Biomedical subjects

R K Kinne

Publications and source records attributed to R K Kinne.

At least 73 records · Page 4Linked to original sources

PubMed1995

Renal inner medullary sorbitol metabolism.

Sorbitol participates in the osmoregulation of several renal cells and has also been found in isolated inner medullary collecting duct (IMCD) cells in primary culture. Therefore, osmotic regulation and distribution of sorbitol and the key enzymes of sorbitol metabolism, aldose reductase and sorbitol dehydrogenase in the renal inner medulla, were investigated in vivo under various osmotic conditions (control, diuresis, antidiuresis). In homogenates of the renal inner medulla of Wistar rats, the sorbitol content correlated with the urine osmolarity [68 +/- 12 mumol/g protein (control), 28 +/- 9 mumol/g (diuresis), 110 +/- 15 mumol/g (antidiuresis)]. Similar results were obtained for the activity of aldose reductase (sorbitol synthesis) [25 +/- 4 U/g (control), 19 +/- 3 U/g (diuresis), and 48 +/- 7 U/g (antidiuresis)]. On the contrary, the activity of sorbitol dehydrogenase (sorbitol degradation) was significantly increased to 1.26 +/- 0.42 U/g under diuretic conditions vs. control (0.84 +/- 0.14 U/g, P < 0.05). These results demonstrate the correlation between the enzymes of sorbitol synthesis and sorbitol degradation in the intact inner medulla and the urine osmolarity in vivo. Whereas the aldose reductase activity was 2.3-fold enriched in IMCD cells, the specific activity of sorbitol dehydrogenase was relatively increased in a preparation of enriched interstitial cells. This distribution was not dependent on the various diuretic conditions. These results indicate that enzymes of synthesis and of degradation of sorbitol are osmotically regulated in vivo. Therefore, the enzymatic activities of sorbitol synthesis appear to be primarily located in epithelial cells, whereas enzymatic activities of sorbitol degradation seem to be localized in interstitial cells of the renal inner medulla.

Aldehyde Reductase↗

PubMed1994

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.

Amino Acid Sequence↗

PubMed1994

Intracellular calcium in primary cultures of rat renal inner medullary collecting duct cells during variations of extracellular osmolality.

There is ample evidence of calcium being an intracellular second messenger during volume regulatory processes in various cells including inner medullary collecting duct (IMCD) cells. Therefore, we measured intracellular calcium concentrations (Cai) under anisotonic conditions in primary cultures of IMCD cells using the Fura-2 technique. Basal steady-state calcium at 600 mosmol/l was found to be 110 +/- 4 nmol/l; n = 119. Exposure to hypotonic medium (300 mosmol/l, reduction of sucrose) resulted, within 1 min, in a strong increase in calcium to 563 +/- 87 nmol/l (n = 7; P < 0.01), followed by a decrease over 4-6 min to twice the initial values. The calcium increase was smaller (260 +/- 14 nmol/l; n = 5; P < 0.05) when the osmotic pressure was decreased by reducing NaCl instead of sucrose. Stepwise reduction of osmolarity to either 500 or 400 mosmol/l increased calcium by a significantly smaller extent, suggesting a threshold for calcium influx between 400 and 300 mosmol/l. In hypotonic calcium-free solutions no significant increase in calcium was observed. Verapamil (40 mumol/l), D-600 (40 mumol/l), diltiazem (40 mumol/l), and nifedipine (40 mumol/l) inhibited the hypotonically induced calcium influx in decreasing order of potency. Lanthanum (La3+) and gadolinium (Gd3+) had no effect. Membrane depolarization by incubation in potassium-rich solution diminished calcium influx. Preincubation with cytochalasin B (50 mumol/l for 30 min) resulted in a lower basal calcium level and attenuated the calcium increase during hypotonic shock. These results demonstrate an increased calcium influx during hypotonic shock in IMCD cells in culture mediated by channels whose nature (stretch activated and/or voltage dependent) remains to be determined. The transient increase in Cai in turn may trigger inorganic and organic osmolyte fluxes observed previously.

Animals↗

PubMed1994

Regulation of ion content and cell volume in isolated rat renal IMCD cells under hypertonic conditions.

