Biomedical subjects

G Stange

Publications and source records attributed to G Stange.

At least 37 records · Page 2Linked to original sources

PubMed1994

Cloning of a Na/Pi cotransporter from opossum kidney cells.

Opossum kidney (OK) cells have been extensively used to study cellular mechanisms of renal proximal tubular Na/P(i) cotransport. We have cloned a cDNA (NaPi-4) most likely encoding an apical Na/P(i) cotransporter from OK cells. The cloning strategy was based on homology to the recently cloned human renal (NaPi-3) Na/P(i) cotransporter (Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5979-5983). Kinetic characterization (P(i) interaction, sodium interaction, and pH dependence) of NaPi-4-induced Na/P(i) uptake showed high similarity to apical Pi transport in OK cell monolayers. The NaPi-4 cDNA is 2548 base pairs long and encodes a protein of 70.5 kDa, containing at least 8 predicted transmembrane domains. Northern blot analysis with OK cell mRNA shows a NaPi-4-related signal (2.5 kilobases) in cells grown on impermeant and permeant supports. Hybrid depletion with NaPi-4 antisense oligonucleotides abolished the mRNA-induced Na/P(i) cotransport in oocytes. Similarly, NaPi-4 antisense oligonucleotides inhibited (up to 70%) Na/P(i) cotransport in OK cell monolayers. We presume that NaPi-4 is closely related to the OK cell apical Na/P(i) cotransporter.

Amino Acid Sequence↗

PubMed1994

cDNA cloning of a rat small-intestinal Na+/SO4(2-) cotransporter.

We have isolated a cDNA (ileal NaSi-1) from rat small intestine by homology screening with a cDNA (renal NaSi-1) encoding rat kidney cortex Na(+)-SO4(2-) cotransport. Ileal NaSi-1 cRNA specifically stimulates Na(+)-dependent SO4(2-) uptake in a time- and dose-dependent manner in Xenopus laevis oocytes, with kinetic parameters almost identical to those of the renal NaSi-1. Ileal NaSi-1 cDNA contains 2722 base pairs (bp), almost 500 bp more than the renal NaSi-1 cDNA; however, it encodes a protein of 595 amino acids identical to the renal NaSi-1 protein. Northern blot analysis shows strong signals in rat lower small intestine and kidney cortex (2.9 x 10(3) and 2.3 x 10(3) bases), with the ileal NaSi-1 corresponding to the longer transcript. We conclude that we have identified a rat ileal cDNA that encodes a membrane protein most likely involved in brush-border Na(+)-SO4(2-) cotransport. It differs to the renal NaSi-1 only in the length of the 3' untranslated region, suggesting that the major difference lies in the differential use of polyadenylation signals.

Amino Acid Sequence↗

PubMed1993

Expression cloning of rat renal Na+/SO4(2-) cotransport.

Injection of rat kidney cortex mRNA into Xenopus laevis oocytes leads to a stimulation of Na(+)-dependent SO4(2-) uptake. Based on this information, we have isolated from a corresponding library a cDNA (NaSi-1) that is most likely related to a Na+/SO4(2-) cotransport system. NaSi-1 cRNA leads in a time- and dose-dependent manner to specific stimulation of Na(+)-dependent SO4(2-) uptake in oocytes. The apparent affinity constants of the NaSi-1 cRNA-expressed transport resemble those of Na+/SO4(2-) cotransport in brush-border membrane. The NaSi-1 cDNA contains 2239 bp [including a poly(A) tail] and encodes a protein of 595 amino acids (66.05 kDa); the hydropathy profile suggests at least eight membrane-spanning regions. In vitro translation of NaSi-1 cRNA results in a protein of the expected size and suggests glycosylation. Northern blot analysis shows signals of 2.3 and 2.9 kb in kidney (more abundant in cortex than in papilla/medulla) and in mucosa of small intestine of rats. The above data indicate that we have structurally identified a membrane protein involved in renal and small-intestinal brush-border membrane Na+/SO4(2-) cotransport.

Amino Acid Sequence↗

PubMed1993

Expression cloning of human and rat renal cortex Na/Pi cotransport.

We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodium-dependent phosphate transport in Xenopus laevis oocytes. Substrate specificity and a detailed kinetic analysis (Na, Pi, H+ concentrations) suggested that expressed uptake activities relate to proximal tubular brush border membrane Na/Pi cotransport. NaPi-2 cDNA contains 2464 bp encoding a protein of 637 aa; NaPi-3 cDNA contains 2573 bp encoding a protein of 639 aa. NaPi-2- and NaPi-3-deduced protein sequences show high homology to each other but are different from the protein sequence deduced from the previously cloned NaPi-1 cDNA (from rabbit proximal tubules). Hydropathy profile predictions suggest at least eight membrane-spanning regions in NaPi-2/3-related proteins. In vitro translation results in proteins of the expected size and suggests glycosylation. Northern blot analysis shows corresponding mRNA species (approximately 2.7 kb) in kidney cortex of various species but no hybridization with RNAs isolated from a variety of other tissues (including intestinal segments); a hybridization signal (approximately 4.8 kb) was observed only in the lung (human). We conclude that we have structurally identified two closely related proteins most likely involved in human and rat renal brush border Na/Pi cotransport.

