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Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

A physics-based model for the sublimation-transport-condensation processes occurring in pharmaceutical freeze-drying by coupling product attributes and equipment capabilities into a unified simulation framework is presented. The system-level model is used to determine the effect of operating conditions such as shelf temperature, chamber pressure, and the load size on occurrence of choking for a production-scale dryer. Several data sets corresponding to production-scale runs with a load from 120 to 485 L have been compared with simulations. A subset of data is used for calibration, whereas another data set corresponding to a load of 150 L is used for model validation. The model predictions for both the onset and extent of choking as well as for the measured product temperature agree well with the production-scale measurements. Additionally, we study the effect of resistance to vapor transport presented by the duct with a valve and a baffle in the production-scale freeze-dryer. Computation Fluid Dynamics (CFD) techniques augmented with a system-level unsteady heat and mass transfer model allow to predict dynamic process conditions taking into consideration specific dryer design. CFD modeling of flow structure in the duct presented here for a production-scale freeze-dryer quantifies the benefit of reducing the obstruction to the flow through several design modifications. It is found that the use of a combined valve-baffle system can increase vapor flow rate by a factor of 2.2. Moreover, minor design changes such as moving the baffle downstream by about 10 cm can increase the flow rate by 54%. The proposed design changes can increase drying rates, improve efficiency, and reduce cycle times due to fewer obstructions in the vapor flow path. The comprehensive simulation framework combining the system-level model and the detailed CFD computations can provide a process analytical tool for more efficient and robust freeze-drying of bio-pharmaceuticals., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Few afflictions have attracted as much attention and impacted on as many societal and biomedical areas as cholera. Dr. John Snow's studies launched the field of epidemiology, were early applications of medical cartography, and promoted the use of statistical methods in medicine. The finding that cholera was due to the ingestion of contaminated water lent to the demise of the prevalent "miasmatic theory of contagion," set the platform for the "germ theory of disease," and promoted the growth of public health concerns for water purification and sanitation. More recent attention to this disease led to the notion of "secretory diarrhea" and the translation of basic principles to the development of oral rehydration therapy and its "spin-offs" (Gatorade and Pedilyte).

Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.

Background: Recent reports suggest that enteral diets enriched with arginine may be harmful by enhancing inflammation. This is consistent with our gut ischemia/reperfusion (I/R) model in which arginine induced the proinflammatory mediator inducible nitric oxide synthase and resulted in injury and inflammation whereas glutamine was protective. We now hypothesize that arginine and glutamine differentially modulate the early proinflammatory transcription factors activated by gut I/R., Methods: At laparotomy, jejunal sacs were filled with either 60 mmol/L glutamine, arginine, or an iso-osmotic control followed by 60 minutes of superior mesenteric artery occlusion and 6 hours of reperfusion and compared with shams. Jejunum was harvested for nuclear factor (NF)-kappaB and activator protein-1 (AP-1) measured by electrophoretic mobility shift assay and c-jun and c-fos (AP-1 family) by supershift., Results: Both NF-kappaB and AP-1 were activated by gut I/R. Arginine and glutamine had no differential effect on NF-kappaB, whereas AP-1 expression (c-jun but not c-fos) was markedly enhanced by arginine and significantly lessened by glutamine., Conclusion: Arginine enhanced expression of the early proinflammatory transcription factor AP-1 but not NF-kappaB. This represents a novel mechanism by which arginine may be harmful when administered to critically ill patients.

Background: Immune-enhancing enteral diets have been shown to improve patient outcome. One contributing mechanism may be via maintenance of gut barrier function. While recent data has shown that glutamine is beneficial, arginine may be harmful. We therefore hypothesized that the immune-enhancing agents, glutamine and arginine, differentially modulate gut barrier function., Methods: At laparotomy, rats had jejunal sacs filled with 10 mmol/L glutamine, arginine, fructose, or magnesium sulfate (osmotic control) followed by 60 minutes of superior mesenteric artery occlusion and 2 hours of reperfusion. Jejunum was harvested for histology, deconvolution microscopy, F:G actin, ATP, and permeability measurements., Results: Glutamine and fructose minimized mucosal injury compared with controls and arginine. Deconvolution microscopy confirmed that glutamine and fructose preserved the actin cytoskeleton but there was disruption by arginine which correlated with F:G actin ratios and tissue ATP levels. Permeability was enhanced by arginine compared with the other groups., Conclusion: Arginine resulted in worsened mucosal injury, disruption of the actin cytoskeleton, decreased tissue ATP and enhanced permeability compared with glutamine which appeared protective. The immune-enhancing agent arginine results in breakdown of gut barrier function which may have important implications for critically injured patients.