The adaptive responses of cell volume and intracellular inorganic electrolytes to hypertonicity were investigated in isolated inner medullary collecting duct (IMCD) cells in vitro. Intracellular water and element content were determined simultaneously by a rapid centrifugation technique and electron-probe microanalysis, respectively. In the absence of adenosine 3',5'-cyclic monophosphate (cAMP), cells isolated at 600 mosM (268 mM NaCl) shrank in 900 mosM buffer (418 mM NaCl) within 100 s to 79 +/- 2% of the control volume and showed no regulatory volume increase (RVI). K+ content decreased steadily during the first 5 min to 18% below control. K+ concentration initially increased 25 +/- 2% above control and was normalized after 5 min to 144 +/- 10 mM (n = 6). Na+ and Cl- content increased slowly during the 60-min incubation period; at this point intracellular Na+ and Cl- concentrations were 97 +/- 6 and 59 +/- 7% (n = 6) above control levels (75 +/- 12 mM Na+ and 143 +/- 29 mM Cl-), respectively. In the presence of 10(-4) M 8-bromo-cAMP (8-BrcAMP; used as a substitute for antidiuretic hormone), IMCD cells showed RVI within 5 min. K+ content initially dropped 18 +/- 2% below control and recovered during the next 4 min but was 12 +/- 4% below control after 60 min. K+ concentration showed similar changes. Na+ and Cl- content increased to a larger extent than in the absence of 8-BrcAMP to 95 +/- 10% (Na+) and 47 +/- 6% (Cl-) above control after 60 min, whereas Na+ and Cl- concentrations were the same as without 8-BrcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cloning and expression of a renal Na-Pi cotransport system from flounder.

Starting with the recently published sequence of the rat renal Na-Pi cotransport system, we have cloned a corresponding cDNA from the kidney of winter flounder (Pseudopleuronectes americanus), designated flounder NaPi-II. Expression of the cognate in vitro transcribed RNA in Xenopus laevis oocytes stimulated Na-dependent Pi transport specifically and in a time- and dose-dependent manner. Apparent affinities of Na and Pi, as well as the pH dependency, were very similar to those found for the mammalian systems. The flounder NaPi-II cDNA is 2,424 base pairs long and encodes a protein of 637 amino acids. The hydropathy plot predicts eight transmembrane spanning domains. In these regions the flounder NaPi-II-deduced protein shows high homology (approximately 80%, identity, approximately 92% similarity) with the amino acid sequences reported for mammalian NaPi-II proteins. However, in the hydrophilic parts of flounder NaPi-II protein, only minimal similarity could be found between fish and mammalian systems (30% homology, 45% similarity). Northern blot analysis with flounder NaPi-II cDNA as a probe confirmed this finding: even under nonstringent washing conditions, no cross-hybridization with mRNA from rat renal cortex was observed. Interestingly, flounder intestine was found to contain high levels of mRNA corresponding to NaPi-II. Supplementary bands of 1.9 and 4.2 kb were observed on Northern blots of renal and intestinal tissue. The close functional relationship of the flounder NaPi-II protein with the previously described Na-Pi cotransport systems and the pronounced differences on the level of their primary structures provide the tools for detailed structure-function analysis of Na-Pi cotransport.

Amino Acid Sequence↗

PubMed1994

Jacob Henle: the kidney and beyond.

The progress in science made by Henle depended on the improvements in light microscopy achieved in the 19th century. The advent of achromatic lenses in particular made it possible for Henle and his contemporary and friend Schwann to uncover the typical structures of cells, thus bringing order at the microscopic level to the world of living tissues. Henle's 'comprehensive' approach--4 basic types of tissue: epithelial, connective, muscular and nervous, contrasted with the accepted doctrine of Bichat of 21 different types of tissues that enter into different combinations in forming the organs of the body--together with rapid progress in chemistry and physics permitted the subsequent intimate probing of cellular physiology. Henle's work was not confined to the description of anatomic structures. After he had observed microorganisms in the excretions of diseased animals, he embraced the unpopular theory of 'contagion' as the source of infection, though he himself was not able to prove that microorganisms were the direct cause of diseases. His discovery of the renal tubule that now bears his name came comparatively late in his career, and although he described its structure in detail he offered no suggestions as to its function.

Germany↗

PubMed1994

Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells.

