Sven Mårdh

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro.

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.

Phosphorylation and dephosphorylation reactions of bovine brain (Na+K+)-stimulated ATP phosphohydrolase studied by a rapid-mixing technique

Abstract 1. 1. In order to allow a study of K + -dependent dephosphorylation of (Na + K + )-stimulated ATP phosphohydrolase (EC 3.6.1.3, (Na + K + )-stimulated ATPase), a rapid-mixing apparatus with two consecutive mixing chambers and four syringes was developed. The shortest incubation time was about 1.5 ms, excluding the quenching time which was about 1 ms. 2. 2. The pseudo first-order rate constant of the Na + -dependent phosphorylation was estimated to be about 11 000 min −1 at 21 °C in the presence of 3 mM MgCl 2 , 120 mM NaCl and 100 μM [γ- 32 P]ATP. This rate seemed to have reached a maximum at this concentration of [γ- 32 P]ATP. The same seemed to hold for the amount of 32 P-labelled enzyme. Since this concentration of [γ- 32 P]ATP only gives half maximal rate of the (Na + K + )-stimulated hydrolysis of ATP the present data suggest a complex role of ATP, which is consistent with reports by others. 3. 3. When studying the disappearance of protein-bound acyl-[ 32 P]phosphate upon the addition of 10 mM KCl, an apparent first-order rate constant of at least 14 000 min −1 was obtained at 21 °C. Within the time resolution of the rapid-mixing apparatus this rate constant seemed to be the same irrespective of which concentration of ATP was used in the range of 5–100 μM. 4. 4. Since the steady-state concentration of the phosphorylatable enzyme form was unknown, the steady-state velocity of the phosphorylation could not be estimated. The concentration of 32 P-labelled enzyme at steady-state was about 0.2 nmole·mg −1 of protein. If it is assumed that all of this phosphoenzyme is dephosphorylated in one step, characterized by a first-order rate constant of at least 14 000 min −1 , the calculated rate of dephosphorylation at steady-state is at least 2800 nmoles·mg −1 ·min −1 . However, the steady-state rate of (Na + K + )-stimulated hydrolysis of ATP at 21 °C was only about 280 nmoles·mg −1 ·min −1 in the presence of 3 mM MgCl 2 , 120 mM NaCl and 10 mM KCl at 100 μM ATP. The consistency of these results with the current hypothesis on two different forms of the phosphorylated intermediate is discussed.

Two-dimensional crystals of membrane-bound gastric H,K-ATPase

H,K‐ATPase protomer. Through stain‐deficient contact regions four adjacent protein units were connected forming a tetrameric structure.

Characterization of proton-transporting membranes from resting pig gastric mucosa.

Membrane vesicles were purified from resting corpus mucosa of pig stomachs by velocity-sedimentation on a sucrose-Ficoll step gradient. Two vesicular fractions containing the (H+ + K+)-ATPase were obtained. One fraction was tight towards KCl, the other was leaky. At 21 degrees C maximal (H+ + K+)-ATPase activities of 0.8 and 0.4 mumol X mg-1 X min-1, respectively, were observed in lyophilized vesicles. The vesicles contained a membrane-associated carbonic anhydrase, the activity of which was in 100-fold excess of the maximal ATPase activity. Both vesicular fractions were rich in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The characteristics of ion permeability and transport in the tight vesicles were in agreement with corresponding data for vesicles of a tubulovesicular origin in the parietal cell. Measurement of the rate of K+ uptake into the vesicles was based on the ability of K+ to promote H+ transport. The uptake was slow and dependent on the type of anion present. The effectiveness in promoting uptake of K+ by anions was SCN- greater than NO3- greater than Cl- much greater than HCO3- greater than SO4(2-). Uptake of K+ was much more rapid at alkaline pH than at neutral or at acidic pH. Addition of CO2 at alkaline pH strongly stimulated the rate of H+ accumulation in the vesicles. The initial part of this stimulation was sensitive to acetazolamide, an inhibitor of carbonic anhydrase. A model how the (H+ + K+)-ATPase and the carbonic anhydrase may co-operate is presented. It is concluded that membrane vesicles of a tubulovesicular origin can produce acid.

