Structural and functional changes in root cells induced by calcium ionophore A23187

The shifts of Ca²⁺, K⁺ and proton homeostasis of wheat (Triticum aestivum L. M. cv Ljuba) root cells induced by the Ca²⁺-ionophore A23187 caused different responses, depending on the time of exposure to the ionophore. Oxygen consumption and heat production by roots were increased when the Ca²⁺-specific effect of A23187 was expressed. Ultrastructural re-organization of cell organelles was found to follow the ion shifts. The endoplasmic reticulum, Golgi apparatus and mitochondria rearranged their membranes following treatment. The increased ion permeability of root cell membranes is proposed to cause an excessive energy expenditure for the restoration of ion homeostasis.     

Ca Shuttling in Vesicles During Tip Growth in Neurospora crassa

Tip-growing organisms maintain an apparently essential tip-high gradient of cytoplasmic Ca²⁺. In the oomycete Saprolegnia ferax, in pollen tubes and root hairs, the gradient is produced by a tip-localized Ca²⁺ influx from the external medium. Such a gradient is normally dispensable for Neurospora crassa hyphae, which may maintain their Ca²⁺ gradient by some form of internal recycling. We localized Ca²⁺ in N. crassa hyphae at the ultrastructural level using two techniques (a) electron spectroscopic imaging of freeze-dried hyphae and (b) pyroantimoniate precipitation. The results of both methods support the presence of Ca²⁺ in the wall vesicles and Golgi body equivalents, providing a plausible mechanism for the generation and maintenance of the gradient by Ca²⁺ shuttling in vesicles to the apex, without exogenous Ca²⁺ influx. Ca²⁺ sequestration into the vesicles seems to be dependent on Ca²⁺–ATPases since cyclopiazonic acid, a specific inhibitor of Ca²⁺ pumps, eliminated all Ca²⁺ deposits from the vesicles of N. crassa.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na⁺/Ca²⁺ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca²⁺ reabsorption in the kidney. (1) Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca²⁺ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca²⁺ reabsorption. (2) The vesicle preparation contained high, digitalis-sensitive (Na⁺+K⁺-ATPase activities indicating its origin from the basolateral portion of plasma membrane. (3) The operation of Na⁺/Ca²⁺ antiport device was excluded by the findings that steep Ca²⁺ gradients formed by ATP-dependent Ca²⁺ accumulation in the vesicles were not discharged by extravesicular Na⁺, and did not drive ⁴⁵Ca²⁺ uptake into the vesicles via a Ca²⁺-⁴⁵Ca²⁺ exchange. (4) The ATP-dependent Ca²⁺ uptake into the vesicles became increasingly depressed with time by extravesicular Na⁺. This was not due to an impairment of the Ca²⁺ pump itself, but caused by Na⁺/Ca²⁺ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca²⁺ accumulation in the vesicles. (5) Earlier observations on Na⁺-induced release of Ca²⁺ from vesicles pre-equilibrated with Ca²⁺, seemingly favoring the existence of a Na⁺/Ca²⁺ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na⁺/Ca²⁺ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca²⁺ reabsorption through tubule epithelium essentially depending on the operation of a Na⁺/Ca²⁺ antiport device.     

Acidic Ca(2+) stores come to the fore

Changes in the concentration of cytosolic Ca²⁺ form the basis of a ubiquitous signal transduction pathway. Accumulating evidence implicates acidic organelles in the control of Ca²⁺ dynamics in organisms across phyla. In this special issue, we discuss Ca²⁺ signalling by these “acidic Ca²⁺ stores” which include acidocalcisomes, vacuoles, the endo-lysosomal system, lysosome-related organelles, secretory vesicles and the Golgi complex. Ca²⁺ release from these morphologically very different organelles is mediated by members of the TRP channel superfamily and two-pore channels. Inositol trisphosphate and ryanodine receptors which are traditionally viewed as endoplasmic reticulum Ca²⁺ release channels can also mobilize acidic Ca²⁺ stores. Ca²⁺ uptake into acidic Ca²⁺ stores is driven by Ca²⁺ ATPases and Ca²⁺/H⁺ exchangers. In animal cells, the Ca²⁺-mobilizing messenger NAADP plays a central role in mediating Ca²⁺ signals from acidic Ca²⁺ stores through activation of two-pore channels. These signals are important for several physiological processes including muscle contraction and differentiation. Dysfunctional acidic Ca²⁺ stores have been implicated in diseases such as acute pancreatitis and lysosomal storage disorders. Acidic Ca²⁺ stores are therefore emerging as essential components of the Ca²⁺ signalling network and merit extensive further study.     

