Alkalinization Stimulates Ca2+-Dependent Spreading During the Activation of Resting Lens Epithelial Cells In Primary Culture

Lens epithelium, when attached to its natural substratum, the lens capsule, can be maintained in culture for more than 2 weeks in a simple HEPES- and EDTA-buffered salt solution (HBS). In HBS, the epithelium shows the same characteristic phenomena of locomotion, initial retraction and respreading which in MEM plus serum precedes the inception of DNA synthesis. These phenomena have been shown to be dependent on extracellular Ca²⁺. 0·05 mM Ca²⁺ is necessary for maintaining cell-to-cell contacts of the in vivo epithelium. Higher concentrations of Ca²⁺ cause the epithelium to retract initially. In contrast, Mg²⁺ greatly favours cell-substratum interactions leading to the formation of lamellopodia and an initial spreading of the epithelium. After some hours in culture the epithelium changes markedly in response to extracellular Ca²⁺ and Mg²⁺; it respreads and flattens in the presence of Ca²⁺, while Mg²⁺ becomes less effective in maintaining cell-to-substratum contacts. Mg²⁺-dependent initial spreading is promoted at pH values near 7·0 but the Ca²⁺-dependent respreading requires an alkalinization of the salt solution.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Mg2+ mediates the cell-substratum interaction of Arg-Gly-Asp-dependent HeLa cell collagen receptors

Three HeLa cell surface collagen receptors of apparent molecular mass 102/58, 87, and 38/33 kDa were eluted from gelatin-Sepharose with salt gradients or Arg-Gly-Asp-containing peptides. To understand how the collagen receptors are involved in HeLa cell spreading on collagen we investigated the effects of divalent cations and Arg-Gly-Asp-containing peptides on adhesion to gelatin, since HeLa cells behave similarly on both native type I collagen and gelatin substrata and also whether Arg-Gly-Asp-containing substrata would substitute for gelatin in facilitating cell spreading. Gly-Arg-Gly-Asp-Ser-containing peptides in solution inhibited HeLa cell spreading onto gelatin and promoted only partial HeLa cell spreading when bound to tissue culture plastic. Both partial spreading of HeLa cells on the Gly-Arg-Gly-Asp-Ser substratum and full spreading on gelatin was dependent on Mg2+, but not on Ca2+. Binding of the 102/58-, 87-, and 38/33-kDa collagen receptors to gelatin-Sepharose was increased fourfold in the presence of Mg2+, and subsequent elution of the collagen receptors and a 45-kDa collagen-binding protein not thought to be involved in HeLa cell spreading was achieved with EDTA. In contrast, affinity chromatography on Gly-Arg-Gly-Asp-Ser-Sepharose eluted predominantly the 45-kDa collagen-binding protein and the 38/33-kDa collagen receptor. In summary, the Mg2(+)-dependent interaction of the collagen receptors with the Arg-Gly-Asp sequence in collagen appears to be essential for the initial events in HeLa cell spreading but is not sufficient for full cell spreading.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Cyclic GMP stimulates Na+/Ca2+ exchange in vascular smooth muscle cells in primary culture.

We examined the effect of cGMP on Na+/Ca2+ exchange in rat aortic smooth muscle cells (VSMCs) in primary culture. The intracellular Ca2+ concentration [( Ca2+]i) was raised by adding ionomycin to VSMCs incubated at high extracellular pH (pH0) (pH0 = 8.8) and high extracellular Mg2+ (Mg2+0) (Mg2+0 = 20 mM), conditions that inhibit activity of the sarcolemmal Ca2+ pump. 45Ca2+ efflux observed under these conditions was mostly extracellular Na+ (Na+0)-dependent and thus presumably catalyzed by the Na+/Ca2+ exchanger. Brief treatment of VSMCs with 8-bromo-cGMP or atrial natriuretic peptide increased this Na+0-dependent 45Ca2+ efflux by about 50%. The 8-bromo-cGMP treatment did not significantly influence total cell Na+, membrane potential, and cell pH. Conversely, when VSMCs were loaded with Na+ and then exposed to a Na+0-free medium, the rate of 45Ca2+ uptake into VSMCs increased as cell Na+ increased. Prior treatment of VSMCs with 8-bromo-cGMP accelerated 45Ca2+ uptake by up to 60% without influencing Na+ loading itself. Treatment of VSMCs with 25 microM 2,5-di-(tert-butyl)-1,4-benzohydroquinone, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, induced a transient elevation of [Ca2+]i. 8-Bromo-cGMP stimulated the rate of recovery phase of this Ca2+ transient measured in the high pHo/high Mg2+o medium. All these results indicate that cGMP stimulates Na+/Ca2+ exchange in VSMCs.     

