Cotransport Rate Research Articles

Direct conversion of monopotassium L-aspartate by electrodialysis metathesis: Stack resistance, co-ion transport, and concentration polarization

The conventional method of synthesizing high-value amino salt from amino acid ammonium involves several steps including acidification, precipitation, and separation. This process not only consumes acids and bases but also generates a substantial amount of wastewater. In this study, monoammonium L-aspartate was directly converted to monopotassium L-aspartate (Asp-K) using electrodialysis metathesis (EDM). To achieve this, K2SO4 was chosen as the optimum potassium salt to supply K+ due to its relatively lower co-transport rate with SO42− ions and lower stack resistance. Additionally, the membrane type was optimized as CIS/AIS membranes due to their low stack resistance, high ion permeability for the transport of Asp− ions, and low concentration polarization. The process resulted in high-purity Asp-K (99.8 %) and low energy consumption (0.14 kWh/kg Asp-K). These results indicate that the EDM process is a promising method for converting amino salts with high purity.

Predictions of burnout times of biomass char using experimentally determined CO to CO2 ratio

The ratio of CO to CO2 produced during char combustion is an important parameter affecting the temperature of the char particle, the rate of mass transfer of gaseous species to and from the surface of the particle, and, consequently, the burnout time. In the present work, the ratio of CO to CO2 from combustion was measured for a biomass char using a thermogravimetric method. A sample of biomass char was combusted in conditions where the process was entirely limited by transfer of oxygen to the external surface of the fuel particles. The experimentally-obtained rate of combustion was compared with the expected rate of CO and CO2 transport, from which the ratio of CO to CO2 was calculated. The CO/CO2 ratio was determined for a char derived from birch wood and the results were compared with those from graphite and activated carbon at the same conditions. The experiments were conducted at various temperatures between 973 and 1173 K, with a range of partial pressures of oxygen in the oxidising gas from 0.0057 to 0.023 bar.The experimental CO/CO2 ratios were used to model the combustion of a single particle of char, predicting the total burnout time. Two models were developed: (1) an analytical solution for the burnout time assuming pseudo-steady state combustion, and (2) a numerical model of a char particle, accounting for gas transport and the combustion reaction inside the porous fuel particle. To validate the models, a separate set of experiments was performed where single particles of birch wood (6 mm dia.) were mounted on the tip of a thin thermocouple, then inserted into a hot reactor through which was flowing N2. The wood particle pyrolysed, leaving a single particle of char (∼4 mm dia.). The char was then burned in a laminar flow of oxidising gas. The burnout times obtained with both models agreed well with the experimental results. Commonly-used empirical expressions for CO/CO2 ratios from the literature were also employed in simulations but were found to under-predict the burnout time.

Mixed gas diffusion and permeation of ternary and quaternary [formula omitted] gas mixtures in Matrimid®, polyetherimide and poly(lactic acid) membranes for [formula omitted] separation

The CO2/CO mixed-gas separation performance of a polyimide (Matrimid®), polyetherimide (PEI) and polylactic acid (PLA) membrane, were characterized in the presence of CO2- rich ternary (CO2/ CO/ O2) and quaternary (CO2/ CO/ O2/ N2) feed gas mixtures mimicking the products of CO2 reforming conversion reactions. The membrane-based separation of this mixture is poorly characterized and original data were obtained in a novel mass spectrometric apparatus that permits to monitor the instantaneous permeate composition, thus allowing to evaluate both mixed gas diffusion and permeability coefficients of all gases. CO2, CO, O2andN2 permeability and diffusivity in single gas tests were measured between 298 and 353 K up to 1 atm feed pressure and relevant activation energies were evaluated. At 298 K Matrimid® exhibits CO permeability of 0.50 ± 0.03 Barrer and an ideal CO2/ CO selectivity of 16 ± 1. PEI and PLA exhibit similar ideal selectivity values but lower CO transport rates. In all examined polymer films the CO2/ CO selectivity has absorption-selective character that favours the permeation of CO2. The ideal CO2/ CO selectivity of all membrane samples decreases with temperature, reaching values of 10 ± 1 at 335 K in Matrimid®. The CO2/ CO selective performances of all examined membrane do not show markable variations exposing the membrane samples to CO2- rich gas mixtures as feed gas. The upper bound correlation among selectivity and permeability for the CO2/ CO gas couple is here for the first time proposed.

What can we learn from erythrocyte Na–K–Cl cotransporter NKCC1 in human hypertension?

