Arabidopsis cyclic nucleotide-gated channel 6 is negatively modulated by multiple calmodulin isoforms during heat shock.

An increased concentration of cytosolic Ca2+ is an early response of plant cells to heat shock. Arabidopsis cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca2+ influx and is activated by cAMP. However, it remains unclear how the Ca2+ conductivity of CNGC6 is negatively regulated under the elevated cytosolic Ca2+ concentration. In this study, Arabidopsis calmodulin isoforms CaM1/4, CaM2/3/5, CaM6, and CaM7 were found to bind to CNGC6 to varying degrees, and this binding was dependent on the presence of Ca2+ and IQ6, an atypical isoleucine-glutamine motif in CNGC6. Knockout of CaM2, CaM3, CaM5, and CaM7 genes led to a marked increase in plasma membrane inward Ca2+ current under heat shock conditions; however, knockout of CaM1, CaM4, and CaM6 genes had no significant effect on plasma membrane Ca2+ current. Moreover, the deletion of IQ6 from CNGC6 led to a marked increase in plasma membrane Ca2+ current under heat shock conditions. Taken together, the data suggest that CNGC6-mediated Ca2+ influx is likely to be negatively regulated by CaM2/3/5 and CaM7 isoforms under heat shock conditions, and that IQ6 plays an important role in CaM binding and the feedback regulation of the channel.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels.

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis.

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Effects of low concentration of dihydroouabain on intracellular calcium in guinea pig ventricular myocytes.

The effects of low concentration of dihydroouabain (DHO) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in guinea pig ventricular myocytes. [Ca(2+)](i) was detected by confocal microscopy and represented by fluorescent intensity. DHO (1 fmol/L~1 mmol/L) increased [Ca(2+)](i), especially at 10 pmol/L. Nisoldipine, egtazic acid, or tetrodotoxin partially inhibited the effect of 10 pmol/L DHO on [Ca(2+)](i). The effects of DHO remained in the absence of extracellular K(+) and Na(+). These results suggest that low concentration of DHO might increase [Ca(2+)](i) via the receptor-operated Ca(2+) channels, TTX-sensitive Na(+) channels or/and triggering of intracellular calcium release; Na(+)/K(+) pump and Na(+)/Ca(2+) exchange seem not involved in the effect of DHO.

Effect of agmatine on intracellular free calcium concentration in isolated rat ventricular myocytes.

The present study was to investigate the effects of agmatine (Agm) on free intracellular calcium concentration ([Ca(2+)]( i )) of isolated rat ventricular myocytes. [Ca(2+)]( i ) was measured by confocal microscopy in single rat ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)]( i ) were represented by fluorescence intensity (FI) or relative fluorescence intensity (F/F(0)%). The results showed that the control level of FI value of single rat ventricular myocytes was 128.8+/-13.8 and 119.6+/-13.6 in the presence of normal Tyrode's solution containing Ca(2+) 1.0 mmol/L and Ca(2+)-free Tyrode's solution, respectively. There was no difference between these two groups (P>0.05). Agm 0.1, 1, and 10 mmol/L significantly reduced the [Ca(2+)]( i ) in both extracellular solutions in a concentration-dependent manner. The similar effect of Agm on [Ca(2+)]( i ) was also observed in the presence of EGTA 3 mmol/L. KCl 60 mmol/L, PE 30 micromol/L, and Bay-K-8644 10 micromol/L, all these substances induced [Ca(2+)]( i ) elevations in ventricular myocytes. Agm (0.1, 1, and 10 mmol/L) markedly inhibited the increase in [Ca(2+)]( i ) induced by KCl, phenylephrine (PE), and Bay-K-8644. When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, 1 mmol/L Agm could block the propagating waves of elevated [Ca(2+)]( i ), and reduce the velocity and duration of propagating waves. These results suggest that Agm possesses an inhibitory effects on [Ca(2+)]( i ) via blocking voltage-dependent Ca(2+) channel, and possibly by alleviating calcium release from SR in single isolated rat ventricular myocytes.

Cholecystokinin octapeptide increases free intracellular calcium of guinea pig cardiomyocytes through activation of Ca2+ channel and tyrosine kinase.

The aim of the present study was to explore the effect of cholecystokinin octapeptide (CCK-8) on [Ca(2+)](i) and its signal transduction mechanism in isolated guinea pig cardiomyocytes. [Ca(2+)](i) was measured by laser scanning confocal microscopy in single ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)](i) were represented by fluorescent intensity (F(i)) or relative fluorescent intensity (F(i)/F(O)%). The results obtained are as follows. (1) In the normal Tyrode's solution containing 1.0 mmol/ L Ca(2+), CCK-8 (1-10(4) pmol/L) elicited a rapid and marked increase in [Ca(2+)](i). (2) When cardiomyocytes were pretreated with the Ca(2+) chelator EGTA (3 mmol/L) and Ca(2+) channel antagonist nisoldipine (0.5 micromol/L) for 5 min, CCK-8 (10(2)pmol/L) caused a slow and small increase in [Ca(2+)](i) (p< 0.01). (3) Pretreatment with the nonselected CCK- receptor (CCK-R) antagonist proglumide (6 micromol/L) or the tyrosine kinase inhibitor genistein (1 micromol/L) for 5 min could inhibit the increase of [Ca(2+)](i) induced by CCK-8 (10(2) pmol/L) (p<0.01). The results suggest that CCK-8 increases the [Ca(2+)](i) via activating the receptor-operated Ca(2+) channel and eliciting the influx of Ca(2+) in isolated guinea pig cardiomyocytes, in which tyrosine kinase may be involved.

