Early Onset Rapidly Progressive Osteoarthritis of the Hip in a Young Athlete.

We report and discuss a very rare case of early-stage rapidly progressive osteoarthritis (RPO) in a 33-year-old female athlete. The etiopathology of RPO remained unclear, although in this case mechanical overloading due to constant joint overuse appeared to be the only significant contributing factor to the very early development of RPO. Key words: rapidly progressive osteoarthritis, rapid destructive arthrosis, hip arthrosis, total hip arthroplasty, athlete, osteoarthritis.

[The bovine cartilage punch model: a tool for the in vitro analysis of biomaterials and cartilage regeneration]. / Das bovine Knorpelstanzenmodell : Ein Werkzeug zur In-vitro-Analyse von Biomaterialien und Knorpelregeneration.

BACKGROUND: The limited regeneration capacity of hyaline articular cartilage requires detailed studies concerning the tissue integration of cartilage transplants with meaningful but time and/or resource-consuming and in part ethically problematic animal models or, alternatively, with in vitro test systems for implant materials. MATERIAL AND METHODS: The present study describes a regeneration model with bovine cartilage rings (outer Ø 6 mm, central defect Ø 2 mm) for insertion, cultivation and biomechanical or histological testing of cartilage replacement materials (HE and safranin O staining). In this study, resorbable polymers composed of polyglycolic acid (PGA) were analyzed. RESULTS: Biomechanical testing showed a continuous decrease of the push-out force for the PGA inserts from the cartilage rings, probably due to the resorbability of the material. Histologically, clear immigration of cells into cell-free PGA was observed even after 4 weeks of culture, but in particular after 10 weeks. In addition, storage of proteoglycans was interpreted as an initial sign of the formation of new matrix. CONCLUSION: Thus, the new regeneration model is in principle suitable for the testing of biomaterials, but shows limitations in assessing the "lateral bonding" of resorbable materials.

[Gait analysis after minimally invasive total hip arthroplasty]. / Ganganalyse nach minimal-invasiver Hüftprothesenimplantation.

Upon implantation of a hip prosthesis by total hip arthroplasty (THA), clinical criteria are not always sufficient for an objective assessment of the functional outcome. Thus, functional improvement of gait behavior was comparatively validated by instrumented 3D gait analysis for a current, minimally invasive surgical approach (MIS; anterolateral approach) and a conventional, transgluteal approach (KONV). In selected cases, disturbed motion sequences were registered by measuring the muscle activity via high-resolution, monopolar surface electromyography (S-EMG) above the operation area. Despite continuous and significant improvement of practically all analyzed kinematic and kinetic gait parameters for both surgical approaches already after 5 weeks but in particular after 6 and 12 months, no significant differences were detected between the 2 procedures for any parameter or time point. The S-EMG demonstrated non-physiological muscle activation on the operated, but also on the non-operated side, even at 6 months after surgery. Advantages of the MIS approach thus seem primarily restricted to early, post-operative results, such as more rapid pain reduction and rehabilitation.

Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop.

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.

Analysis of the pinocytic process in rat kidney. I. Isolation of pinocytic vesicles from rat kidney cortex.

Pinocytosis was induced in rat kidney by exposure to horseradish peroxidase (HRP). Pinocytic vesicle preparations were enriched after homogenization of kidney cortex by differential centrifugation and free-flow electrophoresis with HRP as an exogenous marker. Vesicles were identified by enzymatic analysis and by electron microscopy, including specific staining procedures. Typical brush-border enzymes such as alkaline phosphatase, aminopeptidase, 5'-nucleotidase, lysosomal acid phosphatase, and mitochondrial succinic dehydrogenase were reduced in the vesicular fraction, compared to the kidney cortex homogenate. Glucose-6-phosphatase and Na(+)-K(+)-ATPase were only slightly increased in the fraction. These results indicate that preparations of pinocytic vesicles from rat kidney cortex can be enriched. They have biochemical characteristics that differ from those of the cell organelles and membranes previously purified from renal tissue.

The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings.

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

Discrimination between healthy and degenerated bovine articular cartilage with a fiber Bragg grating based microindenter.