Isolation, characterization and adhesion of calcium-tolerant myocytes from the adult rat heart

High yields of Ca2+ - stable myocytes were obtained by perfusion of adult rat heart with a buffered collagenase medium followed by mincing and three additional digestion periods. Release of lactate dehydrogenase, respiratory control, content of ATP and creatine phosphate, electrical stimulation and attachment to extracellular matrix components indicated that the sarcolemma of the isolated myocytes remained intact and that the cells maintained some of the most basic physiological functions. The myocytes maintained their rod-shape in a medium containing 2.5 mM of Ca2+ and their release of LDH was slow. Some of the myocytes were contracting spontaneously, at a low rate, in an abrupt end-to-end contraction. Other cells appeared quiescent but they were all able to respond to external electrical stimulus. The oxygen consumption was measured by a perifusion method. In different preparations the basal consumption was 14-26 nmol O2/min X 10(5) rod-shaped myocytes. Freshly isolated rod-shaped heart cells attached in 30 minutes to dishes coated with collagen type IV, laminin or fibronectin but did not attach to dishes coated with collagen type I or III or to collagen gels. Attachment occurred at the ends of the cells.

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.

Phosphorylation of bovine brain Na+, K+-stimulated ATP phosphohydrolase by adenosine [32P]triphosphate studied by a rapid-mixing technique

Abstract 1. 1. A bovine brain Na + , K + -stimulated ATPase preparation was incubated with [γ- 32 P]ATP by use of a rapid-mixing technique, the reaction time being 8–40 msec. The phosphorylated material was analysed for 32 P-labelled acyl-phosphate, phosphoserine, phosphothreonine and phospholipids. 2. 2. With 5 μM [γ- 32 P]ATP, 3 mM MgCl 2 and 120 mM NaCl at pH 7.4 and 21° the acyl-[ 32 P]phosphate appeared at a rate of 0.55 μmole/mg protein per min. At the same conditions the enzyme preparation hydrolysed 0.06 μmole of ATP/mg protein per min. This was the rate of ATP hydrolysis irrespective of whether or not 20 mM KCl was present. After 40 msec the acyl-[ 32 P] phosphate comprised more than 99 % of the [ 32 P]phosphate of the high-molecular-weight material. 3. 3. It is concluded that the [ 32 P]phosphorylenzyme, having a similar pH stability to acyl-[ 32 P]phosphate, is formed rapidly enough to be an intermediate of the Na + , K + -dependent ATPase reaction.

Effects of gastrin on cytosolic free Ca2+ in individual, acid-secreting rat parietal cells

The effects of gastrin on cytosolic free Ca2+ ([Ca2+]i) in single, isolated rat gastric parietal cells were investigated using the fluorescent probe Fura-2 and digital image analysis. [Ca2+]i was increased by gastrin (100 nM) in approximately 30% of the parietal cells, which were identified by using either the fluorescent probe acridine orange or a parietal cell-specific monoclonal antibody. In the dominant pattern observed, [Ca2+]i was elevated 50-150% and returned within 1-2 min to a value 30-60% over the basal, which was sustained until withdrawal of the stimulant or addition of the gastrin inhibitor L-365,260 (1 microM). The second, but not the first phase, was abolished in the absence of extracellular Ca2+. The results indicate the existence of functional gastrin receptors in a subpopulation of rat parietal cells.

A method for measuring the Čerenkov radiation produced by 32P in small sample volumes

Abstract A method for analysing 32P in small aqueous samples by measuring the Cerenkov radiation is described. Centering problems with small sample volumes are eliminated by placing the sample in a polystyrene tube in the scintillation vial. The detection efficiency is high, 54.5 ± 0.6% (2 SD) at a sample volume of 25 μl. The reproducibility is good and independent of the sample volume. The detection efficiency of 32P in polyacrylamide gel is shown to be as good as in water.