Bicarbonate-buffered Medium Research Articles

Glycoprotein transport in the surface mucous cells of the rat stomach.

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.

SECRETORY CAPACITY AND ULTRASTRUCTURE OF RAT PANCREATIC ISLETS AFTER PRESERVATION OF PANCREAS IN DIFFERENT CONDITIONS

Experiments were performed with rat pancreas to investigate optimum conditions for obtaining and preserving the pancreas for subsequent isolation of islets that were viable by both functional and morphological criteria. After only 30 min of warm ischaemia, the yield and viability of islets that could be isolated were poor. However, if the pancreas was removed and placed immediately in a cold bicarbonate-buffered medium, which was supplemented with HEPES to maintain the pH and Trasylol to inhibit proteolytic activity, then viable islets could be isolated consistently after over 8 hr of storage. These results demonstrate that even short periods of warm ischaemia will render the pancreas unsuitable for islet isolation. Once the pancreas is placed in a suitable cold medium, however, considerable delay may be permissible without adversely affecting the viability of the islets which can be obtained. The same conditions might prove to be applicable when human pancreas is obtained for the purpose of transplantation into diabetic subjects.

Insulin Biosynthesis in a Broken-Cell Preparation of Islets of Langerhans

Conference Article| December 01 1975 Insulin Biosynthesis in a Broken-Cell Preparation of Islets of Langerhans DAVID G. PARRY; DAVID G. PARRY 1Biochemistry Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. Search for other works by this author on: This Site PubMed Google Scholar KEITH W. TAYLOR KEITH W. TAYLOR 1Biochemistry Group, School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1975) 3 (6): 903–904. https://doi.org/10.1042/bst0030903 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation DAVID G. PARRY, KEITH W. TAYLOR; Insulin Biosynthesis in a Broken-Cell Preparation of Islets of Langerhans. Biochem Soc Trans 1 December 1975; 3 (6): 903–904. doi: https://doi.org/10.1042/bst0030903 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1975 Biochemical Society1975 Article PDF first page preview Close Modal You do not currently have access to this content.

Action of N-methyl-bicuculline on the binding of γ-aminobutyric acid to a synaptosomal fraction of rat cerebral cortex

The interaction of N-methyl-bicuculline and γ-aminobutyric acid (GABA) on a synaptosomal fraction of rat cerebral cortex was examined at 0 C in a balanced bicarbonate-buffered medium using differential centrifugation and double-isotope scintillation spectrometry. The drug, at 10 −4 m, produced a significant decrease in the binding of [ 3H]GABA (10 −8, 10 −7 m) to the particles. Further experiments revealed that N-methyl-bicuculline, at concentrations of 10 −7–10 −3 m, significantly decreased the “binding” of GABA (4.8 × 10 −8 m) to the particles. This bicuculline-induced reduction in GABA binding exhibited linear characteristics over a 10 −10–10 −3 m concentration range of the drug, and when present at 10 −3 m, the drug decreased GABA binding by about 23%. This demonstration of a bicuculline-sensitive component of GABA binding to synaptosomal particles adds support to neurophysiological studies which have indicated that bicuculline may serve as an antagonist to the GABA receptor in vivo.

Growth and metabolism of preimplantation mouse embryos cultured in phosphate-buffered medium.

