Saponin-treated Cells Research Articles

Characteristics of bile acid-mediated Ca2+ release from permeabilized liver cells and liver microsomes

Saponin-treated liver cells and a microsomal fraction were used to characterize the mechanism of the Ca2+ release induced by different bile acids. The saponin-treated cells accumulated 0.8-1 nmol/mg of protein of the medium Ca2+ in a nonmitochondrial, high affinity, and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool. Three of five bile acids tested, lithocholate and the conjugates taurolithocholate and taurolithocholate sulfate, released 85% of the Ca2+ pool within 45-60 s and with ED50 from 16 to 28 microM. Ins(1,4,5)P3 released 80% from the same Ca2+ pool with an ED50 of 0.3 microM. The Ca2+-Mg2+-ATPase inhibitor vanadate (1 mM) had no effect on the Ca2+ released by the bile acids and Ins(1,4,5)P3. The Ins(1,4,5)P3-binding antibiotic neomycin (1 mM) and the receptor competitor heparin (16 micrograms/ml) abolished the releasing effect of Ins(1,4,5)P3 but had no effect on the bile acid-mediated Ca2+ release. The 45Ca2+ accumulated by the microsomal fraction (8 nmol of 45Ca2+/mg of protein) was released by the bile acids within 45-90 s and with an ED50 of 17 microM. In contrast, the bile acids had no effect on the Ca2+ permeability of other natural and artificial membranes. The resting 45Ca2+ influx of intact cells (0.45 nmol/mg of protein/min), the 45Ca2+ accumulated by mitochondria (2-13 nmol of 45Ca2+/mg of protein), and the 45Ca2+ trapped in sonicated phosphatidylcholine vesicles (5 mM 45Ca2+) were not altered by the different bile acids. These results suggest that the Ca2+ release initiated by lithocholate and its conjugates results from a direct action on the Ca2+ permeability of the Ins(1,4,5)P3-sensitive pool. It is not mediated by Ins(1,4,5)P3 or via activation of the Ins(1,4,5)P3 receptor, and it is specific for the membrane of the internal pool.

Visualization and Identification of Cytoskeletal Filaments in Embryonic Chick Heart by the Quick-Freeze Deep-Etch Method Combined with Immunocytochemistry

Cytoskeletal filaments in myocardial cells of chick embryo (stage 18-20, day 3) were studied by immunocytochemical and rapid-freeze deep-etch methods. A three-dimensional network of cytoplasmic filaments surrounding nascent myofibrils was visualized in saponin-treated myocardial cells. The major part of the network was composed of 12 to 14 nm filaments in platinum replicas. To identify the filaments, the myocardial cells were permeabilized with Triton X-100 and treated with myosin subfragment-1 (S1) for actin or immunogold-labeled antibody for desmin. A large number of filaments in myofibrils and a few cytoplasmic filaments were decorated with S1. A loose network surrounding the myofibrils was not decorated with S1 but with gold particles. This finding means that the majority of filaments occupying the interfibrillar space were desmin-containing filaments.

Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery.

We have investigated the mechanism of action of ryanodine on intact and skinned smooth-muscle cells of the rabbit ear artery. The amplitude of the phasic response induced by low noradrenaline (NA) concentrations (less than 30 nM) was inhibited by 10 microM ryanodine, while that elicited by high NA concentrations (greater than 100 nM) was not affected. The phasic contractions induced by both low and high NA concentrations in Ca2+-free solution containing 2 mM EGTA were suppressed by 10 microM ryanodine. The rate of 45Ca efflux in Krebs solution was enhanced by 10 microM ryanodine, while the increased 45Ca efflux induced by 10 microM NA was inhibited by ryanodine. 10 microM ryanodine did not affect the contractile proteins in saponin-treated smooth-muscle cells. The intracellular Ca2+ stores of these skinned cells could be filled by exposing these cells to a solution containing 0.6 microM Ca2+. After a wash in a Ca2+-free solution, a contraction due to a release of the accumulated Ca2+ could be induced by either 25 mM caffeine or 20 microM inositol 1,4,5-trisphosphate (InsP3) or 10 microM A23187. These contractions did not occur if 10 microM ryanodine was present during Ca2+ loading. The addition of ryanodine during the Ca2+-free wash did not affect the subsequent force development. These observations indicate that ryanodine, in the presence of Ca2+, depletes the intracellular Ca2+ stores, and that this depletion is responsible for the inhibition of the component of the NA-induced contraction which depends on the release of Ca2+ from intracellular stores.

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase.

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells.

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.