We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.

Background & Aims: Gastrointestinal function may be impaired after severe injury, hampering tolerance to enteral nutrition. The purpose of this study was to determine how different sodium-coupled solutes modulate gut function after ischemia/reperfusion (I/R) in a rodent model., Methods: At laparotomy, rats had jejunal sacs filled with (glucose + alanine), glucose, glutamine, alanine, or mannitol (osmotic control), followed by superior mesenteric artery clamping for 60 minutes and 30 minutes of reperfusion. After reperfusion, sacs were harvested for morphologic examination, adenosine triphosphate (ATP) assay, or mounted in an Ussing chamber., Results: Small intestinal epithelial absorptive capacity, as assessed by changes in short-circuit current in response to glucose, after gut I/R, was decreased by alanine or (glucose + alanine) but not glucose or glutamine alone and correlated with changes in tissue ATP. Gut I/R caused a significant morphologic injury that was worsened by alanine or (glucose + alanine) but lessened by glucose or glutamine alone., Conclusions: During gut I/R, alanine can enhance gut injury, deplete energy (ATP), and impair gut absorption, whereas glucose or glutamine is protective against injury and can maintain absorptive capacity and ATP. These results suggest that solutes (such as alanine), which further stress an already metabolically stressed bowel, should be cautiously administered to critically ill patients whereas those solutes that contribute to energy production (such as glucose and glutamine) may be safely continued.

It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.

We have previously reported that ATP-inhibitable K(+) channels, in vesicles derived from the basolateral membrane of Necturus maculosus small intestinal cells, exhibit volume regulatory responses that resemble those found in the intact tissue after exposure to anisotonic solutions. We now report that increases in K(+) channel activity can also be elicited by exposure of these vesicles to isotonic solutions containing glucose or alanine that equilibrate across these membranes. We also demonstrate that swelling after exposure to a hypotonic solution or an isotonic solution containing alanine or glucose reduces inhibition of channel activity by ATP and that this finding cannot be simply attributed to dilution of intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K(+) channels may be responsible for the well-established increase in basolateral membrane K(+) conductance of Necturus small intestinal cells after the addition of sugars or amino acids to the solution perfusing the mucosal surface, and we propose that increases in cell volume, resulting in membrane stretch, decreases the sensitivity of these channels to ATP.

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.

86Rb fluxes through ATP-regulated K+ (KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phosphoenolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2, 3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+ pumps to KATP channels in the basolateral membrane, raise the possibility that "cross talk" between those channels and pumps (i.e., the "pump-leak parallelism") may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.

During the past century, the generally accepted view of the function of biological membranes has evolved from that of a static, inert lipoprotein envelope that simply separates the intracellular machinery from the outside milieu, to that of a dynamic structure that is, in fact, an integral part of that machinery actively involved in homeostasis and bioenergetics. This brief review traces that paradigm shift with special emphasis on the evolution of central concepts in epithelial transport physiology.

In all Na(+)-absorbing and Cl(-)-secreting epithelia, an increase in the activity of the Na+,K(+)-pump at the basolateral membrane is accompanied by an increase in the K+ conductance of that barrier and vice versa. We have recently identified an ATP-regulated K+ channel, K(ATP), in basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells that could be responsible for this parallelism between pump activity and leak. Thus, an increase in pump activity would result in a decrease in local ATP activity and an increase in local ADP activity and, in turn, an increase in the open-probability of the channel whereas a decrease in in pump activity would have the opposite effect. Further, the likelihood that the number of pumps far exceeds the number of leaks per unit area of membrane suggests that the ATP and ADP activities that influence K(ATP) channel activity may differ markedly from the "bulk" cytoplasmic values.

K+ channels present in basolateral membrane vesicles isolated from Necturus maculosa small intestinal cells and reconstituted into planar phospholipid bilayers are inhibited by MgATP and sulfonylurea derivatives, such as tolbutamide and glibenclamide, when these agents are added to the solution bathing the inner mouth of the channel. In addition, these channels possess an intrinsic "voltage gate" and are blocked when the electrical potential difference across the channel is oriented so that the inner solution is electrically positive with respect to the outer solution. We now show that increasing the concentration of permeant ions such as K+ or Rb+ in the outer solution reverses channel inhibition resulting from the addition of 50 microM glibenclamide to the inner solution and also inhibits intrinsic voltage gating; these effects are not elicited by increasing the concentrations of the relatively impermeant ions, Na+ or choline, in the outer solution. Furthermore, increasing the K+ concentration in the outer solution in the absence of glibenclamide inhibits voltage gating, and, under these conditions, the subsequent addition of glibenclamide to the inner solution is ineffective. These results are consistent with a model in which the voltage gate is an open-channel blocker whose action is directly reversed by elevating the external concentration of relatively permeant cations and where the action of glibenclamide is to stabilize the inactivated state of the channel, possibly through hydrophobic interactions.

