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R K Kinne

Publications and source records attributed to R K Kinne.

At least 37 records · Page 2Linked to original sources
PubMed1998

High-level expression of Na+/D-glucose cotransporter (SGLT1) in a stably transfected Chinese hamster ovary cell line.

The coding region of the high affinity Na+/d-glucose cotransporter (SGLT1) was inserted into the eukaryotic expression vector GFP-N1 under the control of a CMV promoter. The plasmid was then stably transfected into a Chinese hamster ovary cell line (CHO). Transcription and synthesis of SGLT1 were proved by Northern and Western blot analyses. Transport activities of the transfected cells (G6D3) were examined by measuring the sodium-dependent uptake of alpha-methyl[14C]d-glucoside (AMG). Kinetic analysis revealed a Vmax of 10.3 nmol/min/mg (total cell protein) and a Km of 0.26+/-0.09 mM, respectively. The concentration of phlorizin required to inhibit AMG uptake by 50% in the presence of 0.1 mM AMG was 2.35+/-1.84 microM. Electrophysiological studies showed that AMG induces a significant depolarization of membrane voltage in stably transfected CHO cells, suggesting an electrogenic Na-AMG symport. Immunoprecipitation with an antipeptide antibody yielded a nearly homogeneous polypeptide with a molecular mass of about 72 kDa. The amount of SGLT1 present in the CHO cell plasma membranes represents at least 1% of membrane protein, which is about 30-100 times higher than in natural sources, such as renal brush border membranes. In conclusion, the stably transfected G6D3 cells with a markedly high SGLT1 expression can serve as a promising model for studying cellular events related to Na+/d-glucose cotransport and for analyzing the structure and function of the cotransporter itself.

Animals↗
PubMed1998

Translated anti-sense product of the Na/phosphate co-transporter (NaPi-II).

The homeostasis of Pi in marine teleosts is maintained by renal Pi secretion as well as by Pi reabsorption. A Na/Pi co-transport system belonging to the NaPi-II protein family is instrumental in tightly controlled renal Pi handling in mammals and fish. We have isolated an NaPi-II related cDNA from winter flounder. It was cloned from a female gonad cDNA library and is 624 bp long. The transcript is expressed in female and male flounder gonads as well as in kidney and intestine, although at very low levels. RNase H digestion experiments revealed an opposite orientation of the transcript with regard to NaPi-II-related mRNA. The anti-sense orientation was confirmed by genomic sequence analysis and Southern blotting. Alluding to the sense transcript, the anti-sense transcript was denoted IPAN. The open reading frame of IPAN encodes a basic protein of 68 amino acid residues. Immunohistochemistry confined the anti-sense related protein, Ipan, to a submembranous compartment of immature oocytes, suggesting a role in oocyte development. In kidney and intestine Ipan is partly co-localized with the Na/Pi co-transporter, implying a regulatory function for the anti-sense protein. However, direct protein-protein interaction could not be established. The existence of a putative open reading frame in other species extends the biological significance of the novel protein.

Amino Acid Sequence↗
PubMed1998

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells.

A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special "epithelial cell" medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 omega x cm2. A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an L-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.

Alanine↗
PubMed1998

Regulation of sorbitol content in cultured porcine urinary bladder epithelial cells.

Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 +/- 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 +/- 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 +/- 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 +/- 0.04 mumol.h-1.mg protein-1 at 300 mosmol/kg to 1.81 +/- 0.16 at 600 mosmol/kg and to 3.02 +/- 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 +/- 16 nmol/10 min compared with 389 +/- 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.

Aldehyde Reductase↗
PubMed1998

Na-P(i) cotransport sites in proximal tubule and collecting tubule of winter flounder (Pleuronectes americanus).

Localization of a recently described and cloned Na-Pi cotransport system from flounder was investigated by reverse transcription-polymerase chain reaction (RT-PCR) of microdissected tubules and by immunocytochemistry of kidney of winter flounder. Histological examination showed a small glomerulus, an extremely short proximal tubule PI with a selective affinity to Lens culinaris agglutinin from lentils, and an extensive second proximal tubule segment PII (> 90% of proximal tubules), consisting of cells with numerous apical clear vesicles and extensive amplification of basolateral cell membranes. PII merged with the collecting tubule/ collecting duct (CT/CD) system without a distal segment. By RT-PCR, PII cells revealed high levels of NaPi-II related RNA; low levels were also observed in CTs. Previously characterized antisera against different epitopes of flounder NaPi-II specifically labeled the basolateral regions of PII and the apical cell portion of CT/CD cells and of some PII cells. These results suggest that tubular secretion of P(i) occurs in PII of teleost fish with modulation of urinary P(i) content in the subsequent CT/CD system.

