The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm

The motility of demembranated bull sperm was found to be governed by the concentrations of cyclic adenosine 3', 5'-monophosphate (cAMP) and Ca2+ at low pH (6.6-7.1), and was less sensitive to these variables at higher pH (7.4-7.8). Although motility was generally found to increase with increasing pH in the range from 6.6 to 7.8, the addition of exogenous cAMP markedly and selectively improved the motility at the lower end of the range (pH 6.6-7.1). In the presence of 10 microM cAMP, low Ca2+ (8.0 X 10(-8) M), and a high concentration of Mg-adenosine 5'-triphosphate (ATP, 8 mM), demembranated sperm at pH 6.8 and 7.1 exhibited swimming similar to that of live ejaculated sperm. At a free Ca2+ concentration of 4.4 X 10(-5) M, the motility was rapidly inhibited at pH 6.8-7.1, whereas at pH 7.4-7.8, the activity was not greatly affected. Since calcium is known to antagonize the cAMP pathway by activating Ca2+-dependent phosphodiesterase and Ca2+-dependent phosphatase, this further supports the idea that cAMP-dependent activation is crucial for motility at low pH. Our results demonstrate that the flagellar axoneme can function normally at relatively acidic pH, and produce vigorous swimming at high levels of ATP. The ATP content of live sperm was measured and found to be high enough (approximately 8 mM) to support the vigorous motility seen at pH 6.6-7.1 in the models.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.     

Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s)

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.     

GnRH agonist Buserelin affects colony-forming efficiency of HHUA and Jurkat cells.

In this study, cell proliferation was examined at low and high cell densities, using HHUA and Jurkat cell lines as experimental models for the antiproliferative and proliferation-enhancing effects of GnRH agonist, Buserelin, respectively. For efficient evaluation of Buserelin activity at low cell density, the colony-forming efficiency assay was adopted. Buserelin markedly affected colony-forming efficiency in a dose-dependent manner at low cell density; however, Buserelin had no effect at high cell density. The conditioned medium of HHUA cells inhibited the Buserelin action, whereas that of Jurkat cells mimicked it. These results suggest that each cell line secretes some substances which regulate cell proliferation, and that these substances can also change the effects of Buserelin. The measurement of colony-forming efficiency is a very effective way of eliminating autocrine and/or paracrine effects, and is a highly sensitive method for measuring GnRH activity.     

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities

Intestinal absorption offolates has been characterized as a facilitative process with a low pHoptimum. Studies with intestinal epithelial cells have suggested thatthis activity is mediated by the reduced folate carrier (RFC1). In thispaper, we report on folate transport characteristics in an immortalizedrat IEC-6 cell line that was found to exhibit the predominant influxactivity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs andhigh folic acid uptake only at the low pH. For the two clones with thehighest level of transport activity, relative MTX influx at the two pHswas reversed. Moreover, the low pH MTX influx activity([MTX]_e = 0.5 µM) was markedly inhibited by 20 µM folic acid while influx at neutral pH was not. Furthermore, in thepresence and absence of glucose at low pH, MTX and folic acid influxactivity was inhibited by azide, while MTX influx at pH 7.4 wasstimulated by azide in the absence of glucose but was unchanged in thepresence of glucose and azide. This was contrasted with the results oftransfection of the same RFC1 construct into an L1210 murine leukemiacell line bearing a nonfunctional endogenous carrier. In this case, theactivity expressed was only at pH 7.4. These data indicate that RFC1can exhibit two distinct types of folate transport activities inintestinal cells that must depend on tissue-specific modulators.

     

Stereological-ultrastructural study of pancreatic B cells in metabolic alkalosis

The secretion of insulin in response to glucose and the changes in the B cell at the ultrastructural level were studied in rat pancreas perfused at pH 7.4 and 7.8 with different concentrations of glucose. Raising the extracellular pH from 7.4 to 7.8 significantly inhibits glucose-induced insulin secretion. Coincidentally, morphometric studies showed significant evidences of low secretory activity in B cells from pancreas submitted to high glucose stimulation under alkalosis, namely lower number of emiocytotic figures and microtubules as well as a decrease in the volume density of the granular endoplasmic reticulum and the Golgi complex. On the other hand, a significant increment in the number of images of granulolysis was also demonstrated. These secretory and ultrastructural results confirm the inhibitory effect of pH 7.8 upon B cell secretory activity induced by glucose. Moreover, they lend further support to the role of intracellular hormone degradation as a regulator of B cell insulin content.     

Regulation of passive membrane permeability for potassium ions by cell density of 3T3 and SV40-3T3 cells.

