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Na(+)- Ca(2+) exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca(2+) pool along with the SER Ca(2+) pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca(2+) depletion on NCX-SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na(+)-loaded and then placed in either a Na(+)-containing or in a Na(+)-substituted solution. Subsequently, the difference in Ca(2+) entry between the two groups was examined and defined as the NCX mediated Ca(2+) entry. The NCX mediated Ca(2+) entry in the smooth muscle cells was monitored using two methods: Ca(2+)sensitive fluorescence dye Fluo-4 and radioactive Ca(2+). Ca(2+)-entry was greater in the Na(+)-substituted cells than in the Na(+)-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca(2+) entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na(+)-substituted solution with or without thapsigargin. SER Ca(2+) depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca(2+) entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca(2+) entry may protect the cells against Ca(2+)-overload during ischemia-reperfusion when SERCA2 is known to be damaged., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pig coronary artery smooth muscle expresses, among many other proteins, Na+-Ca²+-exchanger NCX1 and sarcoplasmic reticulum Ca²+ pump SERCA2. NCX1 has been proposed to play a role in refilling the sarcoplasmic reticulum Ca²+ pool suggesting a functional linkage between the two proteins. We hypothesized that this functional linkage may require close apposition of SERCA2 and NCX1 involving regions of plasma membrane like lipid rafts. Lipid rafts are specialized membrane microdomains that appear as platforms to co-localize proteins. To determine the distribution of NCX1, SERCA2 and lipid rafts, we isolated microsomes from the smooth muscle tissue, treated them with non-ionic detergent and obtained fractions of different densities by sucrose density gradient centrifugal flotation. We examined the distribution of NCX1; SERCA2; non-lipid raft plasma membrane marker transferrin receptor protein; lipid raft markers caveolin-1, flotillin-2, prion protein, GM1-gangliosides and cholesterol; and cytoskeletal markers clathrin, actin and myosin. Distribution of markers identified two subsets of lipid rafts that differ in their components. One subset is rich in caveolin-1 and flotillin-2 and the other in GM1-gangliosides, prion protein and cholesterol. NCX1 distribution correlated strongly with SERCA2, caveolin-1 and flotillin-2, less strongly with the other membrane markers and negatively with the cytoskeletal markers. These experiments were repeated with a non-detergent method of treating microsomes with sonication at high pH and similar results were obtained. These observations are consistent with the observed functional linkage between NCX1 and SERCA2 and suggest a role for NCX1 in supplying Ca²+ for refilling the sarcoplasmic reticulum., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Smooth muscle and several non-muscle tissues contain mRNA for an alternative splice of the mRNA for the cardiac sarcoplasmic reticulum (SR) Ca,Mg-ATPase. Based on amino acid composition deduced from cDNA sequences the cardiac isoform (Ic) is 110 kDa while the smooth muscle and the non-muscle isoform (Is) is 115 kDa. This prediction in their molecular masses was tested at the protein level in rabbit stomach, aorta, uterus and vas deferens smooth muscles; stomach mucosa, brain, liver, kidney and heart. The major species of the acylphosphates formed in the presence of Ca2+ and electrophoresed in acid SDS-acrylamide gels were 5 kDa smaller for the heart (Ic) than those for all the other tissues (Is). The size difference was also confirmed in Western blots using a monoclonal antibody which binds to both Is and Ic. Thus consistent with the mRNA splices for the internal Ca2+ pumps previously reported to be present in these tissues, rabbit heart expresses predominantly the Ca2+ pump protein Ic and the various smooth muscle, mucosa, brain, liver and kidney express mainly the isoform Is.

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester (I) and 1 : 14 ester (II). The nitroxide label of I is aligned in the membrane with a very large angle of tilt (47 degrees +/- 1.5 degrees) with respect to the normal to the membrane surface; II does not show such a tilt. I gives spectra corresponding to immobilized label while II gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in I and II are not present at the phosphatidylcholine-water interphase. The data supports a 'horseshoe' configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.

A beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme has been isolated from almond emulsin. The isolated enzyme is a glycoprotein and migrates as a single band on Sephadex G-200 filtration, CM 52 ion exchange chromatography, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focussing. The glucosidase and galactosidase activities traverse together during Sephadex G-200 gel filtration. Polyacrylamide gels stained specifically for the 2 enzymes reveal that the two activities comigrate. The molecular weight of the isozyme has been found to be 135 180 +/- 770, and that of its protomers to be 65 150 +/- 650.

A purified beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme isolated from almond emulsin was found to catalyze hydrolysis of beta-D-glucopyranosides and beta-D-galactopyranosides but not the corresponding alpha-D-derivatives. Hydrolysis of the corresponding beta-D-thioglycopyranosides at rates 10(3)--10(4) times lower than those for the hydrolysis of the beta-D-glycopyranosides was also noted. The enzyme does not exhibit any transferolytic activity using D-glucose or D-galactose as acceptors. D-glucose, p-nitrothiophenyl-beta-D-glucopyranoside, 5-deoxy-5-thio-D-glucose and D-glucono-delta-lactone are shown to exert mainly competitive inhibition on beta-D-galactopyranoside hydrolysis. D-galactose, p-nitrothiophenyl-beta-D-galactopyranside and methylthio-beta-D-galactopyranoside are shown to inhibit the glucopyranoside hydrolysis mainly non-competitively and to exert competitive inhibition of galactopyranoside hydrolysis. The inhibition caused by the antibiotic Nojirimycin (5-amino-5-deoxy-D-glucose) is shown to be more complex. Analysis of the kinetic data indicates that the catalytic site of the enzyme responsible for the beta-D-glucosidase activity is kinetically distinct from the beta-D-galactosidase site.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.