Free Phosphate-buffered Saline Research Articles

Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

Cellular Uptake of Mammalian Heparanase Precursor Involves Low Density Lipoprotein Receptor-related Proteins, Mannose 6-Phosphate Receptors, and Heparan Sulfate Proteoglycans

Mammalian heparanase, strongly implicated in the regulation of cell growth, migration, and differentiation, plays a crucial role in inflammation, angiogenesis, and metastasis. There is thus a clear need for understanding how heparanase activity is regulated. Cells can generate an active form of the enzyme from a larger inactive precursor protein by a process of secretion-recapture, internalization, and proteolytic processing in late endosomes/lysosomes. Cell surface heparan sulfate proteoglycans are the sole known components with a role in this trafficking of the heparanase precursor. Here, we provide evidence that heparan sulfate proteoglycans are not strictly required for this process. More importantly, by heparanase transfection, binding, and uptake experiments and by using a combination of specific inhibitors and receptor-defective cells, we have identified low density lipoprotein receptor-related proteins and mannose 6-phosphate receptors as key elements of the receptor system that mediates the capture of secreted heparanase precursor and its trafficking to the intracellular site of processing/activation.

Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.

Endoglin Null Endothelial Cells Proliferate Faster and Are More Responsive to Transforming Growth Factor β1 with Higher Affinity Receptors and an Activated Alk1 Pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

A Secreted Low--Molecular-Weight Protein From Helicobacter pylori Induces Cell-Cycle Arrest of T Cells

Although Helicobacter pylori is recognized by the human immune system, the bacteria are not eliminated and lead to a chronic inflammation of the gastric mucosa. We investigated the interaction of H. pylori with human lymphocytes. T and B lymphocytes were isolated from H. pylori-infected patients and stimulated with anti-CD3/CD28 or interleukin-6. Proliferation of lymphocytes was abolished on co-incubation with different H. pylori strains (1-5 bacteria/cell) or with protein extracts of culture supernatants. Inhibition of proliferation was independent of known virulence factors. The factor is a protein or protein complex with an apparent molecular weight between 30 and 60 kilodaltons, clearly distinct from VacA. Although antigen-specific activation of T cells (as shown by nuclear factor of activated T cells [NFAT]-activation, interferon-gamma production, and CD25 or CD69 up-regulation) remained intact, cell-cycle analysis showed that S-phase entry of T cells was inhibited completely by H. pylori. Consequently, stimulated T cells arrested in the G1 phase of the cell cycle. Western blot analysis showed markedly reduced phosphorylation of the retinoblastoma protein (pRb), suggesting inhibition of G1 cyclin-dependent kinase activity. In line with this, activities of cyclin D3 and cyclin E were down-regulated, and levels of the cyclin-dependent kinase inhibitor p27Kip1 were increased. Mouse embryonic fibroblasts deficient in p27 showed a decrease in H. pylori-induced inhibition of cell proliferation, suggesting a central role for p27 in mediating H. pylori-induced G1 arrest. Induction of cell-cycle arrest in lymphocytes may be of major significance for the chronic persistence of bacteria in the human stomach.

Genetic Interactions between BLM and DNA Ligase IV in Human Cells

BLM has been implicated in DNA double-strand break (DSB) repair, but its precise role remains obscure. To explore this, we generated BLM(-/-) and BLM(-/-)LIG4(-/-) cells from the human pre-B cell line Nalm-6. BLM(-/-) cells exhibited retarded growth, increased mutation rates, and hypersensitivity to agents that block replication fork progression. Interestingly, these phenotypes were significantly suppressed by deletion of LIG4, suggesting that nonhomologous end-joining (NHEJ) is unfavorable for integrity and survival of cells lacking BLM. We propose that the absence of BLM leads to accumulation of replication-associated, one-ended DSBs, which are deleterious to cells and lead to genomic instability when repaired by NHEJ. In addition, the NHEJ pathway per se was marginally affected by BLM deficiency, as evidenced by x-ray sensitivity and I-SceI-based DSB repair assays. More intriguingly, however, these experiments revealed the presence of an alternative, DNA ligase IV-independent end-joining pathway, which was significantly affected by the loss of BLM. Collectively, our results provide the first evidence for genetic interactions between BLM and NHEJ in human cells.