We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.

Using calmodulin-affinity chromatography, we have isolated a fraction of proteins from solubilized basolateral membranes of rabbit colonocytes which when reconstituted into planar phospholipid bilayers disclosed Ca(2+)-activated single K+ channel activities. The properties of the reconstituted channels are identical to those of native membrane vesicles incorporated into these bilayers with respect to their high selectivity for K+ over C-, high ("maxi") conductance, voltage gating, and inhibition by trifluoperazine. Two-dimensional sodium dodecyl sulfate gel electrophoresis of these proteins revealed three major protein species with molecular masses of 120, 60, and 35 kDa, which constituted 70, 10, and 20%, respectively, of the total protein. The results of other studies strongly suggest that the 35-kDa protein may be the Ca(2+)-activated K+ channel protein in these membranes.

Intracellular sodium activities, (Na)c, were determined in Necturus small intestine before and after addition of galactose to the mucosal bathing solution. In the absence of galactose, (Na)c averaged 12 millimoles per liter. Within 2 minutes after the addition of galactose to the mucosal solution, (Na)c increased to a mean value of 20 millimoles per liter and then declined, in parallel with an increase in transcellular sodium transport, to a value that did not differ significantly from that observed in the absence of the sugar. The final steady state in the presence of galactose was characterized by a three- to fourfold increase in the rate of transcellular Na+ transport in the absence of a significant increase in (Na)c. Thus, the increase in steady-state basolateral pump activity cannot be attributed to an increase in the intracellular sodium transport pool.

An equivalent electrical circuit model for Na transport across epithelial tissues under steady-state conditions, which incorporates the flows and forces across the two limiting membranes and the paracellular pathway, is described. The analysis of electrophysiologic data obtained on rabbit colon within the framework of this model provides information regarding the thermodynamic activity of cell Na and the resistance offered by the mucosal membrane to Na entry. The interpretation of the data dealing with the active extrusion of Na from the cell across the basolateral membrane awaits more detailed information regarding the pump mechanism responsible for this movement; until then, the electromotive force across the basolateral membrane and the resistance of that barrier must be considered phenomenologic parameters that relate the Na current to the observed electrical potential difference.

Previous studies have shown that, immediately after the addition of galactose or alanine to the solution bathing the mucosal surface of Necturus small intestine, there is a rapid depolarization of the electrical potential difference across the mucosal membrane (psi mc). This is followed by a repolarization of psi mc that is paralleled by an increase in the ratio of the effective resistance of the mucosal membrane to that of the basolateral membrane (rm/rs); the latter was shown to be, at least in part, due to a marked increase in the conductance of the basolateral membrane. We now report the following. (i) Exposure of this epithelium to a 12% hypotonic solution results in a hyperpolarization of psi mc and an increase in rm/rs. These effects are blocked by metabolic inhibitors and by the presence of 5 mM Ba2+ in the bathing solution; indeed, in the presence of Ba2+, psi mc depolarizes and rm/rs decreases to low values. (ii) Addition of 15 mM galactose to the mucosal solution when the serosal solution alone contains 5 mM Ba2+ results in a depolarization of psi mc but no subsequent repolarization of psi mc or increase in rm/rs; however, psi mc repolarizes and rm/rs increases when Ba2+ is subsequently removed from the serosal bathing solution. We conclude that (i) the basolateral membrane normally possesses a Ba2+-inhibitable K conductance, which appears to be reduced in the presence of metabolic inhibitors; (ii) after exposure of the tissue to a hypotonic solution or the addition of galactose to the mucosal solution, this conductance increases; and (iii) these responses can be blocked by metabolic inhibitors. These findings suggest that the delayed response of this tissue to the addition of sugars or amino acids to the mucosal solution may be the result of cell swelling resulting from the intracellular accumulation of these solutes in osmotically active forms.