Animals↗
PubMed1998

BCECF in single cultured cells: inhomogeneous distribution but homogeneous response.

Using confocal laser scanning microscopy with a dual-wavelength laser system, the behaviour of BCECF [(2',7'-bis-2-carboxyethyl)-5-(and-6)carboxyfluorescein] was investigated in a variety of cell lines. Selection of a small area for monitoring allowed discrimination between various intracellular organelles, whose identity was established by vital staining. It was found that, after loading the cells with BCECF, both the nucleus and the mitochondria showed a higher level of fluorescence than the cytoplasm. Calibration of the pH-sensitivity of these fluorescence signals using the nigericin method yielded identical curves, as did exposure of the cells to NH4Cl. These studies suggest that BCECF, despite its inhomogeneous intracellular distribution, reports the pH of only one cellular compartment, the cytosol.

Ammonium Chloride↗
PubMed1997

Biochemistry and physiology of carbohydrates in the renal collecting duct.

Using 13C-NMR analysis of cell extracts, enzymatic determination of metabolites and cofactors as well as enzyme assays on cell homogenates aerobic and anaerobic glycolysis, sorbitol formation by aldose reductase, the pentose phosphate shunt, and gluconeogenesis could be identified as the major pathways of D-glucose metabolism in renal inner medullary collecting ducts. In flux studies it was shown that D-glucose enters the collecting duct cells via a sodium-independent, cytochalasin- and phloretin-inhibitable transport system located at the basal-lateral cell side. At the same side sorbitol leaves the cells during regulatory volume decrease in a calcium-calmodulin-dependent fashion. From cell isolation studies it is proposed that sorbitol is taken up by adjacent (interstitial) cells, converted into fructose and then recycled to the collecting duct cells. This cycle might prevent carbohydrate wasting. Thus, IMCD cells exhibit unique aspects of carbohydrate biochemistry and physiology which enable them to function in a surrounding of low oxygen tension, low substrate supply, and extreme changes in extracellular osmolality.

Aldehyde Reductase↗
PubMed1997

Sorbitol uptake in plasma membrane vesicles isolated from immortalized rabbit TALH cells: activation by a Ca2+/calmodulin-dependent protein kinase.

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg2+, Ca2+, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37 degrees C to 4 degrees C. A lower level of uptake was also observed in the presence of 100 microM quinidine and when Ca2+ or ATP were omitted from the medium. Membranes preincubated with Mg2+, Ca2+, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 microM trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca2+/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca2+/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells.

Animals↗
PubMed1997

Hypertonicity-induced alkalinization of rat hepatocytes is not involved in activation of Na+ conductance or Na+,K+-ATPase.

We investigated whether cell alkalinization via activation of Na+/H+ exchange is involved in the stimulation of Na+ conductance and Na+,K+-ATPase in rat hepatocytes under hypertonic stress. Osmolarity was increased from 300 to 400 mOsm/l at constant extracellular pH (7.4), whereas osmotically induced cell alkalinization (0.3 pH units in HCO3-free solutions) was mimicked by increasing extracellular pH from 7.4 to 7.8 in normosmotic solutions. In intracellular recordings with conventional and ion-sensitive microelectrodes, hypertonic stress led to a transient shift in the voltage response to low Na+ solutions (95% in exchange for choline) by -4.3 +/- 0.8 mV and a continuous increase in cell Na+ from 13.7 +/- 1.8 to 18.6 +/- 3.0 mmol/l within 8 min. In the presence of 10(-5) mol/l amiloride, these effects were reduced by 80 and 90%, respectively. In contrast, increasing pH did not change the voltage responses to low Na+ or cell Na+ concentrations significantly. In addition, application of 2 mmol/l Ba2+ pulses revealed that a sustained membrane hyperpolarization of 15.6 +/- 1.4 mV following intracellular alkalinization exclusively reflects an increase in K+ conductance. Increasing osmolarity at pH 7.4 augmented ouabain-sensitive 86Rb+ uptake from 5.5 +/- 1.1 to 8.5 +/- 1.6 nmol mg protein(-1) min(-1). In normosmotic solution at pH 7.8, 86Rb+ uptake equalled 4.9 +/- 1.6 nmol mg protein(-1) min(-1), which is not significantly different from control. We conclude that, in rat hepatocytes, cell alkalinization under hypertonic stress is not responsible for the activation of Na+ conductance and probably does not participate in the stimulation of Na+,K+-ATPase.