Summary. The development of two-cell, eight-cell and morula stages of the mouse embryo in phosphate-buffered medium incubated aerobically was limited to that which occurred during the initial 24 hr of culture. Fewer embryos developed in phosphate- than in bicarbonatebuffered medium. More two-cell embryos developed in 4 mm- than in 1 mm-phosphate-buffered medium but there was no difference in the number of eight-cell embryos and morulae developing in either concentration. Addition of oxaloacetate or malate to phosphate-buffered media did not improve development. With incubation periods of 2 to 12 hr, two-cell embryos were more susceptible to phosphate than eight-cell embryos. Culture for 24 hr in phosphate-buffered medium resulted in a reduced incorporation of pyruvate and lactate carbon into the acidsoluble fraction of embryos at all stages of development. In embryos cultured from the two-cell and eight-cell stage, incorporation of substrate carbon into protein and, to a lesser extent, RNA and DNA was also depressed after culture in phosphate-buffered medium. This reduced synthetic activity could result in less ATP utilization and may be responsible in part for the higher levels of ATP found in embryos at these stages after culture in phosphate- as compared to bicarbonate-buffered medium. At the morula stage, macromolecular synthesis was not affected by culture in phosphate-buffered medium and the ATP levels in these embryos were similar to those in embryos developing in bicarbonate-buffered medium. The oxidation of pyruvate by embryos also indicated that the utilization rather than the production of ATP was more affected by culture in phosphate- than in bicarbonate-buffered medium.

Induction and reversal of contact inhibition of growth by pH modification.

MANY normal mammalian cells in culture are sensitive to a growth-inhibitory effect of cellular interaction, evidenced by an almost complete arrest of macromolecular synthesis and cellular division at relatively low population densities (contact inhibition of growth)1. It has, however, been shown that the environmental pH is an important element in this phenomenon2. In the bicarbonate-buffered media ordinarily used for animal cell cultures, an initial alkalinization to pH 7.8–8.0 as a result of CO2 loss is followed by the metabolic acidification of the medium to pH 6.8–7.0, at a rate determined by the population density and the metabolic activity of the specific cell. If, by the use of appropriate non-volatile buffers3,4, cells are kept at or near the pH optimal for the specific strain (H. E., unpublished work), the early contact inhibition of growth does not develop, and the cells achieve population densities as much as two to four times greater than those ordinarily observed2.

Substitution of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) for bicarbonate in protein-free animal cell culture medium: application to vaccinia virus quantitation and fluorogenic acetylesterase assay in living LM cells.

The buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at 15 mM has been substituted for sodium bicarbonate in the protein-free medium used for the growth of LM mouse fibroblasts. Cells have been cultivated in spinner flasks for up to 6 months with continuous aeration from an air line. The generation time was similar to that of parallel bicarbonate-buffered cells. Cotton-plugged shaken Erlenmeyer flasks produced the same results. HEPES-buffered cells have been grown in cotton-plugged prescription bottles and in glass and plastic Petri dishes in an ordinary humidified incubator. Vaccinia virus plaque production was slightly reduced in cells grown and overlaid with HEPES medium compared to bicarbonate medium. There was little difference in plaque count on cells grown in bicarbonate and overlaid with HEPES medium. These results indicate that HEPES buffer could be used routinely for cell and virus growth in aerated systems where CO2 is not limiting. Acetylesterase (EC. 3.1.1.6), measured by the fluorometric breakdown of fluorescein diacetate by living cells, was lower in HEPES than in bicarbonate-buffered medium, indicating an effect of HEPES on the cell membrane. The ability of HEPES to withstand autoclaving also renders it useful as a buffer for currently available autoclavable Eagle's medium.

Diphenylhydantoin and the cations and phosphates of electrically stimulated brain slices

SUMMARYSlices prepared from guinea pig cerebral cortex were incubated in bicarbonate-buffered media. Slices which were electrically stimulated at 1,000 cps lost K⁺ and gained Na⁺. Contents of ATP and creatine phosphate fell during stimulation. These effects were all diminished by 1 to 5 . 10⁻⁴ molar diphenylhydantoin. Diphenylhydantoin appears to diminish energy consumption by decreasing the downhill movements of monovalent cations that occur upon repeated membrane depolarization.

Effects of progesterone in vitro on glucose and acetate metabolism by the luteinized rat ovary.

SUMMARY Progesterone in vitro decreases the rates of glucose uptake and of acetate uptake and oxidation, of lipogenesis from acetate and of oxygen consumption, and reduces the intracellular concentrations of ATP and citrate in slices of luteinized rat ovary incubated in a bicarbonate-buffered medium. The effect on glucose uptake was shown to be due to inhibition by progesterone of the membrane transport of glucose. In view of the steroid concentrations used to elicit these effects in vitro and the known actions of steroids such as progesterone on biological membranes, these observations are thought to be due to non-physiological lytic effects.

The Na+, K+, and Cl- Content of Goose Salt Gland Slices and the Effects of Acetylcholine and Ouabain

In goose salt gland slices incubated in bicarbonate-buffered medium which contained 170 mEq of Na(+)/liter, net total tissue Na(+), expressed as milliequivalents per kilogram, was, in the presence of either acetylcholine (plus eserine) or ouabain, significantly higher than that of the bathing fluid. Acetylcholine caused an increase in the tissue Na(+) content as compared with untreated slices; there was an approximately equivalent decrease in K(+) and a significant decrease in Cl(-). The calculated net intracellular concentrations of Na(+), expressed as milliequivalents per liter of intracellular water, in unstimulated, acetylcholine-stimulated, and ouabain-treated slices were 2.1, 3.1, and 2.7 times higher, respectively, than the concentration of Na(+) in the bathing fluid. The net intracellular concentration of Na(+), expressed as milliequivalents per liter of intracellular water, in slices incubated in the presence of acetylcholine was 531 mEq/liter; this is approximately the same as the concentration of Na(+) in the secreted fluid of the goose salt gland (515 mEq/liter). The results indicate that the main concentration gradient for Na(+) could be established across the basal membrane. The data do not indicate whether this involves active transport of Na(+) per se. A second stage which might involve Na-K ATPase activity at the luminal membrane is discussed. The sum of the total tissue Na(+) and K(+) was approximately 250 mEq/kg, whereas the Cl(-) content was only approximately 130 mEq/kg.

Regulation of renal citrate metabolism by bicarbonate ion and pH: observations in tissue slices and mitochondria.

The effect of acid-base balance on the oxidation and utilization of citrate and other organic acids has been studied in tissue slices and isolated kidney mitochondria. The results show that: 1) With bicarbonate-buffered media, citrate oxidation and utilization are inhibited in slices of renal cortex and in kidney mitochondria when [HCO(3) (-)] and pH are increased within the physiologic range (pH 7.0 to 7.8; 10 to 60 mumoles HCO(3) (-) per ml). When phosphate or Tris buffers are used, no comparable effect on citrate oxidation occurs when pH is varied. 2) This effect is not demonstrable in heart or liver slices when a physiologic buffer is used. 3) alpha-Ketoglutarate utilization is inhibited in slices of renal cortex under similar conditions. Pyruvate and L-malate utilization are not inhibited in slices or mitochondria. 4) Citrate content in slices of renal cortex incubated with a high [HCO(3) (-)] is considerably greater than the concentration found with a low [HCO(3) (-)] in the medium. This effect is not duplicated by pH change in a nonbicarbonate buffer system. In mitochondria citrate content is also increased markedly at high bicarbonate concentrations. 5) The kinetic characteristics of the inhibition of citrate oxidation are those of a competitive type of inhibition. 6) When pH was varied with a constant [HCO(3) (-)] in the media, citrate oxidation was inhibited by increasing pH in slices of renal cortex but not in mitochondria. On the other hand, when [HCO(3) (-)] was increased without change in pH, no decrease in citrate oxidation occurred in slices, but a marked inhibitory effect was found when mitochondria were used. From a comparison of these results with those previously obtained in intact animal experiments, we conclude that the inhibition of citrate oxidation caused by increasing pH and [HCO(3) (-)] in slices of renal cortex and kidney mitochondria is an in vitro representation of the inhibition of citrate reabsorption in the nephron that occurs in metabolic alkalosis. Thus, citrate clearance increases in metabolic alkalosis because of inhibition of oxidation of reabsorbed citrate within cells of the renal tubules. This inhibition is the result of an inhibitory effect of bicarbonate ion on citrate oxidation in mitochondria.

The effect of insulin, alloxan diabetes, and anoxia on the ultrastructure of the rat heart.

Hearts from normal and alloxan diabetic rats were perfused in vitro with a bicarbonate-buffered medium containing glucose. Transport of glucose through the cell membrane was stimulated with insulin or by induction of anaerobiosis. The organs were rapidly fixed and examined by electron microscopy. Transport stimulation was not associated with any increase in the number of sarcolemmal invaginations or subsarcolemmal cytoplasmic vesicles. It was concluded that glucose transport and the effects of insulin or anoxia do not involve pinocytosis. The relationship of pinocytosis to glucose transport is discussed. The appearance of numerous lipid inclusions at the Z line level of the sarcomeres in the diabetic and anoxic myocardia is described.

WATER DISTRIBUTION IN INCUBATED SLICES OF BRAIN AND OTHER TISSUES

Slices of rat cerebral cortex swell about 40% during one hour's aerobic incubation in bicarbonate-buffered medium. Thiocyanate or sucrose added to the medium equilibrate with part of the tissue fluid leaving a "non-thiocyanate" or "non-sucrose" space equal to about 45% of the total fluid which was present in the fresh unincubated slice. This space does not change with the extent of swelling or the time during which the tracer is present. The swelling thus appears to be extracellular. Inuilin in the medium equilibrates with little more than the water of swelling and docs not appear to enter the rest of the extracellular fluid. The swelling thus seems to involve uptake of fluid which is not continuous with the true tissue fluids. With glutamate or high potassium in the medium, swelling is increased and the non-thiocyanate, non-sucrose, and non-inulin spaces increase correspondingly. This increase is apparently intracellular. Swelling is also increased under anaerobic conditions. Results with sucrose and inulin indicate that this increase is due to intracellular swelling. The space occupied by thiocyanate increases with the time during which the thiocyanate is present in the medium under anaerobic conditions. Swelling can be decreased but not prevented by colloids in the medium. Medium rendered hypertonic with sucrose apparently causes cell shrinkage, as judged by the non-thiocyanate or non-sucrose space, without preventing uptake of "swelling water". Liver and kidney slices swell about 10%. Thiocyanate occupies the whole of the tissue fluid while inulin leaves a constant non-inulin space, equal to about 60% of the total fluid of the fresh unincubated slice. Sectors of diaphragm swell about 15%. Thiocyanate and inulin occupy about equal spaces. The non-thiocyanate and non-inulin spaces are about 49% of the total fluid of the fresh tissues. Diaphragm cut into strips parallel to the circumference swells more and thiocyanate and inulin penetrate most of the fluid. Sciatic nerve swells nearly as much as brain. The swelling is reduced if the ends are tied. Thiocyanate penetrates the tissue progressively with time. Inulin seems to occupy little more than the water of swelling, as in brain.

FACTORS AFFECTING THE POTASSIUM CONTENT OF INCUBATED BRAIN SLICES

When rat cerebral cortex slices are incubated in glucose–saline medium in the presence of oxygen, the slices lose half of their potassium almost immediately and then reconcentrate it to some extent. Uptake of potassium proceeds further if glutamate is added to the medium but, since the apparent intracellular space is also increased by glutamate, the intracellular potassium concentration is not increased by glutamate. The difference between the potassium concentrations in the incubated slice and in the medium reaches a maximum when the medium contains about 5 mM potassium. In the absence of oxygen, under ordinary conditions, slices lose potassium rapidly but the final concentration reached remains higher than that of the medium and this difference is marked if the concentration in the medium is high. The greatly depleted potassium content of slices which have been incubated anaerobically can be largely restored by subsequent aerobic incubation. The rate and extent of this restoration decreases with the length of time during which the slices have been deprived of oxygen. If the medium contains pyruvate or if the slices have been subjected to a previous period of aerobiosis, anaerobic glycolysis is stimulated and concomitantly the potassium concentration in the slices increases. The effects of pyruvate and previous aerobiosis are additive. The sum of sodium and potassium in slices incubated under various conditions does not vary greatly but the sum is significantly increased in the presence of glutamate, in phosphate instead of bicarbonate-buffered medium, and, especially, in medium containing a high potassium concentration. The fluid taken up in swelling has approximately the same total cation content as that of the medium.