Origin of intracellular calcium and quantitation of mobilizable calcium in neutrophils stimulated with chemotactic peptide

The origin and amount of mobilized Ca 2+ in chemotactic peptide-stimulated guinea pig neutrophils were examined using biochemical techniques. The total amount of releasable Ca 2+ by 20 μM A23187 from the unstimulated intact cells was 0.91 nmol 4·10 6 cells , as assessed by change in absorbance of the antipyrylazo III-Ca 2+ complex. Two types of internal vesicular Ca 2+ pool, mitochondrial and non-mitochondrial pool were identified in the saponin-permeabilized cells. The total amount of releasable Ca 2+ was comparable to that accumulated by the non-mitochondrial pool at (1−2)·10 −7 M of a free Ca 2+ concentration. The mitochondrial uncoupler, capable of releasing Ca 2+ from the mitochondrial pool, neither modified the basal cytosolic free Ca 2+ in quin 2-loaded cells nor caused a Ca 2+ efflux from the intact cells. These results suggest that the releasable Ca 2+ may be located in the non-mitochondrial pool of unstimulated intact cells, and the mitochondrial pool contains little releasable Ca 2+. The addition of fMet-Leu-Phe increased the cytosolic free Ca 2+ by two processes: Ca 2+ mobilization from internal stores and Ca 2+ influx through the surface membrane. The Ca 2+ mobilized and effluxed from the intact cells by stimulation with the maximal doses of fMet-Leu-Phe was estimated to be 0.27 nmol 4·10 6 cells . Almost equal amounts were released by the maximal doses of inositol 1,4,5-trisphosphate from the non-mitochondrial pool of saponin-treated cells that had accumulated Ca 2+ at a free Ca 2+ concentration of 1.4·10 −7 M. The mechanism related to the Ca 2+ influx by fMet-Leu-Phe stimulation was also examined. The addition of nifedipine or phosphatidic acid did not affect the change in the cytosolic free Ca 2+ induced by fMet-Leu-Phe, thereby suggesting that the receptor-mediated Ca 2+ channel may be involved in the Ca 2+ influx.

Transmembrane topology and subcellular distribution of the benzodiazepine receptor

The distribution and transmembrane topology of benzodiazepine receptors were investigated in situ using intact and saponin-treated neurons of brain cell cultures. Reversible ligand binding and photoaffinity labeling, using 3H-flunitrazepam as a probe, were employed in conjunction with trypsin-induced exhaustive proteolysis and competition binding using a novel benzodiazepine (Ro7-0213) that contains a quaternary ammonium moiety bearing a full positive charge. About 80% of the benzodiazepine receptors, with apparent subunit molecular weights of 48,000 and 51,000, are located in the surface membrane and contain one or more trypsin-sensitive sites exposed at the extracellular surface. The ligand recognition sites of the surface receptors are orientated toward the extracellular space and are sensitive to both competition by Ro7-0213 and trypsin attack. Approximately 20% of the receptors are intracellular or sequestered and are insensitive to both trypsin and competition by Ro7-0213 in intact cells. Strikingly, all of the 3H-flunitrazepam photolabeled sites inaccessible to competition by Ro7-0213 are also resistant to extracellular trypsin, but are sensitive to trypsin and Ro7-0213 in saponin-treated cells. This result provides strong evidence for protected receptors. Surface receptors are differentially sensitive to trypsin-induced inactivation such that 57% (of total) are inactivated, while 24% (of total) are cleaved but, surprisingly, retain the capacity to bind 3H-flunitrazepam. The observation that the ability of the receptor fragment to bind ligand is unaltered demonstrates that the integrity of the benzodiazepine binding site is not contingent upon the presence of about half of the intact polypeptide chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine secretion from permeabilized mast cells by calcium

A transient increase in the permeability of the mast cell membrane was caused by the exposure of the cells to low concentrations of saponin, 5 or 10 μg/ml. These concentrations had very little effect in the absence of calcium but caused 35 to 50% histamine release, having the character of a secretory response, when 0.25 mM or more calcium was added to the medium. The dose-response curve was steep between 25 μM and 250 μM calcium and tended to flatten with higher concentrations. The release was associated with a pronounced increase in calcium uptake, which was faster than the histamine release. The membrane changes were slight as indicated by only 7 to 12% leakage of lactate dehydrogenase and by the absence of any detectable change in the electron micrographs. The transient nature of the membrane change is shown by the following experiment. When the cells were first exposed to saponin in the absence of calcium, the amount of histamine released by the subsequent incubation with calcium varied inversely with the time interval that elapsed before calcium was added. If calcium was added after 15 minutes no histamine release occured. When calcium uptake was studied in the same manner, the stimulation of calcium uptake in saponin-treated cells also declined progressively with increasing intervals after the exposure to saponin when calcium was added. Stimulation of both histamine release and calcium uptake was inhibited by antimycin A, the inhibition curves with 10 −9M to 10 −7M antimycin A being similar. The effect on the calcium uptake by itself could explain the inhibition of histamine release. But the release was also inhibited by the calmodulin antagonists, W-7 and mepacrine, suggesting that the influx of calcium in the permeabilized cells acts primarily through calmodulin-mediated enzyme activation.