The passive K⁺ permeability of 3T3 and SV40-3T3 cells was evaluated from experiments on passive K⁺ efflux and electrical transmembrane potential measurements at different cell growth densities, external calcium concentrations and temperatures. Passive K⁺ permeability was shown to decrease markedly with increasing cell growth density, to increase with the lowering of external calcium concentration, and at low cell densities to be higher at low temperature (25 °C) than at physiological temperature (37 °C). These and further results taken from the literature are fully consistent with the notion of regulation of proliferation being effected by control of intracellular K⁺ concentrations. The phenomenon of high temperature inactivation of passive K⁺ permeabilities observed at low cell densities is discussed in analogy to recent results on model systems from phospholipid/cholesterol doted with channel-forming antibiotics.     

pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-³H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-³H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-³H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-³H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-³H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-³H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin.

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca2+ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl2 (0.1--2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Lysosomal enzyme secretion from human neutrophils mediated by cyclic CMP: inhibition of cyclic GMP accumulation and neutrophil function by glucocorticosteroids.

The effects of several glucocorticosteroids on cyclic GMP accumulation, guanylate cyclase activity, calcium influx, lysosomal enzyme secretion, and phagocytosis were studied in human neutrophils. Contact between neutrophils and serum-treated zymosan particles, in the presence of calcium at pH 7.4, triggered these cellular events within five minutes. Each of these neutrophil functions was markedly inhibited by methylprednisolone sodium succinate, triamcinolone acetonide hemisuccinate and paramethasone acetate but was unaffected by two mineralo-corticosteroids. Human neutrophil soluble guanylate cyclase activity was not changed by the glucocorticoids. Inhibition of phagocytosis by, and lysosomal enzyme secretion from, neutrophils by glucocorticosteroids may be the result of a reduction in cyclic GMP accumulation within these cells. The data suggest that glucocorticosteroids inhibit cyclic GMP accumulation in neutrophils by reducing the influx of extracellular calcium into the cells, thereby limiting the availability of intracellular calcium for metabolic processes associated with the accumulation of cyclic GMP.     

Influence of pH and sodium on the inhibition of guinea-pig heart (Na+ + K+)-ATPase by calcium.

The inhibition of guinea-pig heart (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) by calcium has been studied at pH 7.4, 6.8 and 6.4. 1. A decrease in pH reduced the threshold inhibitory concentration of calcium and the calcium concentration producing an inhibition of 50% of the enzyme activity. 2. Calcium reduced the apparent affinity of the enzyme of Na+, this effect occurred only at pH 7.4. 3. Calcium increased the apparent affinity of the enzyme for K+, this effect was enhanced at acidic pH. 4. Activation of the enzyme by Na+ for a constant Na+ : K+ ratio has been studied at pH 7.4 and at pH 6.8 in the absence and in the presence of 3.10(-4) M Ca 2+; the results of this experiment indicate that Ca2+ effect at pH 7.4 was not influenced by Na+ -- K+ competition and was probably due to a Na+ -- Ca2+ interaction. 5. At pH 7.4, the calcium inhibitory threshold concentration and the concentration producing 50% inhibition were reduced when Na+ was low; at pH 6.8, the calcium inhibition was not markedly modified by the change of Na+ concentration. 6. The Ca2+ -activated ATPase of myosin B which is related to the contractile behaviour of muscle and the Ca2+ -ATPase of the sarcoplasmic reticulum which is related to the ability of this structure to accumulate calcium were activated in a range of calcium concentration producing an inhibition of (Na2+ + K+) -ATPase. The present results indicate that the increase by acidity of the (Na2+ + K+) -ATPase sensitivity to calcium might be due to a suppression of a Na+ -Ca2+ interaction. On the basis of these observations, it is proposed that calcium might inhibit the Na+ -pump during the repolarization phase of the action potential and that, by this effect, it might control cell excitability.     

The cellular transport of calcium in rat liver

The bidirectional transport of calcium in rat liver was studied using slices labeled with Ca⁴⁷ in a closed two compartment system. Steady-state conditions were observed with influx and efflux transfer coefficients of 0.070 and 0.018 per minute, respectively. The rapidly exchanging cell fraction of calcium existed at a concentration three times higher than the average cell concentration of calcium and occupied cell loci comprising less than 25% of the cell mass, suggesting that calcium associated with the cell membranes, nuclei, and mitochondria participated in the rapidly exchanging fraction. At pH 7.4 and 377deg;C, the influx transfer coefficient was 25% above the steady-state condition and accumulation of calcium by the slices occurred. Studies of the effects of varied physical and chemical conditions revealed that the influx transfer coefficient was increased by elevated pH, strontium, certain metabolic inhibitors, and 2 mM concentrations of cyclic adenosinemonophosphate and adenosinetriphosphate. The influx transfer coefficient was decreased by reduced temperature, decreased pH, magnesium, and 10 mM adenosinetriphosphate. The efflux transfer coefficient was increased by elevated pH, strontium, iodoacetate, and adenosinetriphosphate, and was decreased by reduced temperature and by N-ethylmaleimide. These data support the thesis that cell transport of calcium is accomplished by the attachment of calcium atoms to the cell surface and transport through the plasma membrane bound to either specific carriers or to membrane constituents. Conditions which change the affinities, capacities, and mobilities of plasma membrane ligands that bind calcium or cause extracellular chelation of calcium are capable of altering the rate of calcium transport.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.     

Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration

Even though net fluxes of Pi (orthophosphate) across the cell membrane may be important in clinical disorders involving the abnormal extracellular Pi concentration, in acid-base disturbances, and in the responses of some cells to hormones, relatively few studies have been made of these fluxes, owing to the complexities of interpretation. Here we have studied net fluxes in response to changes in extracellular pH and Pi concentration in the simple case of the human red cell. The permeability of the cell membrane to net Pi fluxes was described in terms of a first-order rate constant, epsilon. By means of a mathematical model, it was possible to discriminate between transmembrane Pi movement, net intracellular generation or consumption of Pi by organic phosphates, and extracellular generation of Pi from the cells lysing during the experiment. We show that net Pi influx into the cell during experimental alkalosis was probably driven by net consumption of Pi by organic phosphates, and that this was reversed during acidosis. Inhibition of net Pi influx by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) suggests that, like Pi self-exchange, net influx is at least partly mediated by the band 3 transport protein. Unexpectedly, epsilon increased from 2 h-1 at extracellular pH 7.4 to approx. 7 h-1 at pH 7.8. From the value of epsilon at pH 7.4, we conclude that the apparent buffering or regulation of steady-state Pi concentrations, previously reported in red cells in vitro, was not an artifact of intracellular generation of Pi from organic phosphates.     

Quantitative morphological assessment of parathyroid activity in response to variations in calcium concentration in vitro

Isolated parathyroid glands from gerbils incubated at low (0.5 mM), normal (1.25 mM) or high (3 mM) calcium concentration for 1 or 6 h were studied with regard to the following stereological parameters: the volume densities of the nucleus, mitochondria, endoplasmic reticulum, Golgi zone, lipid bodies, lysosomes, secretory granules and storage granules, and the surface densities of the cell membrane and endoplasmic reticulum. In glands incubated for 1 h no significant differences were seen between the experimental groups in any of the parameters investigated, however, the volume density of the Golgi zone and the surface density of the cell membrane were significantly increased in glands incubated for 6 h at low calcium concentration, compared with those incubated at high calcium concentration. The enlargement of the Golgi zone in stimulated glands was also evident when the two glands from the same animal incubated at different calcium concentrations were compared. The data suggest that the early response of parathyroid glands to altered concentration of extracellular calcium includes functional activities associated with the Golgi zone.     

The effect of pH on biological activity of plant cytotoxin cauloside C

The effect of plant carboxyl-containing glycoside cauloside C upon eucaryotic cells has been studied. The glycoside interacts with cells as a pH-dependent cytotoxin and increases K+ leakage and Ca2+ uptake with strong action in acidic media Cell viability after glycoside action at acidic pH may be recovered by the shift of medium pH from 5.6 to 7.4. Directed transport of low molecular weight effectors such as cAMP and Ca2+ to human embryo fibroblasts under the action of cauloside C has been demonstrated. Calcium uptake is accompanied by about a twofold stimulation of fibroblast proliferation in serum-free medium. The manifestation of the effect depends on the strictly determined time of the 'open' state of the membrane permeability (2 min) and upon concentration of glycoside in the medium (1 ng/ml) Cauloside C-stimulated Ca-transport is not blocked by Ca-channel blockers such as verapamil, diltiasem, and nitrendipine (all at a concentration of 1 x 10(-6) M) but these blockers inhibit cauloside C-stimulated proliferation of fibroblasts. We conclude that stimulation of fibroblast proliferation is caused by activation of membrane associated Ca-channels at the expense of calcium, incorporated into cells with cauloside C. The use of cauloside C as a new biochemical tool for cell permeabilisation is suggested.     

Contact-dependent regulation of growth of diploid human fibroblasts is dependent upon the presence of terminal galactose residues on plasma membrane glycoproteins

The growth of diploid human fibroblasts has previously been shown to be regulated mainly by the extent of cell-cell contacts [R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361], these contacts being effective only when terminal, beta-glycosidically linked galactose residues were present on plasma membrane glycoproteins. These studies, in which a high cell density in sparse cell cultures has been mimicked by the addition of immobilized plasma membrane glycoproteins, have been further extended to investigate the role of terminal galactose residues directly in cell cultures. The studies presented herein show that (i) culturing human fibroblasts in the presence of beta-galactosidase resulted in an approximately twofold higher saturation density, as well as a twofold higher proliferation rate at high cell densities when compared to the rates found in control cultures. (ii) The presence of alpha-lactalbumin in the culture medium, which acts as a modifier of the activity of galactosyltransferase, had the same effect as beta-galactosidase. (iii) Addition of the lectin I from Bandeiraea simplicifolia (BS I), which is specific for terminal galactose residues, resulted in an increase in the proliferation rate of cell cultures at high cell densities, while the proliferation was not affected at low cell densities. These data show that the presence of terminal, beta-glycosidically linked galactose is vital for the efficient growth control of normal cells.     

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.