycol-bis-(2-aminoethyl) tetraacetic acid (EGTA) or 0.05 mM chlorpromazine (CPZ) were investigated. The results showed that 10 mM CaCl₂ led to an increase in photosynthetic rate (Pn), carboxylation efficiency (CE), ribulose 1,5-biphosphate carboxylase (RuBPCase) activity, chlorophyll content, peroxidase (POD) and catalase (CAT) activity, and proline content of cucumber seedlings under low light intensity and suboptimal temperature, in comparison with the distilled water-pretreated seedlings. However, LaCl₃, EGTA and CPZ were in contrast to CaCl₂. These results suggest that CaCl₂ has beneficial effect on photosynthetic adaptation to low light and suboptimal temperature stress in cucumber seedlings. This might be related to the observed increase in RuBPCase activity, alleviation of lipid peroxidation and enhancement of osmoregulation, with these effects being inhibited by LaCl₃, EGTA and CPZ.