Alkalies↗
PubMed1997

Synthesis of the photoaffinity label [1'-14C]-6C-(azimethyl)octylglucoside and its reaction with isolated renal brush border membranes.

We describe the synthesis of the photolabile analog of n-octylglucoside (OG), 6C-(azimethyl)octylglucoside (diazirino-octylglucoside, DOG). This diazirino derivative (lambda max = 335 nm) can be activated with long-wavelength UV to generate a highly reactive carbene, an intermediate suitable for a covalent ligand-receptor linkage. In inhibitor studies on D-glucose uptake into rabbit renal brush border membrane vesicles (BBMV) DOG showed a competitive inhibition with regard to D-glucose with a Ki of 20 +/- 5 microM, which was not significantly different from that of the template compound OG (Ki = 30 +/- 10 microM). On irradiation (lambda max = 350 nm) of BBMV in the presence of [14C]DOG, proteins of 61, 72 to 78, and 95 kDa were labeled by photoinduction. A band of 73 kDa was also positive in Western blots using an anti-Na+/D-glucose cotransporter antibody. Further investigations revealed that the labeled proteins were presumably mainly glucosidases known to be present in high concentration in renal BBMV. By comparison with authentic material one of the labeled proteins (61 kDa) was identified as trehalase (EC 3.2.1.28). These studies suggest that labels such as DOG carrying the reactive group directly at the D-glucose moiety of the molecule can be used to label proteins with carbohydrate recognition sites.

Affinity Labels↗
PubMed1997

Arachidonic acid as a second messenger for hypotonicity-induced calcium transients in rat IMCD cells.

In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induces Ca2+ transients consisting of an early peak phase caused by a Ca2+ release from intracellular stores and a subsequent plateau phase that involves Ca2+ entry from the extracellular milieu. In the present study, the mechanisms by which cell swelling is transduced into the Ca2+ release were investigated. The free intracellular Ca2+ concentration ([Ca2+]i) was measured using the fluorescent dye fura-2 and cell volume using a confocal laser scanning microscope. In control experiments, after reduction of extracellular osmolarity from 600 to 300 mosmol/l, by omission of sucrose, [Ca2+]i rapidly increased from 106 +/- 9 nmol/l to a peak value of 405 +/- 22 nmol/l (P </= 0.05) and thereafter reached a steady-state of 230 +/- 23 nmol/l. In low-Ca2+ conditions (10 nmol/l), the reduction of osmolarity evoked only a transient increase of [Ca2+]i by 182 +/- 11 nmol/l (P </= 0.05), which reflected Ca2+ release from intracellular stores. Hyposmotic stress had no effect on inositol 1,4,5-triphosphate (IP3) production measured by a [3H]IP3 radioreceptor assay. Preincubation with 100 micromol/l ETYA (a non-metabolisible derivative of arachidonic acid) reduced the Ca2+ response to hyposmotic stress under high and low Ca2+ conditions (87 and 85% inhibition respectively) as well as the regulatory volume decrease (RVD). Extracellular application of arachidonic acid in isotonic medium led to an increase in [Ca2+]i under high and low Ca2+ conditions. Pretreatment of IMCD cells with 50 microg/ml D609 (a phosphatidylcholine-directed phospholipase C inhibitor) or with 200 micromol/l propranolol (a phosphatidate phosphohydrolase inhibitor) reduced the hypotonic Ca2+ response more strongly than pretreatment with 5 micromol/l BPhB (a phospholipase A2 inhibitor). The Ca2+ response was also suppressed after preincubation with 200 micromol/l RHC 80267 (a diacylglycerol lipase inhibitor). Preincubation with 50 ng/ml pertussis toxin (a G-protein inhibitor) reduced the transient component of the Ca2+ response partially. We conclude that G-proteins, phosphatidylcholine-directed phospholipase C, phospholipase A2, diacylglycerol lipase and arachidonic acid, but not IP3, are involved in the mechanisms by which Ca2+ is released from the intracellular stores during RVD in IMCD cells.

Animals↗
PubMed1997

A physiologic in vitro model of the inferior vena cava with a computer-controlled flow system for testing of inferior vena cava filters.