OBJECTIVE: In this study we aim to show that an optical fiber Bragg grating-based microindentation system, which has the potential to be deployed arthroscopically, can differentiate between healthy and degenerated articular cartilage, which represents an important challenge in minimally-invasive surgery. DESIGN: Twenty bovine osteochondral cylinders, extracted from the patellar groove of ten 24 months old animals were subjected to stepwise in vitro stress-relaxation indentation measurements. The indentation procedure comprised 15 indentation steps of 20 µm each, reaching a total depth of 300 µm. Ten samples remained untreated and served as a control group for healthy cartilage. A second group of ten samples was treated for 12 h with an aqueous trypsin solution (concentration 2.5%) to deplete the proteoglycans. For both groups and all indentation depths deeper than 100 µm, the step response functions of a two elements Maxwell-Wiechert model fitted well to the measured relaxation curves. RESULTS: The standard deviations of the identified stiffness parameters within each group were much smaller than the difference of the average stiffness values between both groups. Based on the measured stiffness values, the system was capable to discriminate between healthy and degenerated cartilage with a high level of significance (p < 0.001). The experimental results are also discussed in terms of the biomechanical changes of cartilage under the action of trypsin. CONCLUSION: The fiber Bragg grating microindentation system showed the capability to differentiate intact and proteoglycan depleted cartilage with high significance.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Renal bicarbonate reabsorption in the rat. I. Effects of hypokalemia and carbonic anhydrase.

Free-flow micropuncture studies were carried out on superficial rat proximal and distal tubules to assess the participation of different nephron segments in bicarbonate transport. Particular emphasis was placed on the role of the distal tubule, and micro-calorimetric methods used to quantitate bicarbonate reabsorption. Experiments were carried out in control conditions, during dietary potassium withdrawal, and after acute intravenous infusions of carbonic anhydrase. We observed highly significant net bicarbonate reabsorption in normal acid-base conditions as evidenced by the maintenance of significant bicarbonate concentration gradients in the presence of vigorous fluid absorption. Distal bicarbonate reabsorption persisted in hypokalemic alkalosis and even steeper transepithelial concentration gradients of bicarbonate were maintained. Enhancement of net bicarbonate reabsorption followed the acute intravenous administration of carbonic anhydrase but was limited to the nephron segments between the late proximal and early distal tubule. The latter observation is consistent with a disequilibrium pH along the proximal straight tubule (S3 segment), the thick ascending limb of Henle, and/or the early distal tubule.

Taurocholate transport by rat liver canalicular membrane vesicles. Evidence for the presence of an Na+-independent transport system.

To elucidate the mechanism of vectorial translocation of bile acids in the liver, taurocholate transport was studied in isolated liver canalicular membrane vesicles by a rapid filtration method. The membrane vesicles revealed temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. In the absence of sodium, taurocholate uptake followed saturation kinetics (apparent Km for taurocholate = 43 microM and Vmax = 0.22 nmol/mg protein X 20 s at 37 degrees C) and was inhibited by cholate and probenecid. Transstimulation by unlabeled taurocholate was also demonstrated. When the electrical potential difference across the membranes was altered by anion replacement, a more positive intravesicular potential stimulated, and a more negative potential inhibited, transport of taurocholate by the vesicles. Valinomycin-induced K+-diffusion potential (vesicle inside-positive) enhanced the rate of taurocholate uptake that was not altered by imposed pH gradients. These results indicate that rat liver canalicular plasma membrane contains a sodium-independent taurocholate transport system that translocates the bile acid as an anion across the membrane. In intact hepatocytes, the electrical potential difference across the canalicular membrane probably provides the driving force for taurocholate secretion. The contribution of nonionic diffusion to taurocholate secretion appears to be minimal.

Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen.

Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis.

Cell volume regulation: osmolytes, osmolyte transport, and signal transduction.

In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.

Metabolism of the 'organic osmolyte' glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. I. Pathways for synthesis and degradation.

In isolated inner medullary collecting duct (IMCD) cells the adaptation to changes in extracellular osmolarity involves alterations in intracellular content of organic osmolytes such as glycerophosphorylcholine (GPC), sorbitol and others. To elucidate the basis of such alterations, the metabolism of GPC in IMCD cells was investigated with the labeled GPC precursor [methyl-3H]choline. The lipids phosphatidylcholine (PC), lyso PC (LPC) and sphingomyelin (SM), as well as the non lipids phosphorylcholine (Pcholine), GPC and an unknown water-soluble compound could be identified as intermediates of choline metabolism. In pulse-chase experiments the radioactivity of PC expressed as specific activity was at a higher level than the other metabolites (> 10-fold after 1h). Extended chase incubations caused the specific activity of PC and LPC to decrease significantly. GPC was the only metabolite with a significant increase in specific activity under these conditions, suggesting that PC (via LPC) could be the precursor of GPC. In short-term pulse experiments the specific activity of PC and LPC was always significantly higher compared to the specific activity of GPC. Pulse chase incubations using phosphatidyl[methyl-3H]choline showed a significant decrease in specific activity of PC after 15 h accompanied by a significant increase in specific activity of LPC as well as GPC. Inhibition of the PC hydrolyzing enzyme phospholipase A2 revealed a significant increase in the specific activity of PC. For GPC, a significant decrease in the radioactive labeling could be detected. The total amount of PC decreased by 10% under these conditions whereas the amount of GPC decreased by 22% which was significantly higher because of GPC breakdown. GPC degradation was catalyzed by GPC: choline diesterase generating choline (and phosphoglycerol). Significant activity of GPC:phosphocholine diesterase could not be detected. Betaine synthesis from choline was also not present. The slowest, and probably rate-limiting reaction of GPC synthesis from choline may be the reaction of phosphocholine cytidylyltransferase generating CDP choline, since no radioactive CDP choline could be detected under any conditions. Thus, isolated IMCD cells possess the ability for the synthesis of GPC from choline via PC and LPC, as well as for the GPC degradation to choline (and phosphoglycerol). Significant experimental evidence for the occurrence of de-novo synthesis of GPC from choline or a precursor function of GPC for PC could not be detected. However, although the former possibility seems unlikely, a final proof is still lacking.

Characterization of the renal epithelial cell line, PKE 5, using 31P- and 13C-NMR spectroscopy.

In order to characterize intracellular pH regulation and cellular metabolism in PKE 5 cells, a mutant of the renal epithelial cell line LLC-PK1 supposed to lack Na+-H+ exchanger activity, 31P and 13C-NMR studies were conducted. The 31P studies on intact cell suspensions revealed that these cells have an ATP content and an ATP/ADP ratio similar to the parent cell line. Their intracellular pH, in the presence of 5 mM HCO3-, was 7.17 +/- 0.04 (n = 5) - identical to that of LLC-PK1 cells. After acid loading the cells with 15% CO2, the initial rate of realkalinization was 0.027 pH units/min (n = 6), 50% lower than in the parent cells. The recovery rate was not affected by the removal of extracellular sodium or by the addition of 1 mM amiloride. These results indicate that PKE 5 cells are devoid of Na+-H+ exchange activity, but are able to regulate their intracellular pH by amiloride-insensitive, sodium-independent mechanisms. Extracts prepared from PKE 5 cells incubated with [13C]lactate showed 13C spectra identical to those of the parent cell line. In particular, no synthesis of 13C-labeled D-glucose was observed.

Metabolism of the 'organic osmolyte' glycerophosphorylcholine in isolated rat inner medullary collecting duct cells. II. Regulation by extracellular osmolality.

In isolated inner medullary collecting duct (IMCD) cells requirements for the organic osmolyte glycerophosphorylcholine (GPC) vary with extracellular osmolality. To investigate mechanisms of osmotic adaptation GPC metabolism was studied under different osmotic conditions. In contrast to the GPC precursors choline and phosphatidylcholine (PC) cellular GPC was proportional to the osmolality. Hypotonic decrease in cellular GPC was mediated by fast initial release significantly exceeding the low hypertonic release. In long-term studies the total amount of GPC decreased significantly under hypotonic conditions but remained constant under hypertonic conditions resulting in a significant difference after 15 h. To investigate osmotic influences on GPC synthesis and GPC degradation studies with [methyl-3H]choline were performed. Pulse-chase experiments displayed no significant osmotic differences in PC synthesis or in PC degradation to GPC indicated by a similar specific activity of PC. This suggested that phospholipase A2 (PC degradation) was osmotically insensitive. A small and distinct metabolic PC pool may be responsible for high radioactive labeling of newly synthesized GPC which displayed a significantly higher specific activity under hypotonic conditions accompanied by a decrease in GPC amount. Therefore, a higher activity of glycerophosphorylcholine:choline phosphodiesterase (GPC:choline phosphodiesterase) (GPC degradation) under hypotonic conditions is proposed. Similar conclusions can be drawn from using phosphatidyl[methyl-3H]choline. As further evidence for osmotic regulation of GPC:choline phosphodiesterase the specific activity of choline displayed a significant hypotonic increase with chase time which may be equivalent to increased GPC degradation. Therefore, the in vitro experiments suggest that cellular GPC is regulated by an osmosensitive GPC:choline phosphodiesterase. Such a regulation also seems to be present during long-term in vivo experiments. No evidence was found for a genetic adaptation of GPC:choline phosphodiesterase in vivo.

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles.

The temperature dependence of sodium-dependent and sodium-independent D-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent D-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15 degrees C. The sodium-independent uptake of D-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35-5 degrees C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4 degrees C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias.

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.

Interactions of alkylglucosides with the renal sodium/D-glucose cotransporter.

To study the nature of the glucose binding pocket of the renal Na+/D-glucose cotransporter, we have evaluated the inhibitory potency of various alkylglucosides (AG) on sodium-dependent D-glucose uptake into hog kidney brush border membrane vesicles (BBMV). Inhibition at 0.1 mM AG level was found to be strongly dependent on the anomeric configuration, on the length and on the flexibility of the side chain. Beta-n-AG inhibited transport significantly more effectively compared to the corresponding alpha-anomer (n-octylglucoside: alpha-anomer 15%, beta-anomer 84%) and AG with an unsaturated n-alkenyl side chain were significantly less effective inhibitors than the corresponding saturated compound (cis/trans 3-n-beta-hexenylglucoside 53% and 32%, beta-n-hexylglucoside 76%). A series of beta-n-AG increasing in side chain length from 1 to 13 carbon atoms revealed a global maximum in the inhibition pattern when beta-AG with side chains ranging from 8 to 11 carbon atoms were used, thus beta-methylglucoside inhibited glucose transport by 13%, beta-n-nonylglucoside by 92%, and beta-n-tridecylglucoside showed no effect. Kinetic analysis of inhibition by beta-n-octylglucoside revealed a fully competitive type of inhibition with an apparent K(i) of 10 +/- 2 microM. n-Octylglucoside at 0.1 mM did not inhibit sodium-dependent L-alanine uptake; similarly, n-octylmannoside at 0.1 mM level did not affect D-glucose uptake. These results suggest that the inhibition of sodium-dependent D-glucose uptake was, at least in the concentration range tested (up to 0.1 mM), not due to a detergent effect of AG, but due to interaction with the carrier. Optimum interaction requires a beta-anomer with a glycosidic bond that places the alkyl chain into an equatorial position with regard to the D-glucose molecule and the two main determinants of the sugar recognition site C2 and C3. In addition, the alkyl chain has to be highly flexible. The alkyl chains thus apparently interact with hydrophobic sites at the carrier in a slightly coiled conformation, thereby AG with a chain length up to 6 carbon atoms interact only with one hydrophobic site, AG with higher chain length probably with two sites.

Energetic coupling of Na-glucose cotransport.

(1) Energetic coupling in Na-linked glucose transport in renal brush border membrane vesicles has been studied in terms of various carrier models differing with respect to reaction order (random vs. ordered), and to rate limitation of steps within the routes of carrier-mediated solute transfer (translation across the membrane barrier vs. binding/release between carrier and bulk solution). (2) By computer simulation it was found that effective energetic coupling requires the leakage routes to be significantly, if not predominantly, rate-limited by their (barrier-crossing) translatory steps. This does not apply to the transfer route of the ternary complex, as coupling is possible whether or not this route is rate-limited by the translatory step. (3) The system transports glucose in the absence of Na+ (uniport) and the unidirectional flux is stimulated by unlabeled glucose on the trans side (negative tracer coupling). It is concluded that glucose binds to the carrier on either side without Na, as would be consistent with either a random system or one mode of ordered system with mirror symmetry (glucose binds before Na) but inconsistent with either mode of glide symmetry. The tracer coupling appears to indicate that the rate coefficient of carrier-mediated glucose transfer exceeds that of the empty carrier. (4) The Na-linked zero-trans flow of glucose in either direction is strongly trans-inhibited by Na. This consistent with a random system in which Na blocks or retards the translocation of the glucose-free carrier, thereby reducing 'slipping' through an internal leakage route. It is also consistent with the above mentioned ordered system, (i.e., in the absence of Na-transport without D-glucose) if it is assumed that trans Na interferes with the dissociation of the ternary complex, thereby slowing the release of glucose. (5) Minimum equilibrium exchange of glucose is stimulated in the presence of Na. This appears to indicate that Na expands the flow density of carrier-mediated glucose transfer. This expansion does not result from a 'velocity effect' (the ternary complex moving faster than the binary glucose carrier complex), as Na fails to stimulate maximum equilibrium exchange. It can instead be accounted for by an 'affinity effect' (the affinity of the carrier for glucose being increased by Na) as Na depresses the Michaelis constant of equilibrium exchange.(ABSTRACT TRUNCATED AT 400 WORDS)