The current-voltage relations of the amiloride-sensitive Na entry pathway across the apical membrane of rabbit descending colon, exposed to a high K serosal solution, were determined in the presence of varying mucosal Na activities, (Na)m, ranging from 6.2 to 99.4 mM. These relations could be closely fit to the "constant field" flux equation yielding estimates of the permeability of the apical membrane to Na, PmNa, and the intracellular Na activity, (Na)c. The following empirical relations emerged: (Na)c increased hyperbolically with increasing (Na)m; PmNa decreased hyperbolically with increasing (Na)m and linearly with increasing (Na)c; spontaneous variations in Na entry rate at constant (Na)m could be attributed entirely to parallel, spontaneous variations in PmNa; the rate of Na entry increased hyperbolically with increasing (Na)m obeying simple Michaelis-Menten kinetics; the relation between (Na)c and "pump rate," however, was sharply sigmoidal and could be fit by the Hill equation assuming strong cooperative interactions between Na and multiple sites on the pump; the Hill coefficient was 2-3 and the value of (Na)c at which the pump-rate is half-maximal was 24 mM. The results provide an internally consistent set of relations among Na entry across the apical membrane, the intracellular Na activity and basolateral pump rate that is also consistent with data previously reported for this and other Na-absorbing epithelia.

Segments of descending colon obtained from rabbits, that had been maintained on drinking water containing 25 mM NaCl and an artificial diet which contains 1% Na and is nominally K-free, respond to aldosterone in vitro (after a 30 to 60-min lag period) with a marked increase in the short-circuit current (Isc), an equivalent increase in the rate of active Na absorption (JNa net) and a decline in tissue resistance (Rt). Aldosterone also brings about a marked increase in the unidirection influx of Na into the cells across the mucosal membrane ("zero-time" rate of uptake) which does not differ significantly from the inrease m Isc. Treatment of control tissues with amphotericin B brings about sustained increases in Isc and JNa net to levels observed in aldosterone-treated tissues. However, addition of amphotericin B to the mucosal solution of aldosterone-treated tissues does not result in a sustained increase in Isc or JNa net and these values do not differ markedly from those observed in control tissues treated with amphotericin B. These findings, together with other evidence that Na entry in the presence of amphotericin B is sufficiently rapid to saturate the active Na extrusion mechanism at the baso-lateral membrane, are consistent with the notion that the aldosterone-induced protein increases the permeability of the mucosal membrane to Na but does not increase the "saturation level" of the active Na "pump" within the time-frame of these studies (3 hr). Finally, aldosterone has no effect on the bidirectional or net transepithelial movements of K under short-circuit conditions, suggesting that the enhanced secretion of K observed in vivo is the result of increased diffusion of K from plasma to lumen via paracellular pathways in response to an increased transepithelial electrical potential difference (lumen negative).

Replacement of Cl in the solutions bathing partial mucosal strips of rabbit descending colon with sulfate, isethionate, hydroxypropane-sulfonate and, to a lesser degree, ethanesulfonate stimulates active Na absorption (JNanet) when the baso-lateral pump mechanism is not saturated. These effects are rapid in onset and are readily reversible. Our findings indicate that these stimulatory anions decrease the resistance of the amiloridesensitive Na entry step at the mucosal membrane (RmNa). However, when the active Na pump mechanism at the baso-lateral membrane is saturated these stimulatory anions do not decrease the resistance of the Na entry process. These findings suggest the presence of a negative feedback between the activity of the pump mechanism and the resistance of the Na entry step which may be mediated by the size of the intracellular Na transport pool. In other words, it seems that when the baso-lateral pump is operating at its maximal rate the resistance to Na entry across the mucosal membrane through the amiloride-sensitive pathway is at a minimum and cannot be further decreased.

The addition of 10 mM glycine to a physiological saline bathing Ehrlich ascites tumor cells is followed by a slow increase in cell volume that plateaus between 15 and 30 min at a level approximately equal to 17% greater than the control volume; this increase is not observed when glycine is added to cells suspended in a Na+-free saline. The results of studies using the patch-clamp technique in the cell-attached mode indicate that, 0.5-3 min after the addition of glycine to the bathing solution, there is a marked increase in the activity of single channels, which is almost all instances were previously present and operant in the plasma membrane. Successfully excised patches of membrane that contained a channel stimulated by glycine fell into two categories. Some became inactive within 15 sec in spite of the fact that the G omega seal remained intact. Others persisted for the lifetime of the seal. All of the persistent channels had an 11-fold selectivity for Cl- over K+ and a conductance of 23 pS when bathed by symmetrical 150 mM KCl solutions. Although the ionic specificities of the other channels have not been identified, there is reason to suspect that they might be K+ channels whose activities are dependent on factors lost when the patch is excised. Swelling induced by exposing these cells to a 50% hypotonic perfusate stimulated the activities of Cl- channels whose properties closely resemble those stimulated by the addition of glycine to the perfusate, strongly suggesting that the glycine-induced stimulation of Cl- channel activity is part of a volume-regulatory response to cell swelling. If the increase in channel activity induced by the addition of glycine to the perfusate is indeed a response to cell swelling, then this perfusate is indeed a response to cell swelling, then this volume-regulatory response must be extremely sensitive inasmuch as it appears to be "triggered" by an average increase in cell volume that does not exceed 5%.