[Experimental choroidal neovascularization is inhibited by subretinal administration of Endostatin].

OBJECTIVE: Endostatin is an endogeneous angiogenesis inhibitor. The purpose of this study was to investigated the effect of Endostatin on the eyes of rats with experimental choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by laser photocoagulation. Animals were given subretinal injections of recombinant human Endostatin 20 microl (5 g/L) or 0.9% chlorine sodium. The intensity of fluorescein leakage from the photocoagulated lesions was studied 13 days after photocoagulation. The area of CNV at each rupture site was measured using high molecular weight FITC-dextran (MW 2 x 10(6)) for high resolution angiography in RPE-choroid-sclera flat mounts. In addition, 8 eyes in each group were removed and fixed 14 days after photocoagulation, cut into thin sections. The sections were examined by light microscopy. Immunolocalization of Endoglin (CD105) and factor VIII on sections of CNV lesions was studied by immunohistochemical evaluation. RESULTS: After Endostatin injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes. The average area of CNV at sites of the Bruch's membrane rupture showed significant difference in eyes injected with Endostatin compared with control eyes. Endothelial cells demonstrated strong immunoreactivity of CD105 and factor VIII in CNV lesions of control eyes. CD105-positive cell were not detected in normal chorioretinal tissues. CONCLUSIONS: The development of CNV can be inhibited by injection of Endostatin, which suggest that Endostatin may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.

Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein α subunit and reactive oxygen species.

In recent years, adenosine tri-phosphate (ATP) has been reported to exist in apoplasts of plant cells as a signal molecule. Extracellular ATP (eATP) plays important roles in plant growth, development, and stress tolerance. Here, extracellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness. ADP, GTP, and weakly hydrolyzable ATP analogs (ATPγS, Bz-ATP, and 2meATP) showed similar effects, whereas AMP and adenosine did not affect stomatal movement. Apyrase inhibited stomatal opening. ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor (diphenylene iodonium) or deoxidizer (dithiothreitol), and was impaired in null mutant of NADPH oxidase (atrbohD/F). Added ATP triggered ROS generation in guard cells via NADPH oxidase. ATP also induced Ca(2+) influx and H(+) efflux in guard cells. In atrbohD/F, ATP-induced ion flux was strongly suppressed. In null mutants of the heterotrimeric G protein α subunit, ATP-promoted stomatal opening, cytoplasmic ROS generation, Ca(2+) influx, and H(+) efflux were all suppressed. These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca(2+), and plasma membrane H(+)-ATPase.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.

Impacts of yishen jiangzhuo granule on B lymphocytes and regulatory T-lymphocytes in patients with chronic renal insufficiency / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the impacts of Yishen Jiangzhuo Granule (YJG) on peripheral blood B-cells and regulatory T-cells (Treg) in patients with chronic renal insufficiency (CRI).</p><p><b>METHODS</b>Fifty-three CRI patients were randomly assigned to two groups, the control group and the YJG group. Before and after treatment, the following parameters in blood were detected: the peripheral Treg, percentage (CD19+), activation rate (CD19+ CD69+) and apoptotic rate (AV) of B-lymphocyte by flow cytometry; cytokines (IL-6 and IL-10) by CBA stream protein analyzing system; high sensitivity C-reactive protein (hs-CRP) by scattering turbidimetric analysis; homocysteine (Hcy) by end-point method; hemoglobin (HGB) content by Beckman-Coulter hemo-analyser; blood contents of Ca, phosphate (P), blood urea nitrogen (BUN), creatinine (SCr) and plasma albumin (Alb) by automatic biochemical analyser; and urinary contents of creatinine (UCr) by inverse HPLC. Then the product of calcium-phosphate (Ca x P) was calculated based on blood contents of Ca2 and P and the clearance rate of endogenous creatinine (CCr) was calculated based on blood BUN and SCr.</p><p><b>RESULTS</b>After treatment CD19+ and CCr significantly increased (P < 0.01), but AV and SCr decreased in both groups (P < 0.01), with the changes in the YJG group were more significant than those in the control group (P < 0.05); levels of CD19+ CD69+, Treg, IL-6, IL-10, CRP, BUN, P and Ca x P showed no significant change (P > 0.05); levels of Ca2+, HGB and Alb increased as well as of Hcy in both groups (P < 0.05). Correlation analysis: There were negative correlation in CD19+ with AV and Hcy; Alb with AV and Hcy; CCr with CRP, SCr and BUN, while positive correlation existed in SCr with CRP and BUN; and CRP with BUN.</p><p><b>CONCLUSIONS</b>YJG can improve renal function, and delay the progress of renal failure, and it also shows the regulatory effect on B lymphocytes by lowering the apoptosis rate and improving the percentage of CD19+ in patients.</p>

Ca2+ influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM.

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.

Calmodulin is involved in heat shock signal transduction in wheat.

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.