Modulation of Mouse Paneth Cell α-Defensin Secretion by mIKCa1, a Ca2+-activated, Intermediate Conductance Potassium Channel

Paneth cells in small intestinal crypts secrete microbicidal alpha-defensins in response to bacteria and bacterial antigens (Ayabe, T., Satchell, D. P., Wilson, C. L., Parks, W. C., Selsted, M. E., and Ouellette, A. J. (2000) Nat. Immunol. 1, 113- 38). We now report that the Ca(2+)-activated K(+) channel mIKCa1 modulates mouse Paneth cell secretion. mIKCa1 cDNA clones identified in a mouse small intestinal crypt library by hybridization to human IKCa1 cDNA probes were isolated, and DNA sequence analysis showed that they were identical to mIKCa1 cDNAs isolated from erythroid cells and liver. The genomic organization was found to be conserved between mouse and human IKCa1 as shown by comparisons of the respective cDNA and genomic sequences. Reverse transcriptase-PCR experiments using nested primers amplified mIKCa1 from the lower half of bisected crypts and from single Paneth cells, but not from the upper half of bisected crypts, villus epithelium, or undifferentiated crypt epithelial cells, suggesting a lineage-specific role for mIKCa1 in mouse small bowel epithelium. The cloned mIKCa1 channel was calcium-activated and was blocked by ten structurally diverse peptide and nonpeptide inhibitors with potencies spanning 9 orders of magnitude and indistinguishable from that of the human homologue. Consistent with channel blockade, charybdotoxin, clotrimazole, and the highly selective IKCa1 inhibitors, TRAM-34 and TRAM-39, inhibited (approximately 50%) Paneth cell secretion stimulated by bacteria or bacterial lipopolysaccharide, measured both as bactericidal activity and secreted cryptdin protein, but the inactive analog, TRAM-7, did not block secretion. These results demonstrate that mIKCa1 is modulator of Paneth cell alpha-defensin secretion and disclose an involvement in mucosal defense of the intestinal epithelium against ingested bacterial pathogens.

Cryopreservation extenders affect calcium flux in bovine spermatozoa during a temperature challenge.

Bovine spermatozoa are commercially cryopreserved by diluting the cells in media, known as extenders, followed by slow cooling and freezing. Previous work has shown that this process of cryopreservation alters the cells' ability to control divalent calcium (Ca2+) movement. This study evaluated the effect of a brief exposure to common extenders on bovine spermatozoa during subsequent cooling and rewarming. Three fresh ejaculates from each of three bulls were each split and incubated for 30 minutes at 25 degrees C in milk extender or phosphate-buffered saline (PBS) (control); three other fresh ejaculates from each of three bulls were similarly incubated in egg yolk-Tris extender (EYT) or PBS. Spermatozoa were washed and the fluorescent Ca2+ indicator, indo-1 acetoxymethyl ester, was used to monitor the internal Ca2+ in the spermatozoa in Ca(2+)-free PBS over a continuous temperature gradient of 25 degrees C (15 minutes), cooling to 5 degrees C (32 minutes), at 5 degrees C (15 minutes), rewarming to 25 degrees C (25 minutes), and at 25 degrees C (15 minutes). Milk exposure reduced the initial percentage of missing acrosomes and EYT exposure improved the initial viability and acrosome morphology compared to the controls; only milk immediatetly increased internal Ca2+. The initial rate of Ca2+ uptake at 25 degrees C was greater for milk or EYT-exposed spermatozoa than controls (P < 0.05). During cooling, the rate of Ca2+ uptake in all spermatozoa increased (P < 0.01), and it continued to increase during the 15 minutes at 5 degrees C. During rewarming to 25 degrees C, the internal Ca2+ in all spermatozoa declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.