Inositol 1,4,5-trisphosphate releases Ca2+from a nonmitochondrial store site in permeabilized rat cortical kidney cells

We have recently shown that inositol 1,4,5-trisphosphate (IP3) releases Ca2+ from the endoplasmic reticulum of pancreatic acinar cells and suggested that IP3 may function as a second messenger of hormonal receptors to mobilize Ca2+ from intracellular stores (Streb et al, 1983, Streb et al, 1984). In rat kidney cortical tubules and microdissected mouse proximal tubules, an increased turnover of polyphosphoinositide metabolism following hormonal stimulation with angiotensin II-amide and phenylephrine has been reported (Wirthensohn et al, 1984; Wirthensohn et al, 1985). This suggests that IP3, one of their hydrolysis products, increases during hormonal stimulation. We therefore investigated the effect of angiotensin II-amide and IP3 on intracellular Ca2 stores in saponin-treated cells and homogenate from rat kidney cortex. Saponin-treated isolated cortical kidney cells or homogenate was incubated in a high K+ buffer in the presence of MgATP and respiratory substrates. Ca2+ uptake was determined by measuring the free Ca2+ concentration of the surrounding medium with a Ca2+ specific macroelectrode. Addition of cells or homogenate to the incubation medium resulted in a decrease of the medium free Ca2+ concentration until a steady-state concentration of 5.7 +/- 0.2 X 10(-7) mole/l was obtained. In the presence of mitochondrial inhibitors Ca2+ uptake rate was reduced, whereas the steady-state concentration was unchanged. In contrast, in the presence of the CA2+-ATPase inhibitor vanadate mitochondrial uptake proceeded at the same rate as the control, but the steady-state concentration was higher (6.9 +/- 0.2 X 10(-7) mole/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells.

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.

Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery.

To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.

Calcium mobilization in enzymically isolated single intact and skinned muscle cells of the porcine coronary artery.

The mobilization of 45Ca2+ was investigated in collagenase-treated single smooth muscle cells of the porcine coronary artery. After removal of extracellular 45Ca2+ by 10 mM-EGTA at 0 degree C, the content of exchangeable Ca2+ was estimated to be 0.42 +/- 0.02 nmol/2 X 10(5) cells at rest and 0.62 +/- 0.03 nmol/2 X 10(5) cells in 102.5 mM-external K solution. The efflux of 45Ca2+ into Ca2+-free solution, estimated from the 45Ca2+ remaining in the cells, increased temperature dependently and was reduced by oligomycin. The muscle cells at rest had a substantial amount of stored Ca2+ which was releasable by caffeine or acetylcholine. Saponin-treated (skinned) muscle cells accumulated 45Ca2+ in the presence of Mg ATP. Two mechanisms of ATP-dependent Ca2+ sequestration were observed: one exhibited a low affinity for Ca2+ but a high-capacity uptake which was sensitive to sodium azide; this was thought to be located in the mitochondria. The other had a high-affinity (1.5/microM) and low-capacity uptake (0.92 nmol/2 X 10(5) cells), which was insensitive to sodium azide, potentiated by oxalate and was thought to be mainly mediated via the sarcoplasmic reticulum (s.r.). The minimum concentration of free Ca2+ required for the ATP-dependent Ca2+ uptake in the saponin-treated cells was about 20 nM by the s.r. and 1 microM by the mitochondria. Thus, the mitochondria seem to play a minor role in regulating cytoplasmic Ca2+ during the contraction-relaxation cycle. These results indicate that enzymically isolated muscle cells are functionally intact, and may facilitate direct measurement of Ca2+ movements when attempting to estimate the physiological role of Ca2+ in vascular smooth muscles.

A Pool of Bullous Pemphigoid Antigen(s) Is Intracellular and Associated with the Basal Cell Cytoskeleton-Hemidesmosome Complex

Bullous pemphigoid (BP) antibodies are known to react with an antigen of the basement membrane zone (BMZ) of squamous epithelia and produce, by the indirect immunofluorescence technique, linear fluorescence at the BMZ. Direct and indirect immunoelectron microscopy (IEM) have demonstrated BP antigen to be within the lamina lucida, in close association with the basal cell membrane. Trypsin-dissociated epidermal basal cells bind BP antibodies in a polar distribution, presumably because the BP antigen is restricted to the dermal pole of the basal cell membrane. In this study we have utilized newborn BALB/c mouse skin to obtain both dissociated basal cells (by trypsinization) and epidermal sheets (by dithiothreitol treatment). We show that viable basal cells, which are impermeable to IgG molecules, do not react with BP antibodies. When the basal cell plasma membrane is disrupted by cytospin centrifugation, air drying, freezing and thawing, or hypotonic lysis, or permeated by nonionic detergents (saponin), cells become reactive with BP antibodies. Basal cell cytoskeletons, prepared by sequential treatment with Triton X-100, deoxyribonuclease, and 2 M NaCl continue to react with BP antibodies. Similarly, viable epidermal sheets fail to bind BP antibodies. When epidermal sheets are treated with nonionic detergents, water, or freezing and thawing prior to incubation with BP antibodies, linear BMZ fluorescence is observed. IEM study of saponin-treated basal cells shows the immunoreactants to be localized on intracytoplasmic vacuoles which represent internalized hemidesmosomes. IEM of permeated epidermal sheets shows the immunoreactants as aggregates on the inner surface of the dermal pole of the basal cell membrane. These observations suggest that the BP antigen is intracellular and is in close association with the basal cell cytoskeleton and hemidesmosomes.