Fluxes catalyzed by the human Na–K–Cl cotransporter NKCC1 ( hNKCC1) were extensively investigated in erythrocytes from essential hypertensive patients. Using different techniques, four hNKCC1 abnormalities were described in a significant proportion of hypertensives: (i) low net sodium extrusion, (ii) high unidirectional inward cotransport, (iii) low apparent affinity for internal sodium and (iv) high maximal cotransport rate. All these four hNKCC1 abnormalities are compatible with an increased net inward cotransport. In hypertensive rat models, an increased net inward cotransport drives more chloride inside the cells, favoring membrane depolarization and hypertension. NKCC1 knock out mice are hypotensive and exhibit a compensatory elevation in renin secretion, apparently due to the lack of a functional NKCC1 in juxtaglomerular granular cells, which normally reduces basal renin release. This latter hypothesis is supported by the observation that human hypertensives with high cotransport have low renin hypertension and increased salidiuretic response to furosemide. Therefore, the human erythrocyte data validates animal studies showing increased net inward fluxes by NKCC1 in primary hypertension.

Water transport by Na+‐coupled cotransporters of glucose (SGLT1) and of iodide (NIS). The dependence of substrate size studied at high resolution

The relation between substrate and water transport was studied in Na+-coupled cotransporters of glucose (SGLT1) and of iodide (NIS) expressed in Xenopus oocytes. The water transport was monitored from changes in oocyte volume at a resolution of 20 pl, more than one order of magnitude better than previous investigations. The rate of cotransport was monitored as the clamp current obtained from two-electrode voltage clamp. The high resolution data demonstrated a fixed ratio between the turn-over of the cotransporter and the rate of water transport. This applied to experiments in which the rate of cotransport was changed by isosmotic application of substrate, by rapid changes in clamp voltage, or by poisoning. Transport of larger substrates gave rise to less water transport. For the rabbit SGLT1, 378+/-20 (n=18 oocytes) water molecules were cotransported along with the 2 Na+ ions and the glucose-analogue alpha-MDG (MW 194); using the larger sugar arbutin (MW 272) this number was reduced by a factor of at least 0.86+/-0.03 (15). For the human SGLT1 the respective numbers were 234+/-12 (18) and 0.85+/-0.8 (7). For NIS, 253+/-16 (12) water molecules were cotransported for each 2 Na+ and 1 thiocyanate (SCN-, MW 58), with I- as anion (MW 127) only 162+/-11 (19) water molecules were cotransported. The effect of substrate size suggests a molecular mechanism for water cotransport and is opposite to what would be expected from unstirred layer effects. Data were analysed by a model which combined cotransport and osmosis at the membrane with diffusion in the cytoplasm. The combination of high resolution measurements and precise modelling showed that water transport across the membrane can be explained by cotransport of water in the membrane proteins and that intracellular unstirred layers effects are minute.

Regulation of erythrocyte Na–K–2Cl cotransport by threonine phosphorylation

A method is described to measure threonine phosphorylation of the Na–K–2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na + arsenite. Both fall to 60% control when cell [Mg 2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.

Expression and Regulation of the Na+‐K+‐2Cl− Cotransporter NKCC1 in the Normal and CFTR‐Deficient Murine Colon

Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.

Abnormally fast mobility of CO at electrochemical interfaces

A very fast rate of CO transport at the Pt/electrolyte interface is reported. The mass transport was monitored by following the change of the electrode capacitance caused by H ad displacement during carbon monoxide adsorption at potentials in the hydrogen adsorption region. A diffusion parameter was calculated assuming the validity of Fick’s law. No dependence was found on the surface structure.

CAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1.

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.

Stimulation of Na+-K+-2Cl- cotransport by arsenite in ferret erythrocytes.

