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Biomedical subjects

R Greger

Publications and source records attributed to R Greger.

At least 127 records · Page 7Linked to original sources
PubMed1984

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias). III. Effects of stimulation of secretion by cyclic AMP.

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PDte), transepithelial resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of the lumen membrane (FR1), and the cellular activities for Cl-, Na+, and K+ (alpha cell x) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10(-4, adenosine 10(-4), and forskolin 10(-6) mol x 1(-1) on these parameters were recorded and compared to nonstimulated state (NS). PDte increased from -1.9 +/- 0.2 mV to -11.0 +/- 0.9 mV (n = 51). PDbl depolarized from -86 +/- 1 to -74 +/- 1.4 mV (n =52), Rte fell from 29 +/- 2.8 to 21 +/- 2 omega cm2 (n = 23), and FR1 fell from 0.96 +/- 0.005 to 0.79 +/- 0.04 (n = 9). alpha cell K+ was constant (123 +/- 13 versus 128 +/- 17 mmol x 1(-1) (n = 6), but alpha cell cl- fell significantly from 48 +/- 4 to 41 +/- 3 mmol x 1(-1) (n = 7). alpha cell Na+ increased from 11 +/- 2.1 to 29.5 +/- 6.6 mmol x 1(-1) (n = 4). In series 2 the conductivity properties were examined by rapid K+, and Cl- concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K+ led to a hyperpolarization of PDbl with a mean slope of 28 +/- 1.3 mV/decade (n = 9) (as compared to 19 mV/decade for S-state).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Single channel recordings from basolateral and apical membranes of renal proximal tubules.

A new method is described, which enables the recording of single ionic channels from the basolateral as well as the luminal membrane of renal proximal tubules with the patch-clamp technique. Segments of late proximal tubules of rabbit kidney are cannulated and perfused from one end. The other end is open and freely accessible to a patch pipette. The patch electrode can be moved against lateral cell membranes or can be inserted through the open end into the lumen and brought to contact with the brush-border membrane. Both, in the basolateral as well as in the luminal membrane, giga seals can be achieved. In both membranes, K+ selective channels could be identified.

Animals↗
PubMed1984

Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron.

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50 +/- 12% after 3 min and to 27 +/- 6% (n = 5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (1)] the substrates are taken up. From the 1-side only butyrate (10 mmol X 1(-1) sustained the Isc at 95 +/- 2% (n = 7). All other tested substrates (10 mmol X 1(-1): pyruvate, acetate, beta-OH-butyrate, D-glucose, and L-lactate lead to a marked decline in Isc. From the b-side several substrates (5--10 mmol X 1(-1) sustained the Isc: D-glucose, D-mannose, butyrate, beta-OH-butyrate, acetoacetate, L-lactate, acetate and pyruvate. Other compounds (1--10 mmol X 1(-1): citrate, alpha-ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of the D-glucose, L-lactate, acetate, pyruvate or beta-OH-butyrate concentration with apparent Km's between 0.05--1.0 mmol X 1(-1). Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mumol X 1(-1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition occurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol X 1(-1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. alpha-Cyano-m-OH-cinnamate (10 mmol X 1(-1) inhibited the Isc specifically when L-lactate was the bath substrate. Probenecid (1 mmol X 1(-1) had a similar yet less marked inhibitory effect. The D-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 X 10(-6) mol X 1(-1). We conclude that the cTAL segment of the rabbit utilizes D-glucose and/or small anions such as pyruvate or L-lactate or acetate to energize salt reabsorption.(ABSTRACT TRUNCATED AT 400 WORDS)

Absorption↗
PubMed1984

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias). I. Experiments in isolated in vitro perfused rectal gland tubules.

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PDte), resistance (Rte), the PD across the basolateral membrane (PDbl) and intracellular chloride and potassium activities (a cell Cl-, a cell K+) were measured. In a first series, 67 RGT segments were perfused with symmetric shark "Ringers" solution. The bath perfusate contained in addition db-cAMP 10(-4), forskolin 10(-6), and adenosine 10(-4) mol X 1(-1). PDte was -11 +/- 1 (n = 67) mV lumen negative, Rte 27 +/- 2 (n = 47) omega cm2, PDbl -75 +/- 0.4 (n = 260) mV. a cell K+ and a cell Cl- were 109 +/- 22 (n = 4) and 38 +/- 4 (n = 36) mmol X 1(-1) respectively. These data indicate that Cl-secretion across the RGT must be an uphill transport process, whereas secretion of Na+ could be driven by the lumen negative PDte. Intracellular K+ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl- is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K+ concentration led to a rapid hyperpolarization of PDbl with a mean slope of 19 mV per decade of K+ concentration change. Addition of 0.5 mmol X 1(-1) Ba2+ to the bath solution lead to a marked depolarization and abolished the response to K+ concentration steps. In the lumen a Cl- concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl- concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (Isc = PDte/Rte) was determined as a function of symmetrical changes in Na+ concentration, with Cl- held at 276 mmol X 1(-1), and as a function of symmetrical changes in Cl- concentration, with Na+ held at 278 mmol X 1(-1). Isc was a saturable function of Na+ concentration (Hill coefficient 0.9 +/- 0.1, K1/2 4.4 mmol X 1(-1), n = 7) and also a saturable function of Cl- concentration (Hill coefficient 2.0 +/- 0.1, K1/2 75 mmol X 1(-1), n = 11).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Cellular mechanism of the action of loop diuretics on the thick ascending limb of Henle's loop.

During the passed few years the cellular mechanisms responsible for the NaCl reabsorption in the thick ascending limb of the Henle loop of mammalian nephron and of the early distal tubule of amphibian kidney have been extensively studied. From these studies a new type of secondarily active transport mechanism, i.e. the Na+--2Cl- --K+ symport has emerged. Meanwhile it has been recognized that this system is also present in many other epithelia. All these epithelia share in common that they are sensitive to the so called loop diuretics. The present article summarizes our current knowledge of how the loop diuretics, by reversible interaction with the above cotransport system, inhibit the NaCl reabsorption in the thick ascending limb of Henle's loop. It is shown that these drugs transfer the thick ascending limb cell to a state in which not only transepithelial NaCl reabsorption ceases but in which also very little energy is consumed since then K+ and Cl- "relax" to passive distribution across both cell membranes and Na+ entry into the cell is blocked.

Absorption↗
PubMed1983

Effect of "high ceiling" diuretics on active salt transport in the cortical thick ascending limb of Henle's loop of rabbit kidney. Correlation of chemical structure and inhibitory potency.

The group of "high ceiling" diuretics consists of a variety of chemically different potent diuretic and saluretic substances. Appart from a few exemptions direct evidence for an action of these substances in the thick ascending limb of the loop of Henle (TAL) is still lacking. For furosemide, we have reported recently that it inhibits most likely the Na+-2 Cl--K+ cotransport system present in the lumen membrane of the TAL. The present study tests: 1. whether other "high ceiling" diuretics have a similar site and mode of action, and 2. how modifications of the furosemide molecule alter the inhibitory potency. Isolated cortical TAL (cTAL) segments (n = 185) of rabbit kidneys were perfused in vitro. The equivalent short circuit current (Isc = transepithelial PD/transepithelial resistance), as a measure of active salt transport was correlated to the dose of 64 substances. Several diuretics, such as 2-aminomethyl-4-(1,1-dimethyl-ethyl)-6-iodophenol hydrochloride (MK 447), hydrochlorothiazide, muzolimine, etozoline, tizolimide, amiloride, and triamterene were ineffective both from the lumen and basolateral side at concentrations as high as 10(-4) - 10(-3) mol X 1(-1). The phenoxyacetic acids ethacrynic acid, indacrinone (MK 196), and to less an extend tienilic acid were inhibitory active. They differed from furosemide in one or more of the following criteria: delayed onset, incomplete reversibility, stronger action from the bath, different slope of the dose response curve. Similarly, 1-ozolinone acted stronger from the bath. In contrast, the diuretics of the furosemide type and related compounds (bumetanide and piretanide) showed rapid onset and complete reversibility of inhibition. These substances acted stronger from the lumen. The individual positions in the benzyl ring of the diuretics were differently affected by substitutions, leading to parallel shifts in the dose response curves with halfmaximal inhibition at concentrations ranging between 8 X 10(-8) to greater than 10(-4) mol X 1(-1). For these substances the calculated Hill coefficients were close to unity: 0.96 +/- 0.05. We conclude that the so called "high ceiling" or "loop" diuretics consist of at least 3 groups: 1. drugs that do not interfere with the active salt transport in the cTAL segment, 2. drugs that interfere by so far not characterised mechanisms, and 3. drugs of the furosemide type which inhibit the Na+-2 Cl--K+ cotransport system in the lumen membrane of the cTAL segment.

Animals↗
PubMed1983

Effect of luminal potassium on cellular sodium activity in the early distal tubule of Amphiuma kidney.

From previous studies it is known that a furosemide-sensitive sodium chloride cotransport system is operative in the luminal cell membrane of the early distal amphibian tubule. Since inhibition of sodium chloride cotransport prevents potassium reabsorption in this nephron segment, experiments were carried out to evaluate further the possible relationship between sodium chloride and potassium transport by studying the changes of cellular sodium activity following luminal deletion of potassium ions. Sodium-sensitive liquid ion exchange microelectrodes and conventional microelectrodes were employed to determine the transepithelial potential (PDte), the peritubular cell membrane potential (PDpt) and the intracellular sodium activity (Nai+) in the presence and absence of luminal potassium. The ratio of the luminal cell membrane resistance over the peritubular cell membrane resistance (Rlu/Rpt) was also estimated. When potassium ions are omitted from the luminal perfusate, PDpt hyperpolarizes by some 20 mV, PDte approaches zero and Nai+ decreases by about 40%. Rlu/Rpt is more than doubled in the presence of a potassium-free perfusate. Both potential and resistance changes are fully reversible. Similar results were obtained in experiments in which Barium ions (1 mmol/1 BaCl2) were present during the luminal potassium substitution. Our results indicate that absence of potassium inhibits luminal sodium chloride entry; as a result of continued peritubular sodium extrusion cellular sodium activity falls. The increase of Rlu/Rpt following perfusion with a potassium-free perfusate is interpreted as a decrease of a significant electrodiffusive potassium conductance in the luminal cell membrane.

Animals↗
PubMed1983

Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney.

Rabbit cortical thick ascending limb segments were perfused in vitro, and intracellular Cl- activity was estimated in three types of experiments using conventional and chloride selective microelectrodes. In series 1 Ringer like solutions were present on the two epithelial sides. In series 2 limen Cl- was replaced by gluconate, and in series 3 furosemide, 10-20 . 10(-6) mol . 1-1, was added to the lumen perfusate. It was found that under control conditions intracellular Cl- activity, as estimated from the difference of the reading of the conventional (n = 53) and ion selective electrodes (n = 118) was 26 +/- 1 mmol . 1-1. Thi value is approximately three times higher than expected for passive distribution of Cl-. After removal of lumen Cl- (series 2) intracellular Cl- activity fell to 9 mmol . 1(-1) which is only some 4 mmol . 1(-1) above passive distribution. We argue that these 4 mmol . 1(-1) reflect mainly the interference with the Cl- electrode by other anions, such as phosphate. The above estimates for intracellular Cl- activity, have to be diminished by these 4 mmol . 1(-1), and, thus, are close to 22 mmol . 1(-1). In series 3 a rapid and reversible fall in intracellular Cl- from 23 to 7 mmol . 1(-1) was observed. We conclude that the Cl- activity in cTAL cells is clearly above equilibrium under control conditions and that it falls rapidly to values close to equilibrium when Cl- reabsorption is blocked by either removing lumen Cl- or by blocking the Cl- entry via the Na+-2 Cl--K+-carrier with furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Carbonic anhydrase independent bicarbonate reabsorption.

The present study was designed to define the prerequisites of carbonic anhydrase independent bicarbonate reabsorption. In free flow experiments during systemic application of carbonic anhydrase inhibitor benzolamide (50 mg/kg B. W.) bicarbonate recovery in % of filtered load was found to be 74 +/- 8% in late proximal convoluted tubules, 39 +/- 6% in distal convoluted tubules and 32 +/- 4% in urine, indicating that most of carbonic anhydrase independent bicarbonate reabsorption occurs in tubule segments prior to distal convoluted tubules. In vivo continuous microperfusion experiments in proximal convoluted tubules demonstrated that luminal benzolamide (0.5 mmol/l) virtually abolishes net bicarbonate fluxes, when bicarbonate concentration in the luminal perfusate (25 mmol/l) is close to peritubular plasma concentration (24.4 mmol/l). In contrast, a significant downhill reabsorptive flux occurs, when perfusate bicarbonate concentration is 75 mmol/l and a significant downhill secretory flux is observed, when the perfusate is initially free of bicarbonate. The corresponding apparent permeabilities are 1.0 +/- 0.1 X 10(-6) cm2/s for influx and 1.6 +/- 0.4 X 10(-6) cm2/s for efflux of bicarbonate. Clearance studies reveal that carbonic anhydrase dependent and independent bicarbonate reabsorption are not saturable but depend on the rate of volume reabsorption in the kidney. In conclusion, passive movements of bicarbonate do occur in proximal convoluted tubules and most likely contribute to carbonