pH dependence of protamine action on apical membrane permeability in Necturus gallbladder epithelium.

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

Osmotic properties of mitochondria.

The osmotic behavior of rat liver mitochondria has been studied in a sucrose medium. The mitochondria behave like a two compartment system. One compartment is permeable to sucrose and has a volume of 1.22 microl/(mg mitochondrial dry weight) in a 272 milliosmol sucrose medium; the second, inaccessible to sucrose, has a volume of 0.555 microl/mg dry weight) under the same conditions. Part of the water in the sucrose inaccessible space is apparently not free to participate in osmotic phenomena. This volume is 0.272 microl/(mg dry weight) under the same conditions. It is suggested that the osmotically inactive water corresponds to the water of hydration of the mitochondrial macromolecules. The volume of the remainder of the water in the sucrose inaccessible space depends inversely on the osmolality of the medium, as is to be expected. The volume of water in the sucrose accessible space is constant, independent of the osmolality of the medium, as is the volume of the mitochondrial framework plus the nonvolatile solutes.

Osmotic volume flow in the proximal tubule of Necturus kidney.

Volume changes due to osmotic flow in the distal portion of proximal tubules of Necturi were measured by the split oil drop technique. In agreement with previous findings no volume flow was induced by NaCl concentrations close to 60 mM. The tubule wall was found to be permeable to plasma electrolytes, which have an apparent reflection coefficient of 0.69. The mean apparent hydraulic conductivity was 0.33 x 10(-11) cm(3)/dyne sec, comparable with other epithelia. A number of lipid-insoluble nonelectrolytes of widely varying molecular size had apparent reflection coefficients of about 0.5. In view of the insensitivity to molecular size it seems likely that apparent reflection coefficients determined from tubular volume changes depend primarily on the porosity of the intercellular barrier closest to the lumen and give little information about the subsequent fate of the test substances.

Nodular glomerulosclerosis associated with multiple myeloma. Role of light chain isoelectric point.

A 68-year-old female patient with multiple myeloma exhibited advanced nodular glomerulosclerosis. Immunofluorescence of the kidney showed kappa light chain deposition in the mesangium and in glomerular and tubular basement membrane. Isoelectric focusing and immunofixation of urinary proteins revealed an isolated kappa light chain with an unusually high isoelectric point of 8.4. Most light chain proteins have isoelectric points in the 4.6 to 6.7 range. Since loss of fixed negative charges may precede experimental glomerulosclerosis, it is proposed that this cationic circulating kappa chain may have interacted with glomerular polyanion, thereby inducing a nodular sclerotic reaction leading to irreversible renal damage.

Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha.

The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor-alpha TNF-alpha, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF-alpha. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokine-secreting macrophages--for example, leak flux diarrhea and invasion of bacterial enterotoxins.

Clinical models of intestinal adaptation.

Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na-glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self-filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na-glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor-alpha can specifically downregulate the tight junction, as indicated in the intestinal HT-29/B6 cell model.

Hypertension in the elderly.

Successful control of blood pressure through drug therapy can reduce morbidity and mortality in the elderly population. However, age-related factors also make this group prone to adverse side effects. A cautious approach is therefore recommended in the effort to achieve ideal control of blood pressure.

Permeability changes in Necturus proximal tubule during volume expansion.

The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.

Osmotic water flow pathways across Necturus gallbladder: role of the tight junction.

To explore the quantitative significance of passive water flow through tight junctions of leaky epithelia, transepithelial water flow rates were measured in Necturus gallbladder mounted in chambers. Osmotic flows generated by raffinose gradients were asymmetrical with the greater flow in the mucosal-to-serosal direction. In tissue fixed in situ, intercellular spaces were dilated during mucosal-to-serosal flow and closed during serosal-to-mucosal flow. Tight junctions were focally separated (blistered), which correlated with the magnitude of mucosal-to-serosal flow. Blisters were not observed during serosal-to-mucosal flow or in nontransporting gallbladders. In freeze-fracture replicas, blisters appeared as pockets between intramembranous strands. Protamine, which decreases electrical conductance and increases depth and complexity of the tight junction, reduced osmotic water flow by approximately 30% in the mucosal-to-serosal direction (100 mosmol/kg gradient) without altering serosal-to-mucosal flow. We suggest that in the steady state, at least 30% of osmotically driven water passes transjunctionally in the mucosal-to-serosal direction, but flow is transcellular in the serosal-to-mucosal direction. Directionally divergent pathways may account for flow asymmetry.

Structural alterations in rat kidney proximal tubules perfused with fresh autologous serum.

Two min of intraluminal perfusion of the rat proximal tubules with autologous serum induced marked ultrastructural alterations including extensive cytoplasmic vesiculation due to swelling of rough endoplasmic reticulum cisternae and occasional extrusion of nuclei and cytoplasm into the lumen. Within 4 min pronounced vesiculation of mitochondria was observed. These findings are consistent with the notion that serum-induced inhibition of proximal tubular fluid absorption is due to cell lysis, presumably mediated by complement activation.

Prostaglandin and thromboxane synthesis by highly enriched rabbit proximal tubular cells in culture.