Amino Acid Sequence↗

PubMed1993

Two mRNA transcripts (rBAT-1 and rBAT-2) are involved in system b0,(+)-related amino acid transport.

Previously, we isolated a cDNA clone (rBAT-1) of 2.2 kilobase pairs (kb) from a rabbit kidney cortex cDNA library, encoding a protein involved in sodium-independent transport of L-dibasic amino acids, L-cystine, and some neutral amino acids via a system related to b0,(+)-like activity (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacin, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605). In Northern blot hybridization using an rBAT-1 cDNA probe, 2.2- and 3.9-kb mRNA species were observed. Here we describe the isolation of a 3.9-kb cDNA clone (rBAT-2) by expression cloning using Xenopus laevis oocytes corresponding to the 3.9-kb mRNA species. On the basis of sequence analysis, in vitro translation (major protein of approximately 78 kDa), and functional analysis (expression of transport function), we conclude that rBAT-1- and rBAT-2-related proteins are identical: 677 amino acids in length, with most likely only one transmembrane-spanning domain. There are seven differences in the nucleotide composition within a common overlap of 2189 nucleotides, resulting in 2 amino acid replacements. In comparison with rBAT-1, rBAT-2 has 26 additional nucleotides at the 5'-end, an identical location of the first polyadenylation signal, and approximately 1.7 kb of 3'-untranslated sequence (rich in AT(U) motifs) prior to a poly(A) tail of 63 adenines. We conclude that rBAT-1 and rBAT-2 encode the same protein and that the major difference seems to be related to the use of different polyadenylation signals.

Amino Acid Sequence↗

PubMed1993

'Vector white noise': a technique for mapping the motion receptive fields of direction-selective visual neurons.

A technique is described and tested for mapping the sensitivities and preferred directions of motion at different locations within the receptive fields of direction-selective motion-detecting visual neurons. The procedure is to record the responses to a number of visual stimuli, each stimulus presentation consisting of a set of short, randomly-oriented, moving bars arranged in a square grid. Each bar moves perpendicularly to its long axis. The vector describing the sensitivity and preferred direction of motion at each grid location is obtained as a sum of the unit vectors defining the directions of motion of the bars in each of the stimuli at that location, weighted by the strengths of the corresponding responses. The resulting vector field specifies the optimum flow field for the neuron. The advantage of this technique over the conventional approach of probing the receptive field sequentially at each grid location is that the parallel nature of the stimulus is sensitive to nonlinear interactions (such as shunting inhibition for mutual facilitation) between different regions of the visual field. The technique is used to determine accurately the motion receptive fields of direction-selective motion detecting neurons in the optic lobes of insects. It is potentially applicable to motion-sensitive neurons with highly structured receptive fields, such as those in the optic tectum of the pigeon or in area MST of the monkey.

Animals↗

PubMed1993

Effect of low-phosphate diet on sodium/phosphate cotransport mRNA and protein content and on oocyte expression of phosphate transport.

Recently, we have isolated a complementary DNA most likely related to rabbit kidney cortex brush border membrane sodium/phosphate (Na/Pi) cotransport activity [NaPi-1 (1)]. To further elucidate the cellular mechanisms involved in dietary 'adaptation' of renal Na/Pi cotransport, we have exposed young rabbits for 2 weeks to either a low phosphate (Pi) diet (LPD) or a high Pi diet (HPD). Initial linear uptake of Na/Pi cotransport in isolated brush border membrane vesicles was increased in rabbits on a LPD compared with those on a HPD. Injection of equal amounts of total mRNA isolated from kidney cortex of LPD or HPD rabbits into Xenopus laevis oocytes resulted in a higher stimulation of Na-dependent oocyte Pi uptake in LPD than HPD preparations. No difference in the content of 'specific' mRNA (NaPi-1 cDNA probe, Northern blots) and of the content of the 'specific' brush border membrane protein (NaPi-1 antipeptide antibody, Western blots) between LPD and HPD preparations was observed. We conclude that 'chronic' dietary Pi deprivation leads to a protein synthesis-dependent alteration of Na/Pi cotransport activity which does not involve a change in the total amount of a protein related to the recently cloned NaPi-1 protein.