In response to hypotonic stress, cells of the inner medullary collecting duct (IMCD) undergo swelling followed by a regulatory volume decrease (RVD) and a transient release of organic osmolytes such as sorbitol. In this study, we tested the hypothesis whether membrane recycling is involved in the latter process. Therefore, the state of submembranal actin and the cellular uptake or release of the fluid-phase marker fluorescein isothiocyanate (FITC)-dextran (FD) were investigated as related to changes in membrane permeability for sorbitol. After exposure to hypotonic medium the submembranal actin web rapidly disaggregated but it started to reorganize after 5 min of incubation. The basal-lateral pole of IMCD cells showed a significant uptake of extracellular FD after 100 sec. After 5 min, part of this fluorescence intensity had moved towards the cell center but the main part remained submembranal. Disintegration of the actin network by cytochalasin D diminished the uptake of FD during hypotonicity as did a permanent increase in intracellular calcium induced by ionomycin treatment. During a second osmotic stimulation of IMCD cells preloaded with FD, FD was released in a linear time course reaching a plateau after 1 min. Isotonic ionomycin treatment of preloaded cells also generated a rapid FD release during the first minute but induced a further 2-fold increase during the next 4 min. Under both conditions initial FD release was highly correlated with the simultaneously determined increase in sorbitol permeability. A similar strong correlation was found when different incubation temperatures were used (0 degree C, 15 degrees C, 37 degrees C). These results suggest that during exposure of IMCD cells to hypotonicity the submembranal actin web rapidly disintegrates, and "reserve" vesicles, probably containing sorbitol transporter, move to and fuse with the basal-lateral plasma membrane. The fusion causes a rapid increase in sorbitol permeability. These membrane areas are recovered by internalization, and the transport systems for sorbitol are concomitantly retrieved. In parallel to this internalization the submembranal actin filament network is rearranged. This process seems to be regulated by changes in intracellular calcium.

Actins↗

PubMed1993

Control of sorbitol metabolism in renal inner medulla of diabetic rats: regulation by substrate, cosubstrate and products of the aldose reductase reaction.

Streptozotocin diabetes induces a 4-fold increase in the maximal velocity of inner medullary aldose reductase as determined in vitro but increases sorbitol synthesis in intact inner medullary collecting duct (IMCD) cells only 1.3-fold. In order to resolve this discrepancy we investigated the importance of intracellular factors in controlling the role of cellular sorbitol synthesis. These factors include glucose concentration, sorbitol concentration, the activity of the NADPH-regenerating pentose phosphate pathway, intracellular NADP and NADPH content, and intracellular reduced (GSH) and oxidized glutathione (GSSG). It was found that the apparent Km of cellular sorbitol production for glucose was identical in control and diabetic rats (56 +/- 18 vs. 59 +/- 14 mmol/l D-glucose), whereas Vmax increased by 31% in diabetes. In inner medullary collecting duct cells of diabetic rats containing 146 +/- 5 mumol sorbitol/g protein, sorbitol synthesis was slightly lower (-15%), compared to cells which had been sorbitol-depleted prior to the experiment (87 +/- 4 mumol sorbitol/g protein). However, no inhibitory effect of sorbitol (up to 200 mmol/l) was observed on aldose reductase activity in vitro. In diabetic rats the content of NADPH was about 32% lower than in the control rats (3.8 +/- 0.3 vs. 5.6 +/- 0.4 mumol/g protein) and the ratio of NADPH/NADP was decreased from 25.6 +/- 5.1 to 8.6 +/- 1.7. In homogenates of the inner medulla the activity of 6-phospho-gluconate dehydrogenase (EC 1.1.1.43) was identical in both experimental groups, so the pentose phosphate shunt seems to be unaltered. GSH content in diabetic rats was also diminished (4.02 +/- 0.67 mumol/g protein vs. 7.41 +/- 0.5 mumol/g protein) and the GSH/GSSG ratio fell from 92.6 to 57.4. In enzyme tests in vitro an apparent Km of 7.3 +/- 1.9 mumol/l of the aldose reductase for NADPH was found; NADP acted as competitive inhibitor with an apparent K(i) of 183 +/- 31 mumol/l. Aldose reductase activity was also found to be strongly inhibited by the SH-group reagent p-chloromercurybenzoesulfonate (apparent K(i) = 0.85 x 10(-6) mol/l). Combining the results obtained on the properties of the aldose reductase in vitro and the observation made in the intact cells, the investigators suggest that the decrease in NADPH/NADP ratio, as well as changes in the redox state in the cells of diabetic animals, can play a significant role in the control of sorbitol synthesis.

Aldehyde Reductase↗

PubMed1993

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.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

The role of organic osmolytes in osmoregulation: from bacteria to mammals.

Cells of marine species are known to establish osmotic balance with their environment by adjusting the concentrations of organic osmolytes rather than inorganic osmolytes such as sodium, potassium, and chloride. These organic osmolytes fall into three classes: polyhydric alcohols such as sorbitol, amino acids and amino acid derivatives, and urea and trimethylamines. Substantial evidence is available for a central role of each of these classes in osmoregulation in marine species. In this chapter information on the importance of organic osmolytes is extended to a study of isolated mammalian kidney cells. The intracellular concentration of organic osmolytes in these cells responds dramatically to changes in the osmotic environment. The release of sorbitol following hypoosmotic exposure appears to be triggered by calcium, possibly via a mechanism involving membrane recycling. The summarized experiments provide a basis for further work in marine species.