RATIONALE AND OBJECTIVES: The authors develop a physiologic in vitro model of the inferior vena cava (IVC) for testing of filters. METHODS: The model is driven by a centrifugal pump. A computer-controlled electromagnetic valve is used for generation of different flow patterns. Limitation of the pressure increase in case of IVC occlusion is achieved by a bypass circuit. A glycerin solution is used for perfusion. Artificial clots are made from polyacrylamide gel. Data acquisition includes continuous monitoring of flow and difference pressure over the filter and video recording of the testing events. RESULTS: The model can generate constant and pulsatile flows. The pressure increase can be limited to 70 mm Hg in case of occlusion. Calculation of the flow velocities in the IVC is possible. A classification of thrombus capturing is presented. The testing of most of the results are reproducible. CONCLUSIONS: The in vitro model simulates the physiologic conditions in the IVC. It can be used for comparative testing of different filters and the evaluation of new filter designs.

Blood Flow Velocity↗
PubMed1997

Characteristics of renal Na(+)-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias).

We have investigated the properties of the skate (Raja erinacea) and shark (Squalus acanthias) kidney Na(+)-D-glucose cotransporters (SGLT) in uptake studies of radiolabeled substrates into isolated renal brush-border membrane vesicles (BBMV). Scatchard plot analysis of the substrate dependence revealed that the Na(+)-D-glucose cotransporter population is homogenous within each species. Skate BBMV showed a relatively high affinity for D-glucose [Michaelis constant (K(m)) = 0.12 mM] with an apparent coupling ratio of approximately 2 Na+ to 1 D-glucose, whereas the shark transporter was much lower in affinity (K(m) = 1.90 mM) and had a lower coupling ratio, more like 1 Na+ to 1 D-glucose. These characteristics resemble the properties of SGLT1 and SGLT2, which are known to coexist in the mammalian kidney. Inhibitor studies using sugar analogs and glucosides suggested structural differences of the D-glucose binding site among these transporters, whereas the hydrophobic transporter domains in the vicinity of the D-glucose binding site appeared to be similar. In the high-affinity skate system, D-glucose was recognized by hydrogen bonds to the hydroxy groups at C-2, C-3, and C-4 and by hydrophobic interaction with the C-6 methylene group. In contrast, the low-affinity shark system seemed to lack the hydrophobic recognition motif for the C-6 methylene group of D-glucose.

Animals↗
PubMed1997

Endothelial and epithelial cells: general principles of selective vectorial transport.

Endothelial and epithelial cells are both barriers and bridges between different compartments. This contribution discusses the general principles of paracellular, transcellular, and transmembrane transport with special emphasis on the relation between asymmetry and net movement of small solutes. Asymmetry of cell membrane transport properties is found in both epithelial and endothelial cell layers and provides the basis for transcellular transport. Furthermore, the asymmetry of membrane transporters such as the blood-brain barrier GLUT1 and the renal sodium-glutamate cotransporter is discussed with regard to their different properties at the extra- and intracellular face. These molecular asymmetries play an important role in the efficiency, direction, and regulation of transport processes across the plasma membranes in endothelial and epithelial cells.

Amino Acid Transport System X-AG↗
PubMed1997

Intracellular pH-measurements in rat duodenal mucosa in vitro using confocal laserscan microscopy.

An improved technique was developed to measure intracellular pH-changes in in vitro duodenal mucosa. A confocal laserscan microscope was equipped with a second laser to permit dual wavelength excitation measurements employing BCECF (2'7'-bis-2-carboxyethyl-5-(and-6)carboxyfluorescein), a pH-sensitive fluorescent dye. Intact rat duodenal epithelium was mounted in a microperfusion chamber and loaded with BCECF via submucosal injection. Viability of the epithelial cells could be directly monitored by estimating the nucleus-to-cytoplasm ratio of the fluorescence intensity of BCECF. A calibration procedure using isolated duodenal cells, allowed estimation of the apparent intracellular pH. Initial apparent intracellular pH was 7.32 +/- 0.12, identical to that of isolated duodenocytes. Exposure of the duodenal epithelium to 60 mM NH4Cl led to a steady increase in apparent intracellular pH of 0.46 units within 2 min. Luminal application of 0.01 N HCl led to a steady decrease in apparent intracellular pH of 0.53 pH units within 1 min and was followed by a slow increase to baseline level after acid removal. Thus, confocal laserscan microscopy in combination with BCECF allowed noninvasive monitoring of intracellular pH-changes in single cells of an intact duodenal epithelium.

Acid-Base Equilibrium↗
PubMed1996

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.

Animals↗