Calcium uptake into acini from rat pancreas: Evidence for intracellular ATP-dependent calcium sequestration

Intracellular ATP-dependent Ca2+-sequestration mechanisms were studied in isolated dispersed rat pancreatic acini following treatment with saponin or digitonin to disrupt their plasma membranes. In the presence of 45Ca2+ concentrations less than 10(-6) mol/liter, addition of 5 mmol/liter ATP caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. ADP mimicked the ATP effect by 50 to 60%, whereas other nucleotides such as AMP-PNP, AMP-PCP, CTP, UTP, ITP, GTP, cAMP and cGMP did not. Maximal ATP-promoted Ca2+ uptake was obtained at 10(-5) mol/liter Ca2+. Inhibition of Ca2+ uptake by mitochondrial inhibitors was dependent on the Ca2+ concentration, indicating the presence of different Ca2+ storage systems. Whereas the apparent half-saturation constant found for mitochondrial Ca2+ uptake was approximately 4.5 X 10(-7) mol/liter, in the presence of antimycin and oligomycin (nonmitochondrial uptake) it was approximately 1.4 X 10(-8) mol/liter. In the absence of Mg2+ both ATP- and ADP-promoted Ca2+ uptake was nearly abolished. The Ca2+ ionophore and mersalyl blocked Ca2+ uptake, Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP, which were absent in intact cells and in saponin-cells without ATP or pretreated with A23187. The data suggest the presence of mitochondrial and nonmitochondrial ATP-dependent C2+ storage systems in pancreatic acini. The latter is likely to be located in the rough endoplasmic reticulum.

Analysis of Ca2+ fluxes and Ca2+ pools in pancreatic acini.

45Ca2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45Ca2+. Carbamyl choline (carbachol; Cch) caused a quick 45Ca2+ release that was followed by a slower 45Ca2+ 'reuptake'. Subsequent addition of atropine resulted in a further transient increase in cellular 45Ca2+. The data suggest the presence of a Cch-sensitive 'trigger' pool, which could be refilled by the antagonist, and one or more intracellular 'storage' pools. Intracellular Ca2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45Ca2+ (1 microM), addition of ATP at 5 mM caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca2+ uptake was obtained at 10 microM Ca2+ (half-maximal at 0.32 microM Ca2+). In the presence of mitochondrial inhibitors it was 0.1 microM (half-maximal at 0.014 microM). 45Ca2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45Ca2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive 'trigger' Ca2+ pool and ATP-dependent Ca2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca2+ is taken up into these pools after secretagogue-induced Ca2+ release.U

Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The saponin-treated permeable cell system will serve as a useful system for studying in vitro replication of DNA and chromatin in eukaryotic cells.

The expression of optimum ATPase activities in human erythrocytes: A comparison of different lytic procedures

The exposure of the Na +/K +/Mg 2+- and Ca 2+/Mg 2+-stimulated ATPase activities in human erythrocytes through the use of several different lytic procedures revealed significant variations in the level of activity. Density (age)-separated as well as mixed-age human erythrocytes were subjected to hemolysis in isotonic buffer using saponin or ethylene glycol, to hemolysis in hypotonie buffer using low osmolarity buffers, or to freeze-thaw to allow potential accessibility to the ATPases. The results ranged from maximum exposure of both types of ATPases in saponin-treated cells, to little or no exposure of activity in ethylene glycol-treated cells, to variable responses in membranes derived by hypotonie hemolysis. The inability to elicit maximum exposure of ATPases in young cells by the freeze-thaw treatment was reversed by the use of saponin lysis in isotonic medium. These results illustrate the importance of the lytic conditions of membrane preparations on the recovery of as well as exposure to ATPase activities. It is concluded that saponin lysis in isotonic buffer medium is the preferred lytic technique for preparation of membranes retaining significant levels of the Na +/K +/Mg 2+- and Ca 2+/Mg 2+-stimulated ATPases. These data are also discussed in reference to the degree of retention of the activator protein for the Ca 2+ Mg 2+ ATPase system.