Animals↗

PubMed1992

Expression cloning of a cDNA from rabbit kidney cortex that induces a single transport system for cystine and dibasic and neutral amino acids.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodium-independent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0,+ and carries cystine and dibasic amino acids with high affinity. The rBAT (b0,+ amino acid transporter-related) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b0,+.

Amino Acid Sequence↗

PubMed1992

Expression of Na(+)-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA.

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

Alanine↗

PubMed1990

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters.

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na+/phosphate co-transport and Na+/H+ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na+/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na+/H+ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca2+ concentration do not induce inhibition of Na+/H+ exchange; PTH-dependent inhibition of Na+/H+ exchange is not prevented by ionomycin or by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Ca2+ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40-50% inhibition of Na+/H+ exchange. An analysis of intracellular pH sensitivity of Na+/H+ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced Vmax of Na+/H+ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na+/H+ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).

Animals↗

PubMed1989

Sodium-hydrogen exchange system in brush border membranes from cortical and medullary regions of the proximal tubule.

The Na+/H+ exchange system was studied in brush border membrane vesicles isolated from cortical and medullary regions of the proximal tubule of rabbit kidney. The activity of the exchanger was assessed by measuring hydrogen influx (monitored by acridine orange fluorescence), 22 Na influx and the sensitivity of these fluxes to amiloride and its analogue ethylisopropyl amiloride. In contrast to previously published data (indicating the absence of pH-gradient driven and amiloride sensitive 22Na-influx in medullary site vesicles (13, 15], Na+/H+ exchange activity could be detected in both membrane preparations by sodium tracer and fluorescence detection of hydrogen influx. Amiloride inhibition of 22Na influx was more effectively protected by increasing sodium concentration in cortical than in medullary vesicles, suggesting differences in the action of amiloride in these preparations.

Amiloride↗

PubMed1989

Excitation and suppression of primary auditory fibres in the pigeon.

Spike potentials were recorded from single fibres in the auditory nerve of the pigeon. In fibres with recognizable responses to sound, spontaneous activity and properties of responses to tonal stimuli were studied in quiet background conditions. Mean spontaneous rate in the sample of fibres was 35 spikes/s. Tuning of spike response to tones was manifest as a single peak in rate at each sound pressure level (SPL) in the frequency-intensity plane. The majority of fibres showed only excitation of spike rate above spontaneous rate. Post stimulus time histograms (PSTs) in such cases were typical of excitatory responses, previously described in birds and mammals showing pronounced adaptation and post-stimulus suppression of spike rate. In most cases of excitation-only responses, however, slopes of rate functions depended on stimulus frequency. Close to characteristic frequency (CF), slopes tended to decrease with increasing SPL, whereas away from CF, slopes tended to increase with SPL. In a minority of excitation-only responses, slopes of rate functions were parallel. In some fibres, tones adjacent to the response area caused overt suppression of spontaneous firing. For these fibres, the slopes of rate functions were more-strongly frequency-dependent, being negative at low SPL when rate suppression occurred. Suppression of spontaneous activity at low SPL was non-monotonic and quite different from suppression of spike rate at stimulus intensities above rat saturation. In PSTs of suppressed spontaneous activity, rebound occurred at the termination of the tone. The results clarify previous observations of suppression of primary auditory responses in birds. We conclude that responses in the majority of auditory fibres in the pigeon are the product of opposing excitatory and suppressive influences in the cochlea, generated by single tones in quite.

Acoustic Stimulation↗

PubMed1989

Induced suppression in spike responses to tone-on-noise stimuli in the auditory nerve of the pigeon.

Spike potentials were recorded from single, afferent fibres in the pigeon auditory nerve. Pure-tone stimuli were presented in quiet and in combination with wide band noise. Presented alone, tones produced tuned response areas; noise generally drove spike rate to well above the spontaneous rate measured in quiet. When presented in combination with noise, tones up to 75 dB SPL at frequencies far from the fibre's response area had no effect on the noise-driven spike rate. As the tone frequency was shifted towards the response area, from above or below CF, suppression of the noise-driven spike rate became stronger until the tone reached the edge of the response area. Suppression of the noise-driven rate was directly proportional to the level of the tone. Within the area of response to the tone, tone-driven spike rates generally were unchanged or variably decreased (occasionally slightly increased) by tone-on-noise stimulation, depending on the relation of the tone frequency to CF and the level of the tone relative to that of the noise. Tuning properties were unaffected. It is suggested that in the pigeon, the suppression of driven spike rate during presentation of combination stimuli, which is common to all fibres, depends on the same mechanism as the suppression of spontaneous firing by tones that is observed in a proportion of fibres (Temchin, A.N. (1988), J. Comp. Physiol. A 163, 99-115; Hill et al., (1989) Hear. Res. 39, 37-48).

