Mercuric chloride inhibition of vasopressin release from the isolated neurointermediate lobe of the rat pituitary

The effects of HgCl2 and ouabain on vasopressin release and Ca2+ uptake and distribution was examined in the neurointermediate lobe of the rat pituitary. HgCl2 (0.5 mM) inhibited vasopressin release by approx. 90% in both basal and potassium depolarized states. With 0.1 mM HgCl2 vasopressin release was inhibited by 50% in the depolarized state, but release was not effected in basal state. On the other hand, ouabain (0.5 mM) caused a 3-fold stimulation of vasopressin release in the depolarized state. Both HgCl2 (0.5 mM) and ouabain (0.5 mM) increased net 45Ca+2 uptake by about 80% in groups of neurointermediate lobes. Following 45Ca+2 uptake, HgCl2 (0.5 mM), which is absorbed by the neurointermediate lobe, produced an increase in cytosolic 45Ca+2 content and a decrease in mitochondrial 45Ca+2 content compared to control. In comparison, ouabain (0.5 mM), which does not penetrate the neurointermediate lobe, gave no change in cytosolic 45Ca+2, but an increase in mitochondrial 45Ca+2. These results suggest that HgCl2 inhibits vasopressin release from the neurointermediate lobe of the rat pituitary at a point distal to Ca+2 uptake by the gland.     

Ca<Superscript>2+</Superscript>-mediated Potentiation of the Swelling-induced Taurine Efflux from HeLa Cells: On the Role of Calmodulin and Novel Protein Kinase C Isoforms

The present work sets out to investigate how Ca²⁺ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca²⁺-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation of the release pathway. The accelerated inactivation is not observed in hypotonic Ca²⁺-free or high-K⁺ media. Addition of Ca²⁺-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca²⁺-activated K⁺ channels. The taurine release from control cells and cells exposed to Ca²⁺ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca²⁺-augmented taurine release. Exposure to Ca²⁺-mobilizing agonists prior to a hypotonic challenge also augments a subsequent swelling-induced taurine release even though the intracellular Ca²⁺-concentration has returned to the unstimulated level. The Ca²⁺-induced augmentation of the swelling-induced taurine release is abolished by inhibition of calmodulin, but unaffected by inhibition of calmodulin-dependent kinase II, myosin light chain kinase and calcineurin. The effect of Ca²⁺-mobilizing agonists is mimicked by protein kinase C (PKC) activation and abolished in the presence of the PKC inhibitor Gö6850 and following downregulation of phorbol ester-sensitive PKC isoforms. It is suggested that Ca²⁺ regulates the volume-sensitive taurine-release pathway through activation of calmodulin and PKC isoforms belonging to the novel subclass (nPKC).This revised version was published online in June 2005 with a corrected cover date.     

Interactions between glutamine metabolism and cell-volume regulation in perfused rat liver

1. In the presence of near-physiological glutamine concentrations, exposure of perfused rat liver to hypotonic perfusion media switched glutamine balance across the liver from net release to net uptake. This was due to both stimulation of flux through glutaminase and inhibition of flux through glutamine synthetase. Conversely, during exposure to hypertonic media, net glutamine release from the liver increased due to inhibition of glutaminase flux and slight stimulation of flux through glutamine synthetase. The effect of perfusate osmolarity on glutaminase flux was observed at an NH4Cl concentration (0.5 mM) sufficient for near-maximal ammonia stimulation of glutaminase. This indicates the involvement of different mechanisms of glutaminase flux control by extracellular osmolarity changes and ammonia. The effects of anisotonicity on flux through glutamine-metabolizing enzymes were fully reversible. Glutamine (0.6 mM) stimulated urea synthesis from NH4Cl (0.5 mM) during hypotonic and normotonic conditions. 2. Exposure to hypotonic and hypertonic media led, after initial liver-cell swelling and shrinkage, respectively to volume-regulatory K+ fluxes which largely restored the initial liver-cell volume despite the continuing osmotic challenge. Even after completion of cell-volume regulatory K+ fluxes, the effects of perfusate osmolarity on hepatic glutamine metabolism persisted. This indicates that in anisotonicity the liver cell is left in an altered metabolic state, even after completion of volume-regulatory responses. 3. During perfusion with isotonic media, addition of glutamine (3 mM) led to an increase of liver mass by about 4% within 2 min, which was accompanied by a net K+ uptake by the liver. Thereafter, the new steady state of increased liver mass was maintained throughout glutamine infusion. When the liver mass had reached this new steady state, a net release of K+ from the liver of about 3 mumol/g liver was observed during the following 10 min. Withdrawal of glutamine was followed by a slow reuptake of K+ and the liver mass returned to its initial value. Following exposure to glutamine (3 mM), the intracellular glutamine concentration (as calculated from glutamine tissue levels, taking into account the extracellular space determined with the [3H]inulin technique) rose from about 1 mM to 30-35 mM within about 12 min, indicating a 10-12-fold concentrative uptake of glutamine into the liver cells and an osmotic challenge for the hepatocyte. When intracellular glutamine had reached its steady-state concentration, net K+ efflux from the liver was also terminated.(ABSTRACT TRUNCATED AT 400 WORDS)     

HgCl2-induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells

Fura 2 was used to measure changes in cytosolic [Ca2+] ([Ca2+]i) in cultured rabbit kidney proximal tubule cells exposed to HgCl2. Treatment with 2.5-10 microM HgCl2 resulted in an extracellular [Ca2+] ([Ca2+]e)-independent 2- to 12-fold increase in [Ca2+]i above resting levels of about 100 nM. Treatment with 25-100 microM HgCl2 caused a rapid [Ca2+]e-independent 10- to 12-fold increase in [Ca2+]i within 1 min followed by a recovery to about 2-fold steady state by 3 min. With 25-100 microM HgCl2, both magnitude and rate of Ca2+ increase were similar, but recovery was greater with increasing doses. A slower, secondary increase in [Ca2+]i followed which varied with HgCl2 concentration and required [Ca2+]e. The first increase in [Ca2+]i represents release from intracellular pools. Calcium channel blockers, calmodulin inhibitors, and mitochondrial inhibitors do not alter the patterns of [Ca2+]i changes due to HgCl2. The recovery response with higher HgCl2 concentrations appears to be triggered by Hg2+ and not by the increased [Ca2+]i. Sulfhydryl modifiers N-ethylmaleimide, PCMB and PCMBS produced [Ca2+]e-independent [Ca2+]i increases similar to those induced by low HgCl2 concentrations. Cell killing with HgCl2 was about 50% greater with normal [Ca2+]e than with low [Ca2+]e, suggesting that [Ca2+]e influx is important in accelerating injury leading to cell death.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

A Method to Simultaneously Detect Changes in Intracellular Ca<Superscript>2+</Superscript> Concentration and Cell Volume

A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca²⁺-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca²⁺ concentration were not observed in the examined volume modulation range. The results suggest that Ca²⁺-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.     

Volume Regulation of Nerve Terminals

Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.     

Involvement of Ca2(+)-induced Ca2+ release in the volume regulation of human epithelial cells exposed to a hypotonic medium

Exposure of cultured human epithelial cells (Intestine 407) to a hypotonic solution results in initial osmotic swelling and in a subsequent volume decrease near to the original level. The regulatory volume decrease was inhibited by reduction of the extracellular free Ca2+ concentration to 90 nM. Single epithelial cells responded to a hypotonic challenge with a biphasic increase in the cytosolic free Ca2+ level from about 90 to 200 nM. Both phases of the Ca2+ rise were abolished by reducing the extracellular Ca2+ to 90 nM. In the presence of caffeine (20 mM), the second-phase Ca2+ response to a hypotonic challenge occurred earlier immediately after the first-phase response. The second-phase Ca2+ response was selectively impaired by adenine (10 mM), procaine (1 mM) or ryanodine (5 to 10 microM). These blockers for Ca2(+)-induced Ca2+ release channels inhibited volume regulation after osmotic swelling. It is concluded that Ca2(+)-induced Ca2+ release from a ryanodine-sensitive store is a prerequisite for the volume regulation of human intestinal epithelial cells under hypotonic conditions.     

Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca<Superscript>2+</Superscript>-channels

The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca²⁺-channels was studied on a model of frog polyfunctional nucleated erythrocytes. Both activation and blockade of Ca²⁺-channels has been established to promote swelling of nuclei and an increase of the nucleocytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca²⁺-channels are manifested as a decrease of the cell volume. The blocker of Ca²⁺-channels verapamil produces a transitory increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca²⁺-channels indicates participation of Ca²⁺ ions in mechanisms of the nucleocytoplasmic transfer.     

Ca<Superscript>2+</Superscript> Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells

We have investigated the dynamics of the free [Ca²⁺] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca²⁺-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca²⁺] ([Ca²⁺]_SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca²⁺ pumps with thapsigargin largely blocked Ca²⁺ uptake by the granules in Ca²⁺-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca²⁺ pump inhibitor benzohydroquinone induced a rapid release of Ca²⁺ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca²⁺ pumps is essential to maintain the steady-state [Ca²⁺]_SG. Both inositol 1,4,5-trisphosphate (InsP₃) and caffeine produced a rapid Ca²⁺ release from the granules, suggesting the presence of InsP₃ and ryanodine receptors in the granules. The response to high-K⁺ depolarization was different in both cell types, a decrease in [Ca²⁺]_SG in PC12 cells and an increase in [Ca²⁺]_SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca²⁺]_SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca²⁺ through SERCA-type Ca²⁺ pumps and can release it through InsP₃ and ryanodine receptors, supporting the hypothesis that secretory granule Ca²⁺ may be released during cell stimulation and contribute to secretion.     

Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from barley: Identification and response to salt stress

One of the protective mechanisms used by plants to survive under conditions of salt stress caused by high NaCl concentration is the removal of Na+ from the cytoplasm. This mechanism involves a number of Na+/H+-antiporter proteins that are localized in plant plasma and vacuolar membranes. Due to the driving force of the electrochemical H+ gradient created by membrane H+-pumps (H+-ATPases and vacuolar H+-pyrophosphatases), Na+/H+-antiporters extrude sodium ions from the cytoplasm in exchange for protons. In this study, we have identified the gene for the barley vacuolar Na+/H+-antiporter HvNHX2 using the RACE (rapid amplification of cDNA ends)-PCR (polymerase chain reaction) technique. It is shown that the identified gene is expressed in roots, stems, and leaves of barley seedlings and that it presumably encodes a 59.6 kD protein composed of 546 amino acid residues. Antibodies against the C-terminal fragment of HvNHX2 were generated. It is shown that the quantity of HvNHX2 in tonoplast vesicles isolated from roots of barley seedlings remains the same, whereas the rate of Na+/H+ exchange across these membranes increases in response to salt stress. The 14-3-3-binding motif Lys-Lys-Glu-Ser-His-Pro (371-376) was detected in the HvNHX2 amino acid sequence, which is suggestive of possible involvement of the 14-3-3 proteins in the regulation of HvNHX2 function.     

Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange and regulation of cytoplasmic concentration of calcium in rat cerebellar neurons treated with glutamate

In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

Effects of Purinergic Stimulation,CFTR and Osmotic Stress on Amiloride-sensitive Na<Superscript>+</Superscript> Transport in Epithelia and <Emphasis Type="Italic">Xenopus</Emphasis> Oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.     

Muscarinic Receptor Stimulation Activates a Ca<Superscript>2+</Superscript>-dependent Cl<Superscript>-</Superscript> Conductance in Rat Distal Colon

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.     

Effect of mercury, lead and acetylphenylhydrazine (APHZ) on erythrocyte and bone marrow glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). an electrophoretic study

Male and female rats of Wistar strain have been injected with 0.2% HgCl2, 203HgCl2, PbNO3 and 2% APHZ, in the volume of 0.5 ml saline for 4 days. Subbands of G-6-PD enzyme from erythrocytes and bone marrow cells, were separated by disc polyacrylamide electrophoresis. Four subbands of the enzyme resulted electrophoretic separation. In erythrocytes increase of activity of fraction I. and decrease of fraction II were observed, while in bone marrow opposite reaction was visible. In both tissue following administration of Hg and Pb, fractions III and IV disappeared. In the place of disappeared bands increased radioactivity of 203Hg was detected.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Protoplasmic pH modifies water and solute transfer in beta vulgaris root vacuoles

Volume changes were studied in Beta vulgaris storage root vacuoles, using video microscopy, when exposed to hypotonic conditions. The osmotic gradient was either step-applied or progressively imposed in perfusion experiments. Preincubation at low pH (6.6) or with HgCl2 strongly reduced the vacuoles' water permeability, measured in step experiments. Furthermore, the volumetric response depended on the rate with which the aniso-osmotic condition was established. In perfusion experiments a "plateau value" (osmotic equilibrium or steady-state volume value) was observed, which was significantly lower than the theoretically expected one. Furthermore, if vacuoles were preincubated in presence of HgCl2 or at low pH and then the hypo-osmotic challenge was applied in perfusion experiments, a still lower "plateau value" was observed. This reduction was concentration-dependent and completely reversible. In these conditions, when HgCl2 concentration was 300 mM or medium pH was 6.6, the volume change was abolished. In other experiments, when urea iso-osmotically replaced mannitol, a reversible, pH-dependent volumetric response was observed. These results can be interpreted accepting that 1) mercury-sensitive water channels, present in the studied structure, were blocked by low pH during the hypo-osmotic challenge; 2) modification of water permeability prevents excessive swelling during the osmotic shock; 3) the effectiveness of this last mechanism depended on the osmotic challenge rate; and 4) additionally, urea reflection coefficients were also modified by reduced medium pH.     

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.     

Differences in Ca2+-mediation of hypotonic and Na+-nutrient regulatory volume decrease in suspensions of jejunal enterocytes

We determined differences in the Ca2+ signalling of K+ and Cl- conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95.isotonic) compared with swelling because of the absorption of D-glucose (D-Glc) or L-Alanine (L-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca(2+)-free medium containing EGTA (100 microM) RVD after 0.5 or 0.95.isotonic challenge was prevented. L-Ala swelling and subsequent RVD was influenced in Ca(2+)-free medium. Villus cells were incubated with 10 microM of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N1,N1 tetracetic acid (BAPTA-AM) and RVD after 0.5.isotonic swelling or L-Ala swelling was prevented. Niguldipine (0.1 microM), nifedipine (5 microM), diltiazem (100 microM), Ni2+, and Co2+ (1 mM) all prevented hypotonic RVD but had no effect on RVD after L-Ala addition. Charybdotoxin (25 nM) a potent inhibitor of Ca(2+)-activated K+ channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by D-Glc. We used the calmodulin antagonists, naphthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K+ and Cl- conductance in these two models. L-Ala swelling and subsequent RVD was not influenced by 25 microM W-7; hypotonic RVD was prevented by 25 microM W-7 or 100 microM W-13. The W-13 inhibition of RVD was by-passed with 0.5 microM gramicidin. Our data show that hypotonic RVD requires extracellular Ca2+ and that the K+ conductance activated is not charybdotoxin sensitive but requires calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)