Intracellular chloride activities, (Cl)c, and the electrical potential difference across the mucosal membrane, psimc, were determined in the isolated small intestine of the winter flounder, using Cl-selective and conventional (KCl-filled) microelectrodes. In the presence of a Na-containing buffer psimc averages -69 mV and (Cl)c averages 24 mM, a value that is 3.4 times that predicted for an equilibrium distribution across the mucosal membrane. On the other hand, when the tissue is then perfused with Na-free buffer, (Cl)c slowly falls to a value that does not differ significantly from that predicted for an equilibrium distribution, and psimc depolarizes significantly. Finally, when the tissue is again bathed in the Na-containing buffer, (Cl)c rapidly returns to a value well above equilibrium. These results, together with those of Frizzell et al. (J. Membrane Biol. 46:27, 1979), provide direct evidence that: (1) Cl is accumulated against its electrochemical potential difference (32 mV) by this tissue, and (2) this accumulation is coupled to and energized by the entry of Na down its steep electrochemical potential difference.

The effects of the addition of graded concentrations of amiloride, (A)m, to the mucosal bathing solution on the permeability of the apical membrane of rabbit descending colon to Na (PmNa) were determined when the Na activity in the mucosal bathing solution, (Na)m, was 18, 32 or 100 mM. PmNa was obtained from current-voltage relations determined on tissues bathed with a high-K serosal solution before and after the addition of a maximally inhibitory concentration of amiloride to the mucosal solution as described by Turnheim et al. (Turnheim, K., Thompson, S.M., Schultz, S.G. 1983. J. Membrane Biol. 76:299-309). The results indicate that: (1) As demonstrated previously (Turnheim et al., 1983), PmNa decreases with increasing (Na)m. (2) PmNa also decreases hyperbolically with increasing (A)m. Kinetic analyses of the effect of amiloride on PmNa are consistent with the conclusions that: (i) the stoichiometry between the interaction of amiloride with apical membrane receptors that results in a decrease in PmNa is one-for-one; (ii) there is no evidence for cooperativity between amiloride and these binding sites; (iii) the value of (A)m needed to halve PmNa at a fixed (Na)m is 0.6-1.0 microM; and, (iv) this value is independent of (Na)m over the fivefold range studied. These findings are consistent with the notion that the sites with which amiloride interacts to bring about closure of the channels through which Na crosses the apical membrane are kinetically distinct from the sites with which (Na)m interacts to bring about closure (i.e., "self-inhibition"). In short, the effects of (Na)m and (A)m on PmNa in this tissue appear to be independent and additive.

The results of the present study that NaCl transport by in vitro rabbit gallbladder must be a consequence of a neutral coupled carrier-mediated mechanism that ultimately results in the active absorption of both ions; pure electrical coupling between the movements of Na and Cl can be excluded on the grounds of electrphysiologic considerations. Studies on the unidirectional influxes of Na and Cl have localized the site of this coupled mechanism to the mucosal membranes. Studies on the intracellular ion concentrations and the intracellular electrical potential are consistent with the notion that (a) the coupled NaCl influx process results in the movement of Cl from the mucosal solution into the cell against an apparent electrochemical potential difference; (b) the energy for the uphill movement of Cl is derived from the Na gradient across the mucosal membrane which is maintained by an active Na extrusion mechanism located at the basolateral membranes; and (c) Cl exit from the cell across the basolateral membranes is directed down an electrochemical potential gradient and may be diffusional. Finally, as for the case of rabbit ileum, the coupled NaCl influx process is inhibited by elevated intracellular levels of cyclic 3',5'-adenosine monophosphate. A working model for transcellular and paracellular NaCl transport by in vitro rabbit gallbladder is proposed.