1. Na+-K+-2Cl- cotransport activity was measured in ferret erythrocytes as the bumetanide-sensitive uptake of 86Rb. 2. The Na+-K+-2Cl- cotransport rate was stimulated by treating erythrocytes with sodium arsenite but not by sodium arsenate (up to 1 mM). Stimulation took an hour to develop fully. Arsenite had no effect on bumetanide-resistant 86Rb uptake. 3. In cells stored for 3 days or less, cotransport stimulation by arsenite could be described by assuming arsenite either acts at a single site (EC50, 60+/-14 microM, mean +/- S.E.M., n = 3) or that it acts at both high- (EC50, 35+/-9 microM, mean +/- S.E.M., n = 3) and low- (EC50 >2 mM) affinity sites. 4. Stimulation by 1 mM arsenite was greatest on the day of cell collection (rate about 3 times that of the control), even exceeding that produced by 20 nM calyculin A, and declined during cell storage. Addition of calyculin A to arsenite-stimulated cells resulted in further stimulation of Na+-K+-2Cl- cotransport, suggesting that arsenite and calyculin act synergistically. This was most apparent in stored cells. 5. Stimulation by 1 mM arsenite was not affected by treating cells with the mitogen-activated protein kinase inhibitors SB203580 (20 microM) and PD98059 (50 microM), but was both prevented and reversed by the kinase inhibitors staurosporine (2 microM), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1, 50 microM) and genistein (0.3 mM), and with a combination of 10 microM A23187 and 2 mM EDTA (to reduce intracellular Mg2+ concentration). Only treatment with EDTA and A23187 prevented stimulation by the combination of 1 mM arsenite and 20 nM calyculin, whereas no treatment was able to fully reverse this stimulation once elicited. 6. Our data are consistent with arsenite stimulating (perhaps indirectly) a kinase that phosphorylates and activates the Na+-K+-2Cl- cotransporter.

Regulation of Na+-K+-2Cl- cotransport by protein phosphorylation in ferret erythrocytes.

1. Na+-K+-2Cl- cotransport in ferret erythrocytes was measured as the bumetanide-sensitive uptake of 86Rb. 2. The resting cotransport rate was high but could be increased threefold by treating erythrocytes with calyculin A, a potent inhibitor of serine/threonine phosphatases. Twenty nanomolar was sufficient to maximally and rapidly (within 4 min) stimulate transport. 3. The effects of several kinase inhibitors were tested. High concentrations of K-252a, K-252b, calphostin C and hypericin caused less than 20 % inhibition. Staurosporine (IC50, 0.06 microM) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; IC50, 2.5 microM) were more potent but still only partially (40-50 %) inhibited transport, an effect mimicked by reducing ionized intracellular Mg2+ concentration to submicromolar levels. Genistein may inhibit all transport at a sufficiently high dose (IC50, 0.36 mM) perhaps by directly inhibiting the transporter. 4. Staurosporine, PP1 and the removal of Mg2+ all prevented subsequent stimulation by calyculin A, and all inhibited calyculin-stimulated transport by 20-30 %. The effects of staurosporine, PP1 and Mg2+ removal were not additive. 5. The phosphatase that dephosphorylates the cotransporter is probably Mg2+ (or possibly Ca2+ or Mn2+) sensitive and not the target for calyculin A. The data suggest that this phosphatase is inhibited by phosphorylation, and that it is the regulation of this process which is affected by calyculin A and the kinase inhibitors tested here. Phosphorylation of the phosphatase is probably regulated by members of the Src family of tyrosine kinases.

A putative growth-related renal Na(+)-Pi cotransporter.

The mRNA that encodes for NaPi-2, the renal Na(+)-Pi cotransporter that is upregulated by Pi depletion in the adult rat, is low in the young animal. Yet, renal Na-Pi cotransport rates are higher in rapidly growing than in fully grown rats. The aim of this study was to unravel the molecular basis of this apparent discrepancy. Poly(A) RNA obtained from the renal cortex of young animals induced higher rates of Na(+)-Pi cotransport in oocytes than equal amounts of poly(A) mRNA obtained from the renal cortex of mature rats. Moreover, poly(A) RNA obtained from renal cortex of rapidly growing animals treated with antisense NaPi-2 oligomers or depleted of NaPi-2 transcripts by subtractive hybridization with cDNA generated from the renal cortex of adult rats retained its ability to induce Na(+)-Pi cotransport in oocytes. In addition, renal poly(A) RNA of the young subjected to subtraction hybridization generated a 379-base pair reverse transcriptase-polymerase chain reaction product common to all known type II Na(+)-Pi cotransporters. These observations permit us to surmise that the high rates of Na(+)-Pi cotransport prevailing during growth are due, at least in part, to the expression of a specific mRNA that is only partially homologous to that of NaPi-2.

A molecular view of proximal tubular inorganic phosphate (Pi) reabsorption and of its regulation.