Acoustic Stimulation↗

PubMed1989

Temporal synchronization in the primary auditory response in the pigeon.

Spike potentials were recorded from single fibres in the auditory nerve of the pigeon. In responses elicited by tonal stimuli, the timing of each spike relative to stimulus waveform was measured and period histograms were constructed. Phase locking of spikes was estimated in terms of a synchronicity index obtained by vector addition within the period histogram. A second measure of synchrony in the spike responses was obtained, that of temporal dispersion. For a population of fibres, vector strength of phase locking decreased for frequencies above 1 kHz, as reported for several other species. Temporal dispersion, however, also decreased with frequency, indicating enhanced temporal synchrony as frequency increased within the bandwidth of phase locking. The upper frequency limit of phase locking appears to depend on irreducible jitter of biological origin in the timing of spikes. For individual fibres, the bandwidth of synchronization of spikes consistently exceeds the response area, covering in addition the areas of suppression adjacent to the response area. Spike trains suppressed by a tonal stimulus become synchronized to that stimulus. Phase angles of synchronized responses systematically change as a function of tone level, when tone frequency is above or below CF, as reported for other avian species. Synchronicity and phase angle intensity functions are quite independent of spike rate intensity functions.

Acoustic Stimulation↗

PubMed1989

A model proposing synaptic and extra-synaptic influences on the responses of cochlear nerve fibres.

A unique property of sensory coding in the vertebrate auditory system is the existence of the classical form of excitatory centre-inhibitory surround in relative spike rate along the stimulus frequency dimension, in addition to a representation of temporal fine structure of high frequency periodic stimuli in the discharge pattern of primary afferent spike trains. We present a model which designates three factors that influence rate and temporal synchrony in spike responses; an excitatory factor, a suppressive factor and a synchronizing factor. The model proposes that an essential integration of bioelectric signals occurs in the primary afferent fibre. It is presumed that mean spike rate depends on mean level of membrane depolarization and synchronization depends on periodic modulation of membrane potential at the spike initiating zone. In the model, the excitatory factor is synaptically-mediated, excitatory post-synaptic potential (e.p.s.p.); the suppressive factor is negative DC polarization of the fibre membrane and the synchronizing factor is AC modulation of the fibre membrane potential. It is proposed that both the negatively-polarizing and high-frequency modulating signals are derived from extracellular current flow in the cochlea.

Action Potentials↗

PubMed1989

[Local treatment of Pseudomonas infection of the ear. A small clinical study].

Infections of the middle and external ear caused by the problem-micro-organism Pseudomonas aeruginosa can be cured by local therapy with Ciprofloxacin and Tutofusin very quickly and without any complications. Drum ruptures caused by ear secretions close up again spontaneously. Tympanon tubes can be left in situ. Function disturbances of the middle and internal ear clear up and the functions return to normal.

Audiometry, Pure-Tone↗

PubMed1988

Intracellular cascades in the parathyroid-hormone-dependent regulation of Na+/phosphate cotransport in OK cells.

Parathyroid hormone (PTH) increased intracellular cyclic AMP and reduces Na+/phosphate cotransport activity in OK cells [Malmström & Murer (1986) Am. J. Physiol. 251, C23-C31; Caverzasio, Rizzoli & Bonjour (1986) J. Biol. Chem. 261, 3233-3237]. It was also shown that PTH activates phosphoinositide metabolism in OK cells [Hruska, Moskowitz, Esprit, Civitelli, Westbrook & Huskey (1987) J. Clin. Invest. 79, 230-239]. In the present paper we show that tumour-promoting phorbol esters are effective in reducing Na+/phosphate cotransport. The Ca2+ ionophores A23187 and ionomycin had only a small effect on Na+/phosphate cotransport; added together, A23187 and phorbol esters showed a synergistic action. Phorbol esters and phorbol esters plus ionomycin stimulated prostaglandin synthesis as well as cyclic AMP production; acetylsalicylic acid prevented phorbol-ester-induced prostaglandin synthesis and cyclic AMP production, but had no effect on inhibition of Na+/phosphate cotransport. In suspensions of OK cells, PTH and thrombin produced a rise in intracellular Ca2+. In contrast with PTH, thrombin did not elevate cellular cyclic AMP in suspended OK cells. PTH and thrombin reduced Na+/phosphate cotransport in suspended OK cells. It is suggested that two regulatory cascades are involved in PTH action on Na+/phosphate cotransport: cyclic AMP/kinase A and Ca2+/diacylglycerol/kinase C.

Biological Transport↗

PubMed1987

Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes.

Adenosine Triphosphate↗