Decompaction and biopsy of late mouse morulae: assessment of in-vitro and in-vivo developmental potential

The present study reports a biopsy technique on decompacted late mouse morulae. Zygotes were collected from hyperstimulated F1 hybrids (C57BL6j females x CBAca males) and cultured in modified Earle's balanced salt solution supplemented with 0.5% bovine serum albumin until the late morula stage (92 h post administration of human chorionic gonadotrophin). Decompaction was obtained by exposure of the embryos to Ca(2+)-Mg(2+)-free phosphate-buffered saline (PBS) or an aqueous solution of ethylene-diaminetetraacetic acid (EDTA) and glycine. Using micromanipulation, a single blastomere was aspirated without removal or softening of the zona. The impact of the different decompaction procedures and the biopsy technique was studied by vital staining, by in-vitro culture up to the early egg-cylinder staged and by the recovery of living mice after transfer to pseudopregnant foster mothers. Our investigations revealed no impact of decompaction and biopsy on immediate viability, as assessed by fluorescein diacetate staining. A significant reduction (P less than 0.05) in the number of mouse morulae that reached the early egg-cylinder stage in vitro was observed after the biopsy procedure. We observed that after this microbiopsy technique, successful pregnancies can be obtained but at a lower percentage compared to controls (P less than 0.01).

Dipeptidyl peptidase IV in human lymphocytes: molecular properties

Dipcptidyl peptidase IV (DPP IV; EC 3.4.14.5). an cctocnzyme o n the cell membrane of T-lymphocytes, has bccn shown t o play an important role in the regulation of lymphocyte proliferation and activation. Evidence for this has been provided by the use of specific inhibitors, which revealed (a) a supprcssion of the proliferation of peripheral blood lymphocytes by mitogenic lectins 111, and (b) a decrease in the production of certain lymphokines, namely interleukin 2 and y-intcrferon [2]. In addition, we have reported a rise in the specific activity of DPP IV in lymphocytes stimulated by phytohacmaglutinin (31. Despite the increasing evidence sugesting that DPP IV, like other cellular proteinases, is involved in lymphocytic activation, its exact role remains unknown. Recently, four monoclonal antibodies (TI1 I Y-4-7, 4ELlC7, B1.10.2 and TS145) were found to form a new scrological cluster, named CDw26. Two of them, TI1 19-4-7 (anti-DPP IV) and 4ELlC7 (anti-Tal), recognize the same o r partly idcntical epitopcs on the antigen DPP 1V 141. The use of thcsc monoclonal antibodies will facilitate new invcstigations concerning DPP IV on cell surfaces. In this study we rcvcal some of the biochemical and molecular properties of lymphocytic DPP IV. The information obtained will bc of additional aid in further defining the role of DPP IV in the immune system. For the determination of DPP IV activity we used a scnsitivc fluorometric assay based o n hydrolysis of Gly-Pro4-mcthoxy-2-naphthylamide ( 5 1. Lymphocytes were isolated from huffy coats from healthy volunteers. After diluting twice with physiological saline containing 0.3% tri-sodium citratc and subsequent twofold dilution in Plasmastericl (Fresenius, Bad Hamburg, F.R.G.) to eliminate most of the erythrocytes by sedimentation, the resulting supernatant was used to isolate mononuclear blood cells by Lymphoprep (Nycomcd, Oslo, Norway) gradient centrifugation. The mononuclear cells were aspirated and washed twice with Ca? + and Mg? + -free phosphate-buffered saline. After the last centrifugation step, the cell pellets were stored at 70°C. For the solubilization of DPP IV from the cells, differcnt methods were evaluated, namely sonication, freezing and thawing, and treatment with the non-ionic detergents 1-0-tioctylglucopyranoside, Nonidet-P4O (LKB, Bromma, Sweden) and Triton X-100 (Aldrich Chemical Co., Steinheirn, F.R.G.). Detergent treatment resulted in the highest yields of DPP IV activity, the efficacy of the different detergents being almost the same. Because of its compatibility with most techniques, Triton X-100 was chosen in a final concentration of 1% (v/v). In order to enrich DPP IV, we evaluated different chromatographic procedures (all gels used were from Pharmacia Fine Chemicals, Uppsala, Sweden). Since only small amounts of crude material are available, we were interested in highly selective separation techniques like affinity chromatography. For the purification of DPP IV from other sources, Gly-Pro-AH-Sepharose 4B is often used. After the carbodi-imide method reported earlier [6], we coupled GlyPro t o the AH-Sepharose 4B gel. In comparative studies, at different stages of the purification, we could not detect sig-

Regulatory volume increase in mammalian jejunal villus cells is due to bumetanide-sensitive NaKCl2 cotransport

We assessed ion transport mechanisms operative during regulatory volume increase (RVI) in villus enterocytes isolated in suspension from guinea pig jejunum and examined with electronic cell sizing and 86Rb influx. After validation of the electronic-sizing technique with direct measurements of cell water, the response of cell volume to hypertonic media was evaluated in detail. When shrunk by exposure to hyperosmotic media (455 mosmol/kg medium) cells demonstrated a RVI that was complete in 20 min. RVI required extracellular Na+, K+, and Cl-; this cell swelling showed the following ion sensitivity; Na+ greater than Li+ greater than choline, K+ = Rb+, and Cl- greater than or equal to Br- greater than NO3- = acetate = gluconate. Bumetanide inhibition of villus cell swelling was concentration dependent from 10(-10) to 10(-5) M (7.0 +/- 4.5% vs. 87.8 +/- 0.3%); furosemide (10(-3)M) inhibited RVI (74.1 +/- 9.5%), but amiloride (10(-4) M) had little effect on cell swelling. Disulfonic stilbenes, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (10(-4) M), generated the same inhibition of RVI in either nominally HCO3(-)-free phosphate-buffered saline (PBS) or HCO3(-)-buffered PBS, suggesting anion exchange was not involved. Ouabain (10(-4) M) stimulated cell swelling. Hypertonic shrinkage increased the initial rate of bumetanide-sensitive 86Rb influx (80 +/- 38 vs. 1,011 +/- 241 pmol.mg protein-1.min-1; P less than 0.005) and required extracellular Na+ and Cl- (11 +/- 16 vs. 28 +/- 61 pmol.mg protein-1.min-1). RVI was prevented in low-K+ media (0.2 mM), but the addition of KCl initiated cell swelling. Our data strongly suggest that RVI in jejunal villus enterocytes occurs because of the hypertonic activation of NaKCl2 cotransport.

Ethanol-induced alterations in human erythrocyte shape and surface properties: modulatory role of prostaglandin E1.

Exposure of human erythrocytes to ethanol (1 to 20% by vol) in Ca2+ and Mg2+-free phosphate-buffered saline, pH 7.4, transformed biconcave discs into spiculated echinocytes within 3 min at 25 degrees C. The effects of ethanol were concentration-and time-dependent, but reversible by washing in the incubation buffer system within 60 min of initial exposure to ethanol. After prolonged ethanol exposure (180 min), washing of cells resulted in the formation of stomatocytes (cup-forms). Ethanol-induced echinocytosis was also accompanied by a 30% enhancement in the agglutinability of erythrocytes by ligands with high affinity for negative surface charge (poly-L-lysine and wheat germ agglutinin, 20 microliters/ml) without any alterations in surface charge topography. Concomitant exposure of erythrocytes to prostaglandin E1 (100 nM) selectively prevented the enhancement of ligand-mediated agglutinability, but did not modify cell shape. These data indicate that certain erythrocyte surface properties may not be directly influenced by cell shape and suggest a unique modulatory action of prostaglandin E1 on shape-transformed cells.

Effect of permeabilization of tight junctions on morphology of 3-cell intersection

Tight junctions (TJ) at 3-cell intersections (3Cl) appear in freeze-fracture replicas as channels limited by 2 vertical strands in one cell's membrane, each representing the line of fusion with one of the other cells. Recent observations show in mammary cells exposed to a calcium chelator, retraction of microvilli from cell borders, starting at the 3Cl. Ca2+ depletion is also known to reduce transepithelial resistance. These observations led us to inquire whether 3Cl might be the sites most vulnerable to agents that increase junction permeability. We used the rat mammary adenocarcinoma cell line LA7 (from R. Dulbecco) to ask this question.LA7 monolayers were treated with reagents known to affect transepithelial permeability: Ca2− and Mg2+-free phosphate-buffered saline (PBS-CMF), the Ca2+ chelator EGTA (0.025%), colchicine (0.1mM), cytochalasin B (10 μg/ml), procaine (1 - 10 μg/ml), kinetin (0.4 μM), phalloidin (10-50 μM). Confluent LA7 on impermeable substrates form many domes (Fig I) as fluid transported basally is trapped between the monolayer and the plastic. Their presence is proof of TJ integrity and was the criterion used in this study. Functional effects of the treatments were measured by maintenance or change in dome numbers and morphological effects were followed by freezefracturing.

Growth factor requirements of Chinese hamster lung fibroblasts in serum free media: High mitogenic action of thrombin

We have defined a chemical medium to grow an anchorage and highly serum-dependent Chinese Hamster Lung fibroblast cell line (CCl 39) in the absence of serum. This serum-free medium referred to as DM4, contains Transferrin (5 μg/ml), Insulin (10 μg/ml), Epidermal Growth Factor (10 ng/ml) and Thrombin (0.05 U/ml). Thrombin appeared to be a very potent mitogen for Chinese Hamster Lung cells. DM4 allows exponential growth of CCl 39 cells plated in serum-coated dishes without any lag and with a generation time of 15 hours. Continuous subculturing and clonal growth can be obtained by passaging the cells with Ca ++, Mg ++ free phosphate-buffered saline in fibronectin coated dishes. The entire definition of growth factor requirements for this anchorage dependent cell line is of great value to approach the mechanisms of escape of growth control by these cells.

Particle segregation in chromaffin granule membranes by forced physical contact

Bovine chromaffin granules were exposed to different isotonic non-ionic and ionic solutions (sucrose; Ca 2+ - and Mg 2+-free phosphate-buffered saline; Tris-HCl + NaCl ; Ca 2+- and Mg 2+-free phosphate-buffered saline + sucrose; Tris-HCl + sucrose ) at pH 7 and then frozen either in suspension or as firm pellets. Freezing was performed without prefixation or antifreeze treatments either by ‘standard’ techniques (approx. 1 mm 3 suspended or pelleted material on gold specimen supports dipped into liquid Freon) or with increased cooling rates by spraying suspensions into liquid propane (‘spray-freezing’). Regardless of the freezing method, membrane-intercalated particles were always randomly distributed when chromaffin granules were frozen in suspension. In contrast, forced physical contact between granules produced by centrifugation ( 12 000 × g, 25 min ) provoked dispersal of membrane-intercalated particles, but only in the presence of ions. Sucrose or EDTA in an ionic environment had no inhibitory effect. The following conclusions are derived: (1) Even below the reported phase transition region particle clustering is possible. (2) Chromaffin granule membranes are not liable to thermotropic segregation of membrane-intercalated particles. (3) Although the low freezing rates of ‘standard’ freezing techniques produce large-scale segregation artefacts (by which suspended chromaffin granules are pushed together within the segregated solute) this does not result in intramembraneous particle segregation. (4) Forced physical contact produces a Ca 2+-independent particle segregation, but only when repulsive electrostatic forces of membrane components are partially screened in an ionic environment. (5) This does not invalidate results obtained by others, showing Ca 2+-mediated chromaffin granule agglomeration and segregation of membrane-intercalated particles, but it might indicate the occurrence of another, not directly Ca 2+-dependent particle segregation mechanism in a prefusional stage of close membrane-to-membrane contact during exocytosis.

Isolation and characterization of myocytes from the adult rat heart.

Spontaneously contracting myocytes were isolated from ventricles of the adult rat heart. Hearts were perfused retrogradally via the aorta for 30 minutes at 37 degrees C with Ca2+-free phosphate-buffered saline containing collagenase and hyaluronidase. The venticles were divided into pieces and incubated 15 minutes with the enzymes. Dislodged cells were decanted, diluted with cold buffer and allowed to settle. The washed cells were then sedimented through 3% Ficoll. This procedure yielded approximately 50 mg of protein from 1 gm of heart. Viability measured by trypan-glue exclusion is 90-95%. Approximately 80% of the cells were beating. Scanning electron microscopic studies suggest that the isolated myocytes are morphologically intact. The cells oxidize glucose, pyruvate, citrate and palmitate to CO2 and synthesize protein and RNA. Uptake of glucose, 2-deoxyglucose, leucine and taurine was saturable. Glucose uptake was stimulated by insulin. The cells retained LDH and CPK as well as their capacity to oxidize substrates after 24 hours at 4 degrees C or 4 hours at 37 degrees C. After 24 hours at 4 degrees C the cells resume contracting when returned to room temperature. The procedure reported here for the isolation of spontaneously contracting, adult, rat heart myocytes provides cells with a high index of viability and greater yield than previously reported methods. The cells retain metabolic activity and withstand storage for longer periods than other described preparations.