In recent years, two mammalian proximal tubular brush border membrane Na/Pi cotransporters (types I and II) have been structurally identified by expression cloning techniques. Oocyte expression studies have shown that only the transport characteristics of the type II transporter correspond to the well-known properties of proximal tubular brush border membrane of Pi transport. In studies on physiological regulation by hormonal and non-hormonal factors a direct involvement and determining role of the type II transporter has been documented. Most interestingly, specific membrane retrieval/insertion phenomena participate in acute (minutes/hours) adjustments of brush border membrane Na/Pi cotransport rates; for chronic (hours/days) alterations also specific resynthesis/degradation processes participate. In pathophysiological alterations (e.g. in X-linked hypophosphataemia and in heavy metal-induced nephrotoxicity) the expression of the type II Na/Pi cotransporters is reduced and explains the observed phosphaturia.

Modulation of renal Na-Pi cotransport by hormones acting via genomic mechanism and by metabolic factors

The renal Na-Pi cotransport is subject to multiple regulatory inputs, such as endocrine, paracrine and intracrine. Among lipophilic, long-acting hormones that act via genomic mechanism, thyroid hormones, calcitriol, all-trans-retinoic acid stimulate, whereas glucocorticoids and estradiol inhibit the rate of Na-Pi cotransport across the brush border membrane of proximal tubules in vivo and/or across apical membrane of renal epithelial cells in vitro. Some findings suggest that these hormones may also influence Na-Pi cotransporter by modification of membrane microenvironment. It should be considered that Na-Pi cotransport can be modulated by lipophilic hormones by non-genomic signaling mechanisms such as sphingomyelin-ceramide pathway, NAD-cyclic ADP-ribose-Ca2+i pathway or by Ca2+ influx. Recent studies outline a basis for the putative intracrine signaling mechanisms that utilize Ca(2+)-releasing nucleotides, cyclic ADP-ribose, and nicotinic acid adenosine dinucleotide phosphate (NAADP), as novel second messengers for regulation of Na-Pi cotransport in response to changes of intermediary metabolic processes: gluconeogenesis, pentose phosphate pathway, polyamines and metabolism of fatty acids.

Rate of activation and deactivation of K?Cl cotransport by changes in cell volume in hemoglobin SS, CC and AA red cells

Red blood cells (RBC) of subjects homozygous for hemoglobin A (AA), C (CC) and S (SS) exhibit different cell volumes which might be related to differences in cell volume regulation. We have investigated how rapidly K:Cl cotransport is activated and deactivated to regulate the cell volume in these cells. We measured the time course of net K+ efflux after step changes in cell volume and determined two delay times: one for activation by cell swelling and a second for deactivation by cell shrinkage. Cell swelling induced by 220 mOsm media activated K+ efflux to high values (10-20 mmol/liter cell x hr) in CC and SS; normal AA had a threefold lower activity. The delay time for activation was very short in blood with a high percentage of reticulocytes (retics): (SS, 10% retics, 1.7 +/- 0.3 min delay, n = 8; AA, 10% retics, 4 +/- 1.5 min, n = 3; CC, 11.6% retics, 4 +/- 0.3, n = 3) and long in cells with a smaller percentage of reticulocytes: (AA, 1.5% retics, 10 +/- 1.4 min, n = 8; CC whole blood 6% retics, 10 +/- 2.0 min, n = 10, P < 0.02 vs. SS). The delay times for deactivation by cell shrinking were very short in SS (3.6 +/- 0.4 min, n = 8, P < 0.02) and AA cells with high retics (2.7 +/- 1 min, n = 3) and normal retics (2.8 +/- 1 min, n = 3), but 8-15-fold longer in CC cells (29 +/- 2.8 min, n = 9). Density fractionation of CC cells (n = 3) resulted in coenrichment of the top fraction in reticulocytes and in swelling-activated cotransport (fourfold) with short delay time for activation (4 +/- 0.3 min) and long delay for deactivation (14 +/- 4 min). The delay time for activation, but not for deactivation, increased markedly with increasing cell density. These findings indicate that all CC cells do not promptly shut off cotransport with cell shrinkage and high rates of cellular K+ loss persist after return to isotonic conditions. In summary, (i) K:Cl cotransport is not only very active in young cells but it is also very rapidly activated and deactivated in young AA and SS cells by changes in cell volume. (ii) Delay times for cotransport activation markedly increased with RBC age and in mature cells with low cotransport rates, long delay times for activation were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular mechanisms of acute and chronic adaptation of rat renal P(i) transporter to alterations in dietary P(i).