Striated ciliary root-golgi association in branchial crown epithelial cells ofOwenia. Visualization of Ca2+-binding sites and ATPase activities

Summary Striated Ciliary Roots (SCRs), about 3 m long, are attached to the basal bodies of branchial crown ciliated epithelial cells ofOwenia. These SCRs appear to consist of 5–7-nm diameter filaments organized in a cross-striation pattern with an apparent variable periodicity of 50 to 80 nm. The most exciting observation emerging from this study is the constant and conspicuous close spatial relationship between SCRs and fairly well developed Golgi apparatus. By enhancing contrast and preservation of cell components, the OsFeCN postfixation-staining of material prefixed in glutaraldehyde in the presence of calcium has revealed some fine-structural details within the SCR-Golgi Association. By means of the calcium precipitation method, with antimonate or oxalate in conjunction with X-ray microanalysis, we have identified calcium within SCR dark bands and SCR-associated Golgi bodies. The ability to bind calcium makes the Golgi apparatus a likely candidate for Ca²⁺ regulation of putative contraction of the SCRs and/or ciliary motility. The slight period variability measured in the SCRs and cytochemical localization of Mg²⁺, Ca²⁺-dependent ATPase activities associated with cross striations support the view that theOwenia SCRs may be contractile organelles.The striking and specific close structural association between the Golgi apparatus and the SCR showing Ca²⁺-binding capabilities suggests that some sort of Ca²⁺-mediated functional relationship between these organelles may exist.Abbreviations SCR striated ciliary root - OsFeCN method osmium tetroxide-ferricyanide method - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - ATP adenosine 5-triphosphoric acid - ATPase adenosine triphosphatase - ASW artificial sea-water     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Root apical organization inArabidopsis thaliana ecotype ‘WS’ and a comment on root cap structure

Arabidopsis thaliana roots have closed apical organization with three initial tiers. The dermatogen/calyptrogen tier consists of two parts-the central initials form the columella root cap, and the peripheral initial cells form the protoderm (epidermis) and the peripheral root cap. These peripheral initials divide in a sequence to form a root cap consisting of interconnected cones. the periblem initial tier forms the ground meristem (cortex). For the first week after germination the periblem consists of one layer of initial cells. The peripheral cells of the tier divide periclinally and then anticlinally (a T-division) to form the two-layered cortex (outer cortex and endodermis). After about one week, all the peripheral cells have divided periclinally forming two initials; the outermost produces the outer cortex while the inner initial produces the endodermis and middle cortex layer. The latter two cells arise via a periclinal division. During this time, other cells within the tier divide periclinally to form a two-layered tier. The plerome forms the cells of the procambium (vascular cylinder) by simple anticlinal divisions followed by longitudinal divisions to fill out the cell files of the vascular cylinder. A survey (27 dicot species in 17 families) of roots with closed apical organization revealed that there are three different types of root cap-concentric cylinders of cells (e.g.Linum), interconnecting cones (e.g.Arabidopsis) or overlapping arcs (e.g.Gossypium). H Lambers Section editor     

Calcium signaling in pancreatic β-cells in health and in Type 2 diabetes

Changes in cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) play a crucial role in the control of insulin secretion from the electrically excitable pancreatic β-cell. Secretion is controlled by the finely tuned balance between Ca²⁺ influx (mainly through voltage-dependent Ca²⁺ channels, but also through voltage-independent Ca²⁺ channels like store-operated channels) and efflux pathways. Changes in [Ca²⁺]_c directly affect [Ca²⁺] in various organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus, secretory granules and lysosomes, as imaged using recombinant targeted probes. Because most of these organelles have specific Ca²⁺ influx and efflux pathways, they mutually influence free [Ca²⁺] in the others. In this article, we review the mechanisms of control of [Ca²⁺] in various compartments and particularly the cytosol, the endoplasmic reticulum ([Ca²⁺]_ER), acidic stores and mitochondrial matrix ([Ca²⁺]_mito), focusing chiefly on the most important physiological stimulus of β-cells, glucose. We also briefly review some alterations of β-cell Ca²⁺ homeostasis in Type 2 diabetes.     

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Functional dedifferentiation of parathyroid cells results both from a defective regulation and action of cytoplasmic Ca2+

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca²⁺-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca²⁺ concentration (Ca²⁺) on extracellular Ca²⁺ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Ca _i ²⁺ increased poorly in response to a rise in extracelluiar Ca²⁺. Ionomycin, a Ca²⁺ ionophore, raised Ca _i ²⁺ , but there was only a small inhibition of PTH release and the correlation between Ca _i ²⁺ and secretion was weak. A deteriorated Ca _i ²⁺ regulation and a decreased inhibitory action of cytoplasmic Ca²⁺ on PTH release were also found in ceils from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca²⁺.     

Role of Ca2+ in the activity of rhicadhesin from Rhizobium leguminosarum biovar viciae,which mediates the first step in attachment of Rhizobiaceae cells to plant root hair tips

The first step in attachment of Rhizobiaceae cells to plant root hair tips is mediated by a Ca²⁺-dependent, Ca²⁺-binding protein, rhicadhesin. The possible role of Ca²⁺ in synthesis, anchoring and activity of rhicadhesin was investigated. Growth of Rhizobium leguminosarum biovar viciae cells under Ca²⁺-limitation was found to result in loss of attachment ability. Under these conditions, rhicadhesin could not be usolated from the bacterial cell surface, but was found to be excreted in the growth medium. Divalent ions appeared to be essential for the ability of purified rhicadhesin to inhibit attachment of R. leguminosarum biovar viciae cells to pea root hair tips. Calcium ions were found not to be involved in binding of rhicadhesin to the plant surface, but appeared to be involved in anchoring of the adhesin to the bacterial cell surface. A model for the role of Ca²⁺ in activity of rhicadhesin is presented.     

Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids

AggregatingDictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H⁺ ATPase inhibitor concanamycin A and with the plasma membrane H⁺ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H⁺ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H⁺ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca²⁺-fluxes involves phospholipase A₂ activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca²⁺-efflux. Here we show that arachidonic acid elicited H⁺-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca²⁺-relase channel.     

Mg2+/Ca2+-ATPase Activity Is Not Enriched in Synaptic Vesicles Isolated from Rat Cerebral Cortex

Neuronal ATPases comprise a wide variety of enzymes which are not uniformly distributed in different membrane preparations. Since purified vesicle fractions have Mg²⁺/Ca²⁺-ATPase, the purpose of the present study was to know whether such enzyme activities have a preferential concentration in a synaptic vesicle fraction in order to be used as markers for these organelles. Resorting to a procedure developed in this Institute, we fractionated the rat cerebral cortex by differential centrifugation following osmotic shock of a crude mitochondrial fraction and separated a purified synaptic vesicle fraction over discontinuous sucrose gradients. Mg²⁺/Ca²⁺-ATPase activities and ultrastructural studies of isolated fractions were carried out. It was observed that similar specific activities for Mg²⁺/Ca²⁺-ATPases were found in all fractions studied which contain synaptic vesicles and/or membranes. Although the present results confirm the presence of Mg²⁺ and Ca²⁺-ATPase activities in synaptic vesicles preparations, they do not favor the contention that Mg²⁺/Ca²⁺-ATPase is a good marker for synaptic vesicles.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

N-butyryl-homoserine lactone, a bacterial quorum-sensing signaling molecule, induces intracellular calcium elevation in Arabidopsis root cells

N-acyl-l-homoserine lactones (AHLs) are quorum sensing (QS) signal molecules that are commonly used in gram-negative bacteria. Recently, it has become evident that AHLs can influence the behavior of plant cells. However, little is known about the mechanism of the plants’ response to these bacterial signals. Calcium ions (Ca²⁺), ubiquitous intracellular second messengers, play an essential role in numerous signal transduction pathways in plants. In this study, the cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) was measured by a luminometric method in the excised root cells of Arabidopsis plants that were treated with N-butyryl-homoserine lactone (C4-HSL). There was a transient and immediate increase in [Ca²⁺]_cyt levels, and the highest level (0.4 μM), approximately 2-fold higher than the basal level, was observed at the 6th second after the addition of 10 μM C4-HSL. Pretreatments with La³⁺, verapamil or ethylene glycol tetraacetic acid (EGTA) inhibited the increase in [Ca²⁺]_cyt caused by C4-HSL, whereas it remained unaffected by pretreatment with Li⁺, indicating that the Ca²⁺ contributing to the increase in [Ca²⁺]_cyt was mobilized from the extracellular medium via the plasma membrane Ca²⁺ channels but not from the intracellular Ca²⁺ stores. Furthermore, electrophysiological approaches showed that the transmembrane Ca²⁺ current was significantly increased with the addition of C4-HSL. Taken together, our observations suggest that C4-HSL may act as an elicitor from bacteria to plants and that Ca²⁺ signaling participates in the ability of plant cells to sense the bacterial QS signals.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

Ca2+ ion reversibly inhibits the cytoplasmic streaming ofNitella

Summary When Ca²⁺, K⁺ or Cl^– was injected iontophoretically into the cytoplasm of intactNitella cell, only Ca²⁺ reversibly inhibited the cytoplasmic streaming. However, when an extremely large current was used, the cytoplasmic streaming was reversibly inhibited irrespective of the ion species. This inhibition may be due to a transient increase of free Ca²⁺.     

Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: A patch-clamp study

There is evidence for a role of increased cytoplasmic Ca²⁺ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate—the universality of Ca²⁺ as a component of the signaling chain, and the source of the increased Ca²⁺, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca²⁺, and of internal Ca²⁺ buffers to control levels of cytoplasmic Ca²⁺. We show that ABA-induced reduction of the K⁺ inward rectifier can occur in the absence of external Ca²⁺, but is abolished when Ca²⁺ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca²⁺ is a necessary component of the signaling chain by which ABA decreases the K⁺ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca²⁺ from internal stores is capable of mediating the response, in the absence of any Ca²⁺ influx from the extracellular medium. The work also shows that enhancement of the K⁺ outward rectifier by ABA is Ca²⁺ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790–1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615–644, 1992), is an attractive working hypothesis.     

Ca(2+) signalling in the Golgi apparatus

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments.The Golgi apparatus has been also shown to be involved in Ca²⁺ signalling: it is indeed endowed with Ca²⁺ pumps, Ca²⁺ release channels and Ca²⁺ binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca²⁺ signal within the cell, though this role is still poorly understood.Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca²⁺ handling and selective reduction of Ca²⁺ concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology.In this paper we review the available information on the Ca²⁺ toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.