Animals↗

PubMed1993

Inhibition of Na,K-ATPase by cadmium: different mechanisms in different species.

The mechanism of action of Cd on Na,K-ATPase was investigated in two "classical" model systems, the shark rectal gland and rabbit kidney outer medulla. In lyophilized plasma membranes from dogfish rectal gland Cd inhibited Na,K-ATPase activity after 30 min of preincubation with an I50 of 1.3 x 10(-5) M. K-Dependent p-nitrophenylphosphatase (pNPPase) activity was inhibited 50% by Cd at 9.4 x 10(-6) M. Neither Na nor K altered the interaction of the enzyme with Cd. Decreasing the ATP concentration, however, lowered the apparent sensitivity of Na,K-ATPase to Cd. The inhibitory effect was also significantly reduced when the Mg concentration present during the preincubation was increased from 0.5 to 6.0 mM. The apparent Cd sensitivity of the K-dependent pNPPase is lower at 10 mM Mg than at 1 mM Mg. In initial rate experiments 4 x 10(-5) M Cd increased the apparent Km of the enzyme for Mg significantly from 0.88 +/- 0.29 mM to 1.73 +/- 0.3 mM whereas the Vmax (167 +/- 32 mumol/min x mg protein compared to 140 +/- 16 mumol/min x mg protein) remained essentially unchanged. In lyophilized rabbit kidney outer medulla, Cd was found to inhibit Na,K-ATPase activity with an I50 of 1.9 x 10(-5) M. K-Dependent pNPPase was inhibited 50% under identical conditions by Cd at a nominal concentration of 2.1 x 10(-4) M. Increasing K concentrations protected the enzyme from the inhibitory action of Cd as indicated by a 10-fold decrease in sensitivity of pNPPase when the K concentration was increased from 1 to 20 mM. K, 20 mM, delayed also the onset of inhibition by about 15 min at 37 degrees C. These studies suggest that the mode of action of Cd on Na,K-ATPase varies in different species. In rectal gland Cd competes with a Mg site (or sites) critically involved in ATP and pNPP hydrolysis, whereas in rabbit renal medulla Cd interacts with a potassium-binding site. Differences in the protein sequence, protein conformation, and/or in the kind of protein membrane-lipid interaction might contribute to this diversity observed in the inhibitory mechanisms.

4-Nitrophenylphosphatase↗

PubMed1993

Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy.

Confocal laserscan microscopy with a dual-excitation device was used to record intracellular pH (pHi) regulation in rat proximal convoluted tubules microperfused in vivo. Cells were loaded with the pH-sensitive, fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Single cells could be distinguished within the tubules and separate measurements were possible. Application of an NH4Cl pulse by peritubular perfusion caused an immediate increase in intracellular pH. Intraluminal injection of NH4Cl led to a slower increase in intracellular pH. In both cases, cessation of perfusion led to an immediate acidification. Peritubular perfusion with 300 microM 4,4'-diisothiocanatodihydrostilbene-2,2'-disulphonic acid (H2DIDS) caused an intracellular alkalinisation. Microperfusion, pH-sensitive dyes and confocal laserscan microscopy provide a new non-invasive method to measure intracellular pH effectively in individual cells of near-surface structures of the intact kidney.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Ion content and cell volume in isolated collecting duct cells: effect of hypotonicity.

On isolated inner medullary collecting duct (IMCD) cells of the rat kidney the capability of osmoregulatory adaptation was investigated in vitro. IMCD cells were isolated by differential centrifugation at 600 mOsm (268 mM NaCl) and subsequently exposed to hypotonic buffers (300 mOsm, 118 mM NaCl). The alterations of ion content and cell volume following this change in extracellular osmolarity were studied by electron probe microanalysis and determination of intracellular water. After swelling within 40 seconds to 152 +/- 15% of control (P < 0.001; N = 9) cell volume was restored after 15 minutes. This regulatory volume decrease (RVD) was observed irrespective whether extracellular osmolarity was changed by using NaCl or mannitol as the major osmolyte. During RVD the cells lost sodium (48 +/- 11%) and chloride (14 +/- 5%), and the potassium content remained nearly unchanged. Correspondingly, sodium and chloride concentrations were progressively lowered, whereas the potassium concentration changed only transiently. RVD was diminished by 10(-4) M NPPB, 10(-3) M SITS and in the absence of HCO3-. Twenty millimoles of ouabain or 5 mM barium also inhibited RVD with little additive effect. A total of 10(-3) M amiloride and 10(-4) M bumetanide showed no effect on the hypoosmotic volume response. The experiments show that in isolated IMCD cells exposed to hypotonic conditions, rapid reversible changes in cell volume and sustained alterations in cell inorganic ion content occur, and thereby transmembrane sodium and potassium gradients are maintained. Since the loss in inorganic electrolytes does not account for RVD, the major part of volume regulation seems to occur via changes in organic osmolytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

In vitro permeability of PBCA nanoparticles through porcine small intestine.