Recently, the cDNA for a Na-P(i) cotransport system of rat kidney cortex (NaPi-2) has been identified by expression cloning. Using polyclonal antibodies raised against this renal Na-P(i) cotransport system, and using the polymerase chain reaction after reverse transcription of mRNA in microdissected nephron segments, we recently demonstrated that NaPi-2-related mRNA and protein is expressed in the brush-border membranes (BBM) of the proximal tubules of rat kidney. The purpose of the present study was to study the cellular mechanisms involved in adaptation of rat renal Na-P(i) cotransporter to acute and chronic alterations in dietary P(i). Compared with rats fed chronically (7 days) a high-P(i) diet (1.2%), in rats fed chronically a low-P(i) (0.1%) diet the 3.4-fold increase in BBM Na-P(i) cotransport rate (chronic upregulation) was associated with a 2.2-fold increase in renal cortical NaPi-2 mRNA and a 4.9-fold increase in BBM NaPi-2 protein abundances. In contrast, compared with rats fed chronically (7 day) a high-P(i) diet, in rats fed acutely (2 h) a low-P(i) diet the 1.5-fold increase in Na-P(i) cotransport rate (acute upregulation) was associated with a 1.8-fold increase in NaPi-2 protein but no change in NaPi-2 mRNA abundance. Similarly, compared with rats fed chronically a low-P(i) diet, in rats fed acutely (2 h) a high-P(i) diet the 1.9-fold decrease in Na-P(i) cotransport rate (acute downregulation) was associated with a 3.8-fold decrease in NaPi-2 protein but no change in NaPi-2 mRNA abundance.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells.

PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Nao and 0.7 mM for Ko, is absolutely dependent on Clo and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 approximately 650 nM) or calyculin A (EC50 approximately 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF on cotransport rate is biphasic, with an initial rapid approximately 2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 approximately 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (> 2 days) leads to neurite formation and a maintained approximately 2-fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.

Pancreatic extract and the intestinal uptake of vitamin B12. II. Inhibitory effect of trypsin and trypsinogen.

Pancreatic extract (PE) contained small-molecular, thermo-stable as well as macro-molecular, thermo-labile factors capable of reducing the uptake of 57CoB12 bound to rat intrinsic factor by perfused rat intestinal segments (p less than 0.01 and p less than 0.01). Neither non-radioactive vitamin B12 nor non-pacreatic protein reduced the 57CoB12-uptake (p greater than 0.5 and p greater than 0.1) Crystalline trypsin and trypsinogen, but not chymotrypsin, also inhibited the uptake (p less than 0.05, p less than 0.02 and p greater than 0.05). The tryptic inhibition was abolished by soybean trypsin inhibitor (p greater than 0.05).

The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway.

Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al-Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).

Internal magnesium, 2,3-diphosphoglycerate, and the regulation of the steady-state volume of human red blood cells by the Na/K/2Cl cotransport system.

This study is concerned with the relationship between the Na/K/Cl cotransport system and the steady-state volume (MCV) of red blood cells. Cotransport rate was determined in unfractionated and density-separated red cells of different MCV from different donors to see whether cotransport differences contribute to the difference in the distribution of MCVs. Cotransport, studied in cells at their original MCVs, was determined as the bumetanide (10 microM)-sensitive 22Na efflux in the presence of ouabain (50 microM) after adjusting cellular Na (Nai) and Ki to achieve near maximal transport rates. This condition was chosen to rule out MCV-related differences in Nai and Ki that might contribute to differences in the net chemical driving force for cotransport. We found that in both unfractionated and density-separated red cells the cotransport rate was inversely correlated with MCV. MCV was correlated directly with red cell 2,3-diphosphoglycerate (DPG), whereas total red cell Mg was only slightly elevated in cells with high MCV. Thus intracellular free Mg (Mgifree) is evidently lower in red cells with high 2,3-DPG (i.e., high MCV) and vice versa. Results from flux measurements at their original MCVs, after altering Mgifree with the ionophore A23187, indicated a high Mgi sensitivity of cotransport: depletion of Mgifree inhibited and an elevation of Mgifree increased the cotransport rate. The apparent K0.5 for Mgifree was approximately 0.4 mM. Maximizing Mgifree at optimum Nai and Ki minimized the differences in cotransport rates among the different donors. It is concluded that the relative cotransport rate is regulated for cells in the steady state at their original cell volume, not by the number of copies of the cotransporter but by differences in Mgifree. The interindividual differences in Mgifree, determined primarily by differences in the 2,3-DPG content, are responsible for the differences in the relative cotransport activity that results in an inverse relationship with in vivo differences in MCV. Indirect evidence indicates that the relative cotransport rate, as indexed by Mgifree, is determined by the phosphorylated level of the cotransport system.