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R K Kinne

Publications and source records attributed to R K Kinne.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Localization and activity of renal carbonic anhydrase (CA) in CA-II deficient mice.

A null allele at the mouse Car 2 locus was induced by ethylnitrosurea; mice homozygous for the new allele lack the carbonic anhydrase (CA)-II isoenzyme. The expression of this genetic lesion was investigated by: (1) using tissue fractionation techniques to determine localization and activity of CA in the kidney, and (2) examining renal response to CA inhibition in CA-II deficient mice (CAD), in normal (N) mice and in heterozygous litter mates (LM). N and LM mice had CA activity in proximal tubule brush border membranes and cytosol. CA activity was also localized to membranes and cytosol of the outer medullary region. CAD mice lacked cytosolic activity but had normal CA activity in all membranes examined. All membrane associated CA had 2-8-fold lower sulfonamide sensitivity than cytosolic CA. These inhibition characteristics suggest that the membrane enzyme is CA-IV. Baseline urinary excretion of Na+, K+, and HCO3- was similar in all groups. Urine pH and Cl- excretion were higher and titratable acid output was lower in CAD mice. Inhibition of CA (methazolamide, 25 mg/kg) led in all groups to equivalent increments of urine pH, urine flow, and HCO3-, Na+, and K+ excretion. Cl- excretion was unchanged. Thus the extent of the genetic deficiency of CA-II mice extends to the kidney cytosol but does not alter membrane localization or levels of CA, probably CA-IV. The similar response to CA inhibition in CAD mice suggests that CA-IV, the membrane bound isoenzyme is the important isoenzyme in proximal tubule HCO3- reabsorption.

Animals↗
PubMed1991

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)

Animals↗
PubMed1991

Sequence comparison of the sodium-D-glucose cotransport systems in rabbit renal and intestinal epithelia.

Using an isolated 1.2 kbp PCR product as specific probe, the rabbit renal cortex sodium-D-glucose cotransport system has been found and cloned from a renal cortex cDNA library. Two overlapping cDNA clones were found, one with a length of 2.1 kbp and one of 1.1 kbp resulting in a total sequence of 2.2 kbp without the poly(A) tail. Northern blot hybridization indicated a transcript of approx. 2.4 kb in size. Tissue distribution in the rabbit kidney and intestine indicates that this sequence is present in renal cortex, renal medulla and small intestine in different proportions. It is proposed that this clone codes for a member of the sodium-D-glucose cotransporter systems.

Amino Acid Sequence↗
PubMed1991

Studies on the phenotype and karyotype of immortalized rabbit kidney epithelial cell lines.

Differentiated mammalian cell lines can be isolated by immortalizing primary cells by transfection with DNA from plasmids containing sequences from SV40 early region. These cell lines show cytogenetic abnormalities but the degree of aneuploidy is considerably less than that observed in other established cell lines. No correlation was observed between the degree of differentiation of a clone and the extent of chromosomal damage.

Aneuploidy↗
PubMed1991

Selectivity and direction: plasma membranes in renal transport.

Studies using isolated renal plasma membranes are now considered to be essential methods for elucidating renal transport processes in health and disease. This review first describes the development of methods and concepts in working with isolated plasma membranes some 20 years ago, when few people believed that it was actually possible to isolate plasma membranes at all. The concepts we developed at that time are still applicable today, and I believe they are therefore worth reiterating. The description of the past leads to a presentation of current ideas about the interrelationship between selectivity of renal plasma membranes and direction of transepithelial transport. Finally, the future direction of kidney research is discussed in view of recent physiological, biochemical, and genetic studies.

Adenosine Triphosphate↗
PubMed1990

Role of Ca2+ in sorbitol release from rat inner medullary collecting duct (IMCD) cells under hypoosmotic stress.