Peroral nanoparticle-mediated drug absorption was studied using a laser scanning confocal microscope. Additional diffusion studies in side-by-side diffusion cells with radiolabelled polybutylcyanoacrylate (PBCA) nanoparticles were carried out to confirm the results of this study. Fluorescence-labelled PBCA nanoparticles were incubated in vitro in the lumen of freshly excised intestine. Computer-aided optical sectioning of thick samples with dramatically improved resolution and the possibility of rejecting out-of-focus noise enabled tracking of the fluorescence-labelled PBCA nanoparticles in the intestinal tissue after incubation of the particles in freshly excised porcine small intestine. The results of this study suggest that the nanoparticles are absorbed by the surface of the gut wall, creating a high concentration gradient, thereby enhancing the absorption of drugs that may be loaded to the nanoparticles. A significant amount of particles was found in hot (very fluorescent) spots that were assumed to be Peyer's patches. No particles, however, traversed the entire gut wall over a period of 2 to 4 h. These results were confirmed by the diffusion study. No radioactivity permeated through Peyer's-patch-free intestine within 4 h, whereas the amount of radioactivity that was transported through intestine with Peyer's patches during this time was 1.1% of the total amount in the donor chamber.

Animals↗

PubMed1993

Epithelial transport of magnesium in the kidney of fish.

That the kidneys of marine fish have powerful renal mechanisms for the excretion of magnesium (Mg) from the body has been known since the early 1930s, but it took another 40 years before the first renal Mg transport model was suggested by Natochin and Gusev. Since rates of net renal sodium (Na) reabsorption were closely correlated with rates of net renal Mg secretion in scorpion fish, Natochin and Gusev proposed tubular Na/Mg exchange transport. However, confirmation of Na/Mg exchange in other fish kidneys has been elusive. Detailed renal clearance studies in sea water rainbow trout have shown that bladder reabsorption of Na and water, the process which concentrates Mg in the bladder, accounts for much of Natochin's original observation. Nevertheless, studies of isolated perfused proximal tubules of the flounder and killifish do show inverse relationships between the concentrations of Na and Mg in the tubule lumen, consistent with Na/Mg exchange. Unfortunately, large paracellular Na permeabilities in renal proximal tubules do not clarify whether paracellular Na fluxes of Na/Mg exchange transport across the brush border membrane are responsible for generating inverse concentrations of Na and Mg in the tubule lumen. These uncertainties have led the authors to their present use of brush border membrane vesicles to look for evidence of Na/Mg exchange transport.

Adaptation, Physiological↗

PubMed1992

Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR.

The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.

Amiloride↗

PubMed1991

Photoaffinity labeling of plasma membrane proteins involved in the transport of loop diuretics into hepatocytes.

To identify proteins involved in the hepatocellular uptake of loop diuretics, [3H]bumetanide was photoactivated by light flash in the presence of either intact isolated rat hepatocytes, rat liver basolateral plasma membranes or integral membrane proteins extracted from the basolateral plasma membranes. Proteins of 52-54, 48, 33, 27, 25 and 23 kDa in sodium dodecyl sulfate (SDS) gel electrophoresis were radiolabeled on intact hepatocytes. On liver basolateral plasma membranes a 50-52 kDa protein was the most intensely labeled protein. After separation into integral and associated membrane proteins by extraction with Triton X-114, radioactive labeling was only found in integral membrane proteins with a molecular weight of 50-52 kDa. Photoactivated bumetanide irreversibly inhibited the hepatocellular uptake of cholate, taurocholate but not of serine. Binding proteins for photoactivated bumetanide were absent on AS 30-D ascites hepatoma cells. Labeling of all proteins was sodium dependent in intact hepatocytes but was sodium independent in plasma membranes. Labeling was prevented by non-labeled bumetanide and by the loop diuretics piretanide and furosemide. Labeling protection was further achieved with organic anions such as bromosulfophthalein, rifampicin, probenecid and by the bile acids taurocholate, deoxycholate and dehydrocholate. The radiolabeled proteins did not belong to the bumetanide-sensitive NaCl/KCl co-transport system which apparently does not occur in intact isolated rat hepatocytes.

Affinity Labels↗