WATER DISTRIBUTION IN INCUBATED SLICES OF BRAIN AND OTHER TISSUES

Slices of rat cerebral cortex swell about 40% during one hour's aerobic incubation in bicarbonate-buffered medium. Thiocyanate or sucrose added to the medium equilibrate with part of the tissue fluid leaving a "non-thiocyanate" or "non-sucrose" space equal to about 45% of the total fluid which was present in the fresh unincubated slice. This space does not change with the extent of swelling or the time during which the tracer is present. The swelling thus appears to be extracellular. Inuilin in the medium equilibrates with little more than the water of swelling and docs not appear to enter the rest of the extracellular fluid. The swelling thus seems to involve uptake of fluid which is not continuous with the true tissue fluids. With glutamate or high potassium in the medium, swelling is increased and the non-thiocyanate, non-sucrose, and non-inulin spaces increase correspondingly. This increase is apparently intracellular. Swelling is also increased under anaerobic conditions. Results with sucrose and inulin indicate that this increase is due to intracellular swelling. The space occupied by thiocyanate increases with the time during which the thiocyanate is present in the medium under anaerobic conditions. Swelling can be decreased but not prevented by colloids in the medium. Medium rendered hypertonic with sucrose apparently causes cell shrinkage, as judged by the non-thiocyanate or non-sucrose space, without preventing uptake of "swelling water". Liver and kidney slices swell about 10%. Thiocyanate occupies the whole of the tissue fluid while inulin leaves a constant non-inulin space, equal to about 60% of the total fluid of the fresh unincubated slice. Sectors of diaphragm swell about 15%. Thiocyanate and inulin occupy about equal spaces. The non-thiocyanate and non-inulin spaces are about 49% of the total fluid of the fresh tissues. Diaphragm cut into strips parallel to the circumference swells more and thiocyanate and inulin penetrate most of the fluid. Sciatic nerve swells nearly as much as brain. The swelling is reduced if the ends are tied. Thiocyanate penetrates the tissue progressively with time. Inulin seems to occupy little more than the water of swelling, as in brain.

FACTORS AFFECTING THE POTASSIUM CONTENT OF INCUBATED BRAIN SLICES

When rat cerebral cortex slices are incubated in glucose–saline medium in the presence of oxygen, the slices lose half of their potassium almost immediately and then reconcentrate it to some extent. Uptake of potassium proceeds further if glutamate is added to the medium but, since the apparent intracellular space is also increased by glutamate, the intracellular potassium concentration is not increased by glutamate. The difference between the potassium concentrations in the incubated slice and in the medium reaches a maximum when the medium contains about 5 mM potassium. In the absence of oxygen, under ordinary conditions, slices lose potassium rapidly but the final concentration reached remains higher than that of the medium and this difference is marked if the concentration in the medium is high. The greatly depleted potassium content of slices which have been incubated anaerobically can be largely restored by subsequent aerobic incubation. The rate and extent of this restoration decreases with the length of time during which the slices have been deprived of oxygen. If the medium contains pyruvate or if the slices have been subjected to a previous period of aerobiosis, anaerobic glycolysis is stimulated and concomitantly the potassium concentration in the slices increases. The effects of pyruvate and previous aerobiosis are additive. The sum of sodium and potassium in slices incubated under various conditions does not vary greatly but the sum is significantly increased in the presence of glutamate, in phosphate instead of bicarbonate-buffered medium, and, especially, in medium containing a high potassium concentration. The fluid taken up in swelling has approximately the same total cation content as that of the medium.