A highly Ca2+-sensitive pool of granules is regulated by glucose and protein kinases in insulin-secreting INS-1 cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca(2+) to directly measure the Ca(2+) sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca(2+) sensitivity of release in that a small proportion of granules makes up a highly Ca(2+)-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca(2+). A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca(2+) are not located close to Ca(2+) channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca(2+)-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca(2+), suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca(2+) sensor that is poised to respond to modest, global elevations of [Ca(2+)](i).     

Astrocyte swelling leads to membrane unfolding, not membrane insertion

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.     

Ca2+ ionophores trigger membrane remodeling without a need for store-operated Ca2+ entry

Calcium (Ca2+) ionophores are the most effective agents able to elicit rapid membrane remodeling in vitro. This process exposes aminophospholipids at the surface of platelets and blood cells, thus providing a catalytic surface for coagulation. To explore the underlying mechanism, we examined if cytosolic Ca2+ ([Ca2+]i) increase through store-operated Ca2+ entry (SOCE) was necessary for the potent effect of ionophores. Recent studies have demonstrated that the Ca2+-ATPase inhibitor thapsigargin, although able to elevate [Ca2+]i through SOCE, does not trigger the rapid membrane remodeling. However, it was not known if the additional effect of ionophores to promote the process required SOCE or could it occur independently. We took advantage of two mutant B lymphoblast cell lines, characterized either by defective SOCE or altered membrane remodeling, to simultaneously assess [Ca2+]i increase and membrane remodeling in the presence of ionophores or thapsigargin. Results imply that ionophores trigger membrane remodeling without the requirement for a functional SOCE.     

A Study of Membrane Activity in Rat Prostate Cancer Cells: An Evaluation of the FM1-43 Dye Technique

A study was initiated to test whether the FM1-43 dye technique could beapplied to the study of endocytic membrane activity in two rodent prostatecancer (MAT-LyLu and AT-2) cell lines of markedly different metastaticability. The lipophilic dye FM1-43, which has frequently been used tomonitor endo/exocytic activity in excitable cells was employed. We found,as in excitable tissues, that both strongly metastatic (MAT-LyLu) andweakly metastatic (AT-2) cells in culture take up FM1-43 to give vesicularstaining of a variable pattern, which appeared to differ between the twocell lines. However, unlike excitable tissues, neither cell linesubsequently released the dye. Indeed, both cell lines retained the dyethrough several rounds of cell division suggesting that dye incorporatedby cells does not enter the endo/exocytotic cycle. Uptake of dye wasindependent of temperature, Na⁺/K+ gradients, pH or metabolism. Wesuggest that passive accumulation of FM1-43 can occur in cancer cells andshould not, automatically, be interpreted as evidence of endocytosis.     

Membrane recycling and calcium dynamics during settlement and adhesion of zoospores of the green alga Ulva linza

Recruitment of individuals of the marine alga Ulva linza on to a suitable habitat involves the settlement of motile zoospores on to a substratum during which a preformed adhesive is secreted by vesicular exocytosis. The fluorescent styryl dye FM 1-43 and fluorescent Ca(2+) indicators were used to follow membrane cycling and changes in cytosolic Ca(2+) ([Ca(2+)](cyt)) associated with settlement. When swimming zoospores were exposed continuously to FM 1-43, the plasma membrane was preferentially labelled. During settlement, FM 1-43-labelled plasma membrane was rapidly internalized reflecting high membrane turnover. The internalized membrane was focused into a discrete region indicating targeting of membrane to an endosome-like compartment. Acetoxymethyl (AM)-ester derivatives were found to be unsuitable for monitoring [Ca(2+)](cyt) because the dyes were rapidly sequestered from the cytoplasm into sub-cellular compartments. [Ca(2+)](cyt) was, however, reliably measured using dextran-conjugated calcium indicators delivered into cells using a biolistic technique. Cells loaded with Oregon Green BAPTA-1 dextran (Invitrogen, Paisley, UK) showed diffuse cytosolic loading and reliably responded to imposed changes in [Ca(2+)](cyt). During settlement, zoospores exhibited both localized and diffuse increases in [Ca(2+)](cyt) implying a role for [Ca(2+)](cyt) in exocytosis of the adhesive.     

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, approximately 11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.     

Turgor pressure, membrane tension and the control of exocytosis in higher plants

Both turgor pressure and differences in membrane tension are capable of providing an energy input into exocytosis, the process of fusion of Golgi vesicles with the cell membrane in plants. It is shown that the contribution of turgor pressure is much larger than that of membrane tension, so that the exocytotic process is not likely on thermodynamic grounds to be reversible unless another source of energy is made available. However, recycling of membrane material as flattened, empty vesicles is energetically possible and is likely to be favoured when the magnitude of membrane tension in the cell membrane is low. Thus the outward flows of membrane and cell wall material are in principle linked to turgor, whereas membrane tension influences the inward flow of membrane material.     

Protamine-induced permeability changes in the neutrophil plasma membrane as the basis of activation of exocytosis.

Protamine induces a gradual change in plasma membrane permeability in rabbit neutrophils, which is evident from the increase of indol fluorescence, and the leakage of quin2 from quin2-loaded neutrophils. The influx of extracellular Ca2+ into the neutrophil provides an explanation for exocytosis which occurs in the presence of Ca2+ and protamine. The dependence of exocytosis on Ca2+ concentration follows the same pattern as is observed in neutrophils permeabilized by other means. In the absence of Ca2+, and in the presence of protamine, La3+ has an activating effect on exocytosis. At higher concentrations La3+ inhibits exocytosis that occurs in the presence of Ca2+ and protamine, as do some other metal ions. The resemblance between the membrane effects of a number of toxins, as reported in literature, and protamine-induced membrane damage suggests that they occur via the same mechanism.     

Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca²⁺ activity curve comparable to exocytosis (CV–PM fusion). Here we show that Sr²⁺ and Ba²⁺ also trigger CV–CV fusion, and agents affecting different steps of exocytotic fusion block Ca²⁺, Sr²⁺, and Ba²⁺-triggered CV–CV fusion. The maximal number of active fusion complexes per vesicle, <n\>_Max, was quantified by NEM inhibition of fusion, showing that CV–CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>_Max and the Ca²⁺ sensitivity of fusion complex activation were comparable to that determined for CV–PM fusion. Using Ca²⁺-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV–CV and CV–PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca²⁺, Sr²⁺, and Ba²⁺, the complex remains Ca²⁺- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca²⁺ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca²⁺-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca²⁺ sensitivity of fusion.     

Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.     

Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K-channel KAT1

The relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of fluorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a fluorescent chimaera of the K(+)-inward rectifying channel from Arabidopsis thaliana and the green fluorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspecific labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were fluorescently labelled. The identification is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K(+)-channel KAT1 from the PM via endocytic vesicles.     

Ca(2+) and Mg(2+)/ATP independently trigger homotypic membrane fusion in gastric secretory membranes

Exocytic activation of gastric parietal cells represents a massive transformation. We studied a step in this process, homotypic fusion of H,K-ATPase-containing tubulovesicles, using R18 dequenching. Ca²⁺ and Mg²⁺/ATP each caused dramatic dequenching, reflecting a change in R18 distribution from 5% to 65–90% of the assay's membranes in 2.5 min. These stimuli also triggered fusion between tubulovesicles and liposomes. Independent confirmation that dequenching represented membrane fusion was established by separating tubulovesicle–liposome fusion products on density gradients. Only agents that trigger fusion allowed the transmembrane H,K-ATPase to move to low-density fractions along with R18. EC₅₀ for Ca²⁺-triggered fusion was 150 n m and for Mg²⁺/ATP-triggered fusion 1 m m , the latter having a Hill coefficient of 2.5. ATP-triggered fusion was specific for Mg²⁺/ATP, required ATP hydrolysis, and was insensitive to inhibition of NSF and/or H,K-ATPase. Fusion initiated by either trigger caused tubulovesicles to become resistant to subsequent challenge by either trigger. Ca²⁺-and Mg²⁺/ATP-triggered fusion required protein component(s) in tubulovesicles, though this was required in only one of the fusing membranes since tubulovesicles fused well with liposomes containing no proteins. Our data suggest that exocytosis in parietal cells is triggered by separate but interacting pathways and is regulated by self-inhibition.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

The intracellular Ca2+ channels of membrane traffic

Regulation of organellar fusion and fission by Ca²⁺ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca²⁺-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca²⁺ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca²⁺ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.     

The plasma membrane transport systems and adaptation to salinity

Salt stress represents one of the environmental challenges that drastically affect plant growth and yield. Evidence suggests that glycophytes and halophytes have a salt tolerance mechanisms working at the cellular level, and the plasma membrane (PM) is believed to be one facet of the cellular mechanisms. The responses of the PM transport proteins to salinity in contrasting species/cultivars were discussed. The review provides a comprehensive overview of the recent advances describing the crucial roles that the PM transport systems have in plant adaptation to salt. Several lines of evidence were presented to demonstrate the correlation between the PM transport proteins and adaptation of plants to high salinity. How alterations in these transport systems of the PM allow plants to cope with the salt stress was also addressed. Although inconsistencies exist in some of the information related to the responses of the PM transport proteins to salinity in different species/cultivars, their key roles in adaptation of plants to high salinity is obvious and evident, and cannot be precluded. Despite the promising results, detailed investigations at the cellular/molecular level are needed in some issues of the PM transport systems in response to salinity to further evaluate their implication in salt tolerance.     

Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D Ascites hepatocarcinoma plasma membrane

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca²⁺, Mg²⁺)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 µmoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 µmoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca²⁺/mg/10min, respectively). Interestingly, the ATP Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca²⁺, Mg²⁺)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP Pi/mg/min, similar to the one observed for the enzyme from normal liver (30 nmoles ATP Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.Dedicated to the memory of Catalina Mas Oliva and Valentín Mas Morera.     

Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca²⁺] and [Cd²⁺], or [Ca²⁺] and [Ba²⁺], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba²⁺] was added to a B cell line that had been stimulated with anti-immunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca²⁺], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba²⁺. The conductance for Ba²⁺ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca²⁺] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba²⁺.