Calcium-dependent,slow desensitization distinguishes different types of glutamate receptors

1. L-Glutamate, the most likely transmitter of rapid excitatory synaptic interactions in the brain and spinal cord, is a potent neurotoxin. Mechanisms that terminate the action of glutamate are, therefore, likely to be important for maintaining the integrity of glutaminoceptive neurons. In this study, we show that glutamate currents evoked in voltage-clamped chick motoneurons fade during prolonged or repeated application of glutamate by pressure ejection from nearby pipettes. 2. The magnitude of the decline depends on the Ca2+/Mg2+ ratio in the extracellular medium. With Ca2+ = 10.0 mM and no added Mg, the steady-state glutamate current amounted to 50% of the initial value. 3. Single-channel measurements indicate that the fade is due to receptor desensitization rather than to agonist-induced channel blockade, as the mean channel open time within bursts is independent of the agonist concentration. 4. Application of more selective agonists showed that Ca2+-dependent slow desensitization involved only G1 (NMDA) receptors. G2 responses (activated by kainate and quisqualate) did not exhibit this slow phase of desensitization under the same conditions.     

ATPase activities, Ca2+ transport and phosphoprotein formation in sarcoplasmic reticulum subfractions of fast and slow rabbit muscles.

Subfractionation of sarcoplasmic reticulum from fast-twitch and slow-twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca2+,Mg2+)-dependent (extra) ATPase, Mg2+-dependent (basal) ATPase, Ca2+ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electrophoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples. In fast-twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca2+,Mg2+)-dependent ATPase, negligible activity of Mg2+-dependent ATPase, high initial rate and high capacity of Ca2+ uptake, high amount of phosphorylated 115000-Mr polypeptide, and appeared morphologically as thin-walled vesicles covered with particles of 4 nm in diameter. Low density vesicles had little (Ca2+,Mg2+)-dependent ATPase but high Mg2+-dependent ATPase. Although the initial rate of Ca2+ uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000-Mr polypeptide was detectable at low concentrations. Instead, 57000 and 47000-Mr polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg2+. Negatively stained, these vesicles appeared to have smooth surfaces. It is suggested that low density vesicles represent a Ca2+ sequestering system different from that of high density vesicles and that Mg2+-dependent (basal) ATPase as well as the 57000 and 47000-Mr polypeptides are part of the Ca2+ transport system within the low density vesicles. According to the results from slow-twitch muscle, Ca2+ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the low density vesicles.     

Modulation of endothelial cell shape by SPARC does not involve chelation of extracellular Ca2+ and Mg2+.

SPARC (secreted protein, acidic and rich in cysteine) is an extracellular, Ca(2+)-binding protein that inhibits the spreading of newly plated cells and elicits a rounded morphology in spread cells. In this study, I investigated whether the rounding effect of SPARC depends on the ability of the protein to chelate Ca2+ at the cell surface. Bovine aortic endothelial cells were plated in the presence of different concentrations of SPARC and Ca2+; control experiments were performed with 1 mM EGTA and with Mg2+. Quantitative estimates of cell rounding were calculated according to a rounding index. SPARC, at concentrations between 0.15 and 0.58 microM, elicited rounding (or prevented spreading) of cells cultured for 16-38 h in 0.5-2.0 mM Ca2+. Addition of 0.5-2.0 mM Mg2+ to cells previously rounded in the presence of SPARC did not abrogate the effect of SPARC. When the levels of extracellular Ca2+ were adjusted with 1 mM EGTA to maximum values ranging from 7.1 to 320 microM, cells displayed a rounded morphology in the presence of exogenous SPARC. Although the rounding induced by 1 mM EGTA was essentially reversed by the inclusion of 2 mM Ca2+, cultures containing these reagents together with SPARC maintained the rounded phenotype. These results do not support a mechanism that involves the abstraction of Ca2+ from proteins at the cell surface or the provision of Ca2+ from native extracellular SPARC to cells. Therefore, SPARC does not appear to act as a local chelator of extracellular Ca2+ and Mg2+ and presumably exerts its function as a modulator of cell shape via a different pathway.     

The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Salinity-induced calcium deficiencies in wheat and barley

Salinity-calcium interactions, which have been shown to be important in plants grown in dryland saline soils of the Canadian prairies, were studied in two species differing in salt tolerance. In solution culture, wheat showed a greater reduction in growth and a higher incidence of foliar Ca deficiency symptoms than barley when grown under MgSO₄ or Na₂SO₄ plus MgSO₄ salt stress. Amendment of the saline solution with Ca to increase the Ca/(Na+Mg) ratio ameliorated the effects of salt, but more so in wheat than in barley. At least part of the difference in salt tolerance between the two species must therefore relate to species differences in the interaction of salinity and Ca nutrition. The greater response of wheat to Ca was not due to a lower Ca status in leaf tissue; on the contrary, although Ca amendments improved tissue Ca/(Na+Mg) ratios in both species, salinized wheat had equivalent or higher Ca content, and higher Ca/(Na+Mg) ratios than did barley. The higher Ca requirement of wheat is apparently specific to a saline situation; at low salinity, wheat growth was not reduced as extensively as that of barley as Ca/(Na+Mg) ratio was decreased. High night-time humidity dramatically improved wheat growth under saline conditions, but increasing the Ca concentration of the saline solution had no effect on growth in the high humidity treatment. Membrane leakage from leaf tissue of wheat grown under saline conditions was increased compared to tissue from non-saline plants. Plants grown in Ca-amended saline solutions showed no increase in membrane leakage. These results confirm the importance of Ca interaction with salinity stress, and indicate differences in species response.     

A (Ca2+, Mg2+)-ATPase activity in plasma membrane fragments isolated from squid nerves

A (Ca2+, Mg2+)-ATPase activity and a (Ca2+, Mg2+)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca2+, Mg2+)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca2+ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na+-Ca2+ exchange system but an ATP-driven uncoupled Ca2+ pump.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

alpha(1)-Adrenoceptor-induced Mg2+ extrusion from rat hepatocytes occurs via Na(+)-dependent transport mechanism

The stimulation of the alpha(1)-adrenergic receptor by phenylephrine results in a sizable extrusion of Mg2+ from liver cells. Phenylephrine-induced Mg2+ extrusion is almost completely abolished by the removal of extracellular Ca2+ or in the presence of SKF-96365, an inhibitor of capacitative Ca2+ entry. In contrast, Mg2+ extrusion is only partially inhibited by the Ca2+-channel blockers verapamil, nifedipine, or (+)BAY-K8644. Furthermore, Mg2+ extrusion is almost completely prevented by TMB-8 (a cell-permeant inhibitor of the inositol trisphosphate receptor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (an intracellular Ca2+-chelating agent), or W-7 (a calmodulin inhibitor) Thapsigargin can mimic the effect of phenylephrine, and the coaddition of thapsigargin and phenylephrine does not result in an enlarged extrusion of Mg2+ from the hepatocytes. Regardless of the agonist used, Mg2+ extrusion is inhibited by >90% when hepatocytes are incubated in the presence of physiological Ca(2+) but in the absence of extracellular Na(+). Together, these data suggest that the stimulation of the hepatic alpha(1)-adrenergic receptor by phenylephrine results in an extrusion of Mg2+ through a Na(+)-dependent pathway and a Na(+)-independent pathway, both activated by changes in cellular Ca2+.     

Intracellular-free magnesium in the smooth muscle of guinea pig taenia caeci: a concomitant analysis for magnesium and pH upon sodium removal

This study is concerned with the regulation of intracellular-free Mg2+ concentration ([Mg2+]i) in the smooth muscle of guinea pig taenia caeci. To assess an interaction of Ca2+ on the Na(+)-dependent Mg(2+)- extrusion mechanism (Na(+)-Mg2+ exchange), effects of Na+ removal (N- methyl-D-glucamine substitution) were examined in Ca(2+)-containing solutions. As changes in pHi in Na(+)-free solutions perturb estimation of [Mg2+]i using the single chemical shift only of the beta-ATP peak in 31P NMR (nuclear magnetic resonance) spectra, [Mg2+]i and pHi were concomitantly estimated from the chemical shifts of the gamma- and beta- peaks. When extracellular Na+ was substituted with N-methyl-D- glucamine, [Mg2+]i was reversibly increased. This increase in [Mg2+]i was eliminated in Mg(2+)-free solutions and enhanced in excess Mg2+ solutions. ATP content fluctuated little during removal and readmission of Na+, indicating that [Mg2+]i changes were not induced by Mg2+ release from ATP, and that Mg(2+)-extruding system would not be inhibited by fuel restriction. A slow acidification in Na(+)-free solutions and transient alkalosis by a readmission of Na+ were observed regardless of the extracellular Mg2+ concentration. When the extracellular Ca2+ concentration was increased from normal (2.4 mM) to 12 mM, only a marginal increase in [Mg2+]i was caused by Na+ removal, whereas a similar slow acidosis was observed, indicating that extracellular Ca2+ inhibits Mg2+ entry, and that the increase in [Mg2+]i is negligible through competition between Mg2+ and Ca2+ in intracellular sites. These results imply that Na(+)-Mg2+ exchange is the main mechanism to maintain low [Mg2+]i even under physiological conditions.     

Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Endonuclease activation during apoptosis: the role of cytosolic Ca2+ and pH.

An axiom of apoptosis is that increases in cytosolic Ca2+ activate a Ca2+/Mg(2+)-dependent endonuclease. However, when HL-60 human promyelocytic leukemia cells were incubated with the Ca2+ ionophore ionomycin in varied extracellular Ca2+, DNA digestion was independent of extracellular Ca2+. Under these conditions, intracellular Ca2+ concentrations did not correlate with the observed DNA digestion. In contrast, intracellular acidification correlated well with DNA digestion. These data indicate that increased intracellular Ca2+ is not the primary signal for endonuclease activation in all forms of apoptosis, but that intracellular acidification may be involved. The observed intracellular acidification is consistent with the involvement of deoxyribonuclease II in apoptosis.     

Stimulating effects of dopamine on chloride transport across the rat caudal epididymal epithelium in culture

The present study investigated the effects of dopamine on chloride transport across cultured rat caudal epididymal epithelium. The results showed that dopamine induced a biphasic short-circuit current (Isc) in a concentration-dependent manner. The dopamine-induced response consisted of an initial rapid spike followed by a sustained phase. The alpha and beta adrenoreceptor inhibitors, phentolamine and propranolol, inhibited the initial spike and the sustained phase, respectively, suggesting a contribution of adrenergic receptors. The response was almost abolished by removing the extracellular Cl-, suggesting that the dopamine-induced short-circuit current is primarily a Cl- current. The response was inhibited by the apical Cl- channel blocker, diphenylamine-dicarboxylic acid, and the Ca2+-activated Cl- channel blocker, disulfonic acid stilbene, indicating that Cl- may pass through two types of Cl- channels on the apical side. Preloading monolayers with the intracellular Ca2+ chelator BAPTA/AM abolished the initial spike and greatly reduced the second phase in the Isc response to dopamine. Pretreating the monolayers with an adenylate cyclase inhibitor, MDL12330A, inhibited all of the second Isc response and part of the initial spike. Also, characteristics of the Cl- currents induced by dopamine were observed in whole-cell patch-clamp recording. The increases of intracellular cAMP and Ca2+ induced by dopamine were also measured. The results suggest that extracellular dopamine activates Ca2+-dependent and cAMP-dependent regulatory pathways, leading to activation of both Ca2+-dependent and cAMP-dependent Cl- conductances in epididymal epithelial cells.     

Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion

Transition of leukocyte function-associated antigen-1 (LFA-1), from an inactive into an activate state depends on the presence of extracellular Mg2+ and/or Ca2+ ions. Although Mg2+ is directly involved in ligand binding, the role of Ca2+ in LFA-1 mediated adhesion remained obscure. We now demonstrate that binding of Ca2+, but not Mg2+, directly correlates with clustering of LFA-1 molecules at the cell surface of T cells, thereby facilitating LFA-1-ligand interaction. Using a reporter antibody (NKI-L16) that recognizes a Ca(2+)-dependent epitope on LFA-1, we found that Ca2+ can be bound by LFA-1 with different strength. We noticed that weak binding of Ca2+ is associated with a dispersed LFA-1 surface distribution on T cells and with non- responsiveness of these cells to stimuli known to activate LFA-1. In contrast, stable binding of Ca2+ by LFA-1 correlates with a patch-like surface distribution and vivid ligand binding after activation of LFA- 1. Mg(2+)-dependent ligand binding does not affect binding of Ca2+ by LFA-1 as measured by NKI-L16 expression, suggesting that Mg2+ binds to a distinct site, and that both cations are important to mediate adhesion. Only Sr2+ ions can replace Ca2+ to express the L16 epitope, and to induce clustering of LFA-1 at the cell surface. We conclude that Ca2+ is involved in avidity regulation of LFA-1 by clustering of LFA-1 molecules at the cell surface, whereas Mg2+ is important in regulation of the affinity of LFA-1 for its ligands.     

Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.