The role of Ca2+ was studied in the release of the organic osmolyte sorbitol from rat IMCD cells in response to hypoosmotic stress. When cells were exposed to hypoosmotic media, sorbitol release was greatly reduced in Ca-free media which, on readmission of Ca2+, returned to control values. Under isoosmotic conditions, the ionophore A23187 stimulated sorbitol release without any effect on cell volume. Addition of trifluoperazine, a calmodulin inhibitor, but not the protein kinase C inhibitor H-7, inhibited the osmotically-activated sorbitol release. These results suggest that sorbitol release is a calmodulin-dependent event, possibly activated by a rise in intracellular calcium as a result of cell swelling.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Mechanisms of mercurial and arsenical inhibition of tyrosine absorption in intestine of the winter flounder Pseudopleuronectus americanus.

Effects of HgCl2 (100 microM) para-chloromercuribenzene sulfonate (PCMBS) (1 mM), and oxophenylarsine (OPA) (250 microM) were determined on (a) the rate of Na pump activity in intact winter flounder intestine; (b) activity of Na-K-ATPase in tissue homogenates; and (c) Na-dependent and Na-independent uptake of tyrosine in brush border membrane vesicles. Initial rate of uptake (influx) of 86Rb from the serosal solution of tissues mounted in Ussing chambers, a measure of Na-K-ATPase activity in the intact cell, was inhibited by all three agents with differing time courses. Rapidly permeating HgCl2 inhibited influx to the same degree as ouabain at 30 min, whereas the effects of PCMBS and OPA required 90 min. Cell potassium was also measured as an indirect indicator of ATPase activity and cell membrane permeability. All three agents decreased cell K, although effects on cell K lagged behind those for inhibition of the ATPase. At the concentrations used in the Ussing chamber (or at one-tenth concentration), all agents completely inhibited Na-K-ATPase activity in enzyme assays performed with tissue homogenates. In contrast, only HgCl2 decreased Na-dependent uptake of tyrosine by brush border membrane vesicles. These results suggest that mercurial and arsenical effects on tyrosine absorption are due to inhibition of the Na-K-ATPase thus decreasing the driving force for the cellular uptake by the Na-tyrosine cotransport system. Direct effects on Na-tyrosine cotransport may play a role in the inhibition observed with HgCl2, but not for PCMBS or OPA.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Further characterization of adenosine transport in renal brush-border membranes.

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.

Adenosine↗
PubMed1990

Characterization of a Na(+)-K(+)-2Cl- cotransport system in oocytes from Xenopus laevis.

In order to characterize the transport systems mediating K+ uptake into oocytes, flux studies employing 86Rb were performed on Xenopus oocytes stripped of follicular cells by pretreatment with Ca2(+)-Mg2(+)-free Barth's medium. Total Rb+ uptake consisted of an ouabain-sensitive and an ouabain-insensitive flux. In the presence of 100 mmol/l NaCl and 0.1 mmol/l ouabain the ouabain-insensitive flux amounted to 754.7 +/- 59.9 pmol/oocyte per h (n = 30 cells, i.e., 10 cells each from three different animals). In the absence of Na+ (Na+ substituted by N-methylglucamine) or when Cl- was replaced by NO3- the ouabain-insensitive flux was reduced to 84.4 +/- 42.9 and 79.2 +/- 12.1 pmol/oocyte per h, respectively (n = 50 cells). Furthermore, this Na(+)- and Cl(-)-dependent flux was completely inhibited by 10(-4) mol/l bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransport system. These results suggest that K+ uptake via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport system represents a major K+ pathway in oocytes.

Animals↗
PubMed1990

[Study of kidney function using isolated cells].

After summarizing the progress which has been made with regard to the isolation and characterization of homogeneous cell populations from the kidney, a brief survey of current techniques available for the analysis of intracellular parameters is given. Special emphasis is thereby placed on the use of electron probe X-ray microanalysis to determine intracellular elements and on "in vivo" nuclear magnetic resonance to define metabolic pathways in isolated cells. These methods have been applied to study ion and substrate fluxes in isolated collecting duct cells and the response of these cells to changes in osmolality of the extracellular medium. This response involves initially fast water movements accompanied by changes in intracellular sodium and chloride but not potassium concentration. Longterm adaptation is achieved by the adjustment of the intracellular concentration of "organic osmolytes" such as sorbitol, myoinositol, glycerophosphorylcholine, and betaine through changes in the rate of efflux of these metabolites from the cell. In the last section the effect of experimentally induced diabetes mellitus on the osmoregulation in isolated collecting ducts is described.

Animals↗
PubMed1990

Choline transport in collecting duct cells isolated from the rat renal inner medulla.

Glycerophosphorylcholine (GPC) plays an important role in the osmoregulation of the renal inner medulla. Under hyperosmotic conditions, a striking increase in cellular GPC content is observed. In order to characterize the cellular events involved in GPC metabolism, we have studied the uptake of choline, a precursor of GPC, by freshly isolated rat inner medullary collecting duct (IMCD) cells at 300 mosmol/l. Choline uptake occurred by a single transport system with an apparent affinity (Km) of 80 microM and a maximal velocity (Vmax) of 120 pmol/microliter cell water/min. Hemicholinium-3, ethanolamine and N,N-dimethylethanolamine were potent inhibitors, but betaine had no effect. Choline uptake was not altered by the replacement of Na+ with N-methylglucamine+, suggesting a sodium-independent process. Addition of 50 mM KCl to the incubation medium to reduce the cell membrane potential inhibited choline uptake by 19 +/- 4% after 10 min. Increasing the extracellular osmolarity to 600 or 900 mosmol/l had no effect on the kinetic parameters of choline uptake. These results suggest that choline uptake into IMCD cells occurs by a sodium-independent transport system driven by the inside negative cell membrane potential. Furthermore, the increase in the GPC content under hyperosmotic conditions is not associated with increased activity of the transport systems of biosynthetic precursors.

Animals↗
PubMed1990

Alterations of bile acid and bumetanide uptake during culturing of rat hepatocytes.

Uptake by the multispecific bile acid transport system of [3H]taurocholate, [14C]cholate, and [3H]-bumetanide into primary cultures of rat hepatocytes was compared with their uptake into freshly isolated rat hepatocytes. The uptake maximum velocity (Vmax) of all compounds declined in primary culture, whereas the Michaelis constant (Km) values remained stable. Loss of uptake was not due to the reduction of driving forces as evaluated from the level of ATP and the activity of Na(+)-K(+)-ATPase. No alpha-fetoprotein was detectable in culture supernatants. Neither growth factors (glycylhistidyl-lysine, epidermal growth factor), peroxisome and cell proliferators (nafenopin, dimethyl sulfoxide), nor bile acids prevented the loss of transport in hepatocyte culture. However, addition of dibutyryl adenosine 3'5'-cyclic monophosphate protracted the transport activity significantly. When cultured rat hepatocytes with reduced transport were detached by trypsin, cells rounded up and showed the same uptake capacity for bumetanide, cholate, and taurocholate as seen in freshly isolated hepatocytes. "Cryptic" transport activity in the lower basolateral membrane facing the support was found using an incubation chamber for cultured hepatocytes, which allowed us to distinguish simultaneously between uptake via the upper and lower basolateral membrane of the cultured cells.

Adenosine Triphosphate↗
PubMed1989

Pathways for the synthesis of sorbitol from 13C-labeled hexoses, pentose, and glycerol in renal papillary tissue.

Suspensions of rabbit renal papillary tissue were incubated with D-[6-13C]glucose, D-[1-13C]fructose, D-[1-13C]ribose, and [2-13C]glycerol. The perchloric acid extracts of the above incubations were investigated with 13C NMR spectroscopy. All 13C-labeled substrates give rise to 13C-labeled D-sorbitol. D-[6-13C]Glucose and D-[1-13C]fructose are converted directly into D-sorbitol via the aldose reductase and sorbitol dehydrogenase pathway, respectively, whereas D-[1-13C]ribose and [2-13C]glycerol give rise to labeling of the D-glyceraldehyde pool which on its turn causes a labeling of D-sorbitol. Label exchanges observed from incubations with glycerol and D-ribose indicate that the pentose shunt plays a role in this synthesis of D-sorbitol.

Animals↗