Prostaglandin synthetic profiles were studied in monolayers of highly enriched rabbit renal proximal tubular cells cultured in serum-free, hormone-supplemented, defined media. The cultures were initiated from glomeruli-free cortical suspensions. Cells in culture demonstrated morphologic and functional characteristics highly suggestive of proximal tubular cells. The basal and stimulated synthesis of immunoassayable prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in response to various agonists, as well as the effect of two cyclooxygenase inhibitors, was assessed. Under both basal and stimulated conditions, PGE2 was the major product synthesized. PGF2 alpha and 6-keto-PGF1 alpha were synthesized to a lesser extent, and TxB2 was undetectable. The basal synthesis of PGE2 and PGF2 alpha in cultured cells was found to be higher than in isolated proximal tubular fragments by sevenfold and fivefold, respectively. Exogenous arachidonate, angiotensin II, and the divalent cation ionophore A23187 stimulated all three immunoassayable prostaglandins in a dose-dependent manner. Arginine vasopressin (10(-5) mol/L) had no stimulatory effect. In Ca++-free media or in the presence of 10(-5) mol/L Ca++ channel blocker, verapamil, the stimulatory effects of angiotensin II and A23187 were ameliorated. The stimulatory effect of angiotensin II was inhibited by saralasin (10(-5) mol/L), indicating that receptor binding could mediate PGE2 synthesis. Both indomethacin and sulindac sulfide (10(-5) mol/L) reversibly inhibited PGE2 synthesis.

Effect of protamine on the permeability and structure of rat peritoneum.

The effect of protamine, a polycationic protein, on the mesothelial permeability and ultrastructure was evaluated in a rat model of peritoneal dialysis. The peritoneal permeability to urea and inulin were measured after the intraperitoneal instillation of protamine sulfate in varying concentrations. A functional correlation was made with the ultrastructure of omentum. Protamine concentrations between 5 and 30 micrograms/ml decreased the permeability to inulin without significantly altering that of urea. At concentrations from 30 to 75 micrograms/ml, protamine increased permeability to urea by 50% and to inulin by 20%. Mesothelial cells revealed a loss of microvilli and minor degrees of disorganization of submembranous, cytoplasmic microfilaments without significant changes in the intramembranous structure of occluding junctions. Effects were partially reversible. At a concentration of 100 micrograms/ml, there was an irreversible doubling in permeability to inulin without a comparable effect on permeability to urea associated with the disruption of occluding junctions in focal areas. This complex response to protamine suggests at least two transperitoneal diffusion pathways, transcellular and paracellular. The mesothelial cell occluding junctions may be a diffusion barrier in the latter pathway.

Emergency department hemodialysis in a case of severe ethylene glycol poisoning.

A 36-year-old man with a history of depression presented to the emergency department after ingesting approximately 3,000 mL of ethylene glycol antifreeze in a suicide attempt. The patient's ethylene glycol concentration, 1,889 mg/dL, was higher than any level previously documented in the medical literature. Although his course was complicated by nausea, emesis, lethargy, metabolic acidosis, and kidney failure, the patient survived without persistent kidney failure or other chronic problems. Sustained hemodialysis and ethanol infusion were instituted in the ED, on the basis of the patient's history, before laboratory confirmation of the ingestion was obtained.

Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane.

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture.

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.

Epithelial tight junction structure in the jejunum of children with acute and treated celiac sprue.

Tight junction morphology was analyzed in freeze fracture electron micrographs from biopsies at two locations along the surface-crypt axis in the jejunum of children with treated and untreated sprue and in control subjects. In control jejunum, strand number, meshwork depth, and total depth of the tight junction decreased from surface to crypt, consistent with the concept of the crypt being more permeable than the surface epithelium. In acute sprue, strand number was reduced in all regions along the surface-crypt axis, from 5.5+/-0.2 to 3.4+/-0.3 (surface) and from 4.7+/-0.2 to 3.6+/-0.1 (crypt). Meshwork depth was also reduced at all regions along the surface-crypt axis. Strand discontinuities were more frequent in acute sprue. Aberrant strands appeared below the main meshwork of crypt tight junctions in acute sprue. In asymptomatic children treated with the gluten-free diet, jejunal tight junctional structure only partially recovered. Strand number was restored to normal at the surface, but was still decreased in the crypts, from 4.7+/-0.2 to 3.9+/-0.3. We conclude that the epithelial barrier function of the small intestine is seriously disturbed by structural modifications of the tight junction in acute symptomatic celiac disease, thereby accounting for increased ionic permeability noted in a parallel study on identical specimens. This epithelial barrier defect may contribute to diarrhea in celiac disease by a "leak flux mechanism." In children with sprue treated with a gluten-free diet, barrier dysfunction was only partly recovered, suggesting a level of "minimal damage."

Peritoneal permeability in the rat: modulation by microfilament-active agents.

A model of peritoneal dialysis in the rat was used to determine the effects of cytochalasins on ultrastructure and peritoneal permeability to molecules of varying molecular weight. The permeability to urea, inulin, and plasma albumin were determined after intraperitoneal administration of cytochalasin B (2 to 10 X 10(-6) M) and cytochalasins D and E (2 X 10(-6) M). Cytochalasin B (20 X 10(-6) M) increased the permeability to inulin, urea, and albumin by 30, 60, and 150%, respectively. These effects were, to a large degree, reversible. Cytochalasins D and E produced greater increments in permeability for all molecules; this increase was only partially reversible. Ultrastructure analysis by scanning electron microscopy revealed extensive development of membrane protuberances (zeiotic knobs) on mesothelial cells exposed to cytochalasin B. A return to a normal apical cell surface was apparent although incomplete at 24 hr. Tight junctions were not grossly altered and major changes in intramembranous junctional strands were not observed. The major effect of cytochalasins on the cell surface may be responsible for the increased permeability to urea, predominately a transcellular probe. Inulin, which follows a paracellular route, was less affected. Altered protein permeability may be due to the action of cytochalasin on the exposed capillary endothelium in subdiaphragmatic areas where the mesothelium is discontinuous.