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.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity

Single Ca²⁺ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl₂ gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca²⁺ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca²⁺ channel antagonist). Extracellular AlCl₃ (70 M, pH 4.9) almost completely blocked the channel.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Genes for calcium-permeable channels in the plasma membrane of plant root cells

In plant cells, Ca²⁺ is required for both structural and biophysical roles. In addition, changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) orchestrate responses to developmental and environmental signals. In many instances, [Ca²⁺]_cyt is increased by Ca²⁺ influx across the plasma membrane through ion channels. Although the electrophysiological and biochemical characteristics of Ca²⁺-permeable channels in the plasma membrane of plant cells are well known, genes encoding putative Ca²⁺-permeable channels have only recently been identified. By comparing the tissue expression patterns and electrophysiology of Ca²⁺-permeable channels in the plasma membrane of root cells with those of genes encoding candidate plasma membrane Ca²⁺ channels, the genetic counterparts of specific Ca²⁺-permeable channels can be deduced. Sequence homologies and the physiology of transgenic antisense plants suggest that the Arabidopsis AtTPC1 gene encodes a depolarisation-activated Ca²⁺ channel. Members of the annexin gene family are likely to encode hyperpolarisation-activated Ca²⁺ channels, based on their corresponding occurrence in secretory or elongating root cells, their inhibition by La³⁺ and nifedipine, and their increased activity as [Ca²⁺]_cyt is raised. Based on their electrophysiology and tissue expression patterns, AtSKOR encodes a depolarisation-activated outward-rectifying (Ca²⁺-permeable) K⁺ channel (KORC) in stelar cells and AtGORK is likely to encode a KORC in the plasma membrane of other Arabidopsis root cells. Two candidate gene families, of cyclic-nucleotide gated channels (CNGC) and ionotropic glutamate receptor (GLR) homologues, are proposed as the genetic correlates of voltage-independent cation (VIC) channels.     

Arbuscular mycorrhizal fungi induce differential activation of the plasma membrane and vacuolar H+ pumps in maize roots

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.     

Possible mechanism of ciliary stimulation by extracellular ATP: Involvement of calcium-dependent potassium channels and exogenous Ca2+

Summary Ciliary motility was examined optically in tissue cultures from frog palate epithelium and frog's esophagus as a function of extracellular concentration of adenosine 5-triphosphate (ATP) and related compounds. The addition of micromolar concentration of ATP caused a strong enhancement of frequency and wave velocity in the direction of the effective stroke. Since adenosine 5-[, imido]-triphosphate (AMP-PNP), a nonhydrolyzable analog of ATP, produces the same effects, ATP hydrolysis is not required. The overall potency is ATP AMP-PNP>ADP adenosine>AMP. It is suggested that both the phosphate and the base moieties are involved in ATP binding.The enhancement of ciliary activity by extracellular ATP is dependent on the presence of extracellular Ca²⁺, which can be replaced by extracellular Mg²⁺. The effect of a number of potent inhibitors of the voltage-gated calcium channels on the stimulation of ciliary activity by ATP were examined. No effect was detected in the concentration range within which these agents are specific. On the other hand, quinidine, a potent inhibitor of K⁺ (calcium-dependent) channels, inhibits the effect of ATP.The following model is suggested: exogenous ATP interacts with a membrane receptor in the presence of Ca²⁺, a cascade of events occurs which mobilizes intracellular calcium, thereby increasing the cytosolic free Ca²⁺ concentration which consequently opens the calcium-activated K⁺ channels, which then leads to a change in membrane potential. The ciliary response to these changes is the enhancement of ciliary activity.This work was supported by a grant from the Fund for Basic Research administered by the Israel Academy of Science and Humanities.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K⁺ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K⁺ channel blocker TEA⁺ at a high concentration (20 mm). Barium, another classical K⁺ channel blocker, inhibited K⁺ currents with a K _i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca²⁺ level proved to be ineffective in regulating the K⁺ conductance at hyperpolarization. External Ca²⁺ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K⁺ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca²⁺ to control K⁺ influx in xylem parenchyma cells is interpreted in physiological terms.     

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.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Properties of potassium uptake by seedling roots of grape cultivars

Uptake rates of (⁸⁶Rb)K⁺ by seedling roots of six cultivars were measured and compared with K⁺ content of the root, K⁺ leakage, H⁺ efflux, and K⁺-ATPase activity of a partially purified plasmalemma fraction.Different cultivars showed significantly different rates of (⁸⁶Rb)K⁺ uptake. The uptake rates of the first (0–5 min) period did not correlate with K⁺ content of the seedling roots.The rates of uptake in the 10 to 30 min period, supposed to be active, were negatively correlated with K⁺ content of the root. Roots consistently leaked K⁺ during the first 5 min. This leakage was positively correlated with the endogenous K⁺ content of the tissue.H⁺ efflux was significantly different among the cultivars and correlated with the K⁺-ATPase activity of a microsomal fraction partially purified on discontinuous (18/34%) sucrose gradient. The relationships among transport parameters are discussed.Part of the present research was carried out at the Department of Agricultural Biotechnology of University of Padova, Italy.Part of the present research was carried out at the Department of Agricultural Biotechnology of University of Padova, Italy.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Proton pumps of the vacuolar membrane in growing plant cells

Plant growth results from the division, enlargement and specialization of cells. The two processes of the enlargement and the differentiation of cells are not spatially separated in plant tissue. We focus our attention here on the enlargement and elongation of cells. In most cases, growing plant cells contain a large central vacuole. The acid growth theory is based on the space-filling function of the large vacuole. The active transport systems in the vacuolar membrane are essential for maintenance of high osmotic pressure and for the expansion of the vacuole. The secondary active transport systems of the vacuole for sugars and ions are driven by the proton-motive force which is generated by the vacuolar H⁺-ATPase and H⁺-translocating inorganic pyrophosphatase. In this review, the relationship between cell elongation and these enzymes of the vacuolar membrane is emphasized.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

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.