A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by ⁴⁵Ca²⁺-overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca²⁺-dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ∼ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ∼ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca²⁺-CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca²⁺- or a Ca²⁺-CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.     

Structural basis for the red light induced repolarization of tip growth in caulonema cells ofCeratodon purpureus

Summary Two dynamic changes are associated with the phytochrome-regulated phototropic response in tip cells of the mossCeratodon purpureus: a tip-located gradient shift of chlortetracy-cline (CTC)-stained calcium and a structural reorganization of apical microfilaments (MFs). We examined the interdependence of these processes. Cells were treated with the antimicrotubule drug oryzalin, the antimicrofilament drug cytochalasin-D, and the calcium channel blocker nifedipine. respectively. The effects on phototropic growth, on the structural alignment of the cytoskeleton (microtubules, MTs; microfilaments) and on the distribution of CTC-stained calcium were studied under each of these conditions. In gravitropically growing tip cells the apical MFs form a cortical collar-like structure, consisting of actin bundles with a parallel axial alignment. These MFs point towards the presumptive growing point, a weakly stained region in the tip of the cell from which bundles are absent. MTs are present in the cortex and in the endoplasm of the tip, predominantly oriented longitudinally. The MTs converge within the central apex. The cells show a steep tip-to-base CTC-calcium gradient with its highest signal in the central apex. Destruction of MTs by 1 M oryzalin induces several translocational effects: (i) the growing zone and phototropic outgrowth shift from the apex to subapical parts of the cell; (ii) the structural integrity of the apical MFs and the tip-to-base alignment of the CTC-calcium gradient are disturbed; and (iii) the red light induced gradient shift and the reorientation of MFs proceed in an expanded area spanning from the tip to subapical parts of the cell. Cytochalasin-D (10 g/ml) destroys the MFs. Under these conditions tip growth stops and the phototropic outgrowth is suppressed. The apical MT-structure and the CTC-calcium gradient are not influenced by the agent. Unilateral red light still induces the light-directed translocation of the gradient. Tip cells memorize a unilateral irradiation applied during growth inhibition with cytochalasin-D. After recovery in darkness the cells start to grow in the former light direction. The restoration of the MFs precedes the outgrowth. The structural alignment of the rebuilt actin bundles indicates the future growth direction. The calcium channel blocker nifedipine (10 M) also inhibits tip growth and concurrently phototropic outgrowth. Nifedipine destroys the CTC-calcium gradient and apical MFs; MTs are not influenced by the channel blocker.Abbreviations CTC chlortetracycline - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N-N-tetraacetic acid - MBS 3-maleimidobenzoic acid N-hydroxysuccimide ester - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - RP rhodamine labeled phalloidin     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

Polyanion-mediated mineralization — a kinetic analysis of the calcium-carrier hypothesis in the phytoflagellatePleurochrysis carterae

Summary Polyanions are postulated intermediates in biomineralization because they sequester large numbers of calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. In this study mineral ion and polyanion metabolism was examined inPleurochrysis carterae to determine whether polyanions function as intermediate calcium-carriers during coccolith (mineralized scale) formation. In this organism mineralization occurs intracellularly in coccolith-forming saccules, and mature coccoliths are extruded through the plasma membrane into the coccosphere. The polyanions (acidic polysaccharides known as PS-1 and PS-2) are synthesized in medial Golgi cisternae and transported to the coccolith-forming saccule prior to the onset of mineral deposition; they also cover the mineral surface of mature coccoliths. Pulse-chase experiments with⁴⁵Ca²⁺ and¹⁴CO₃ ^– show the calcium uptake into the coccolith-forming saccule is much slower than carbonate uptake. The extended intracellular half-life of calcium ions destined for the coccosphere suggests that calcium is initially sequestered in more distal Golgi elements (perhaps in association with the polyanions) and enters the coccolith-forming saccule only after passage through the endomembrane system. This is consistent with previous cytochemical studies showing that the polyanions are complexed with calcium prior to mineral deposition. It has been suggested that polyanions may be degraded at the mineralization front in order to free calcium ions for precipitation with available carbonate or phosphate ions. However, this study demonstrates that the polyanions are not degraded; essentially all PS-1 and PS-2 are eventually secreted with the mineral phase into the coccosphere. The kinetics of mineral ion and polyanion secretion are consistent with a polyanion-mediated calcium transport; however, the manner in which calcium might be sequestered by and freed from the polyanions is still obscure.Abbreviations PS-1/2/3 polysaccharide 1/2/3 - EDTA ethylenediaminetetraacetic acid - TCA trichloroacetic acid     

Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures

Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K⁺ (using ⁸⁶Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of ⁸⁶Rb release but an increase in d -[³H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K⁺ channels, and inhibition of calmodulin all inhibit RVD. Because d -[³H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [³H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K⁺ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K⁺ and taurine release in RVD.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation

Abstract: In rat hippocampal slices and in neurons in primary culture, K⁺-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca²⁺-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca²⁺and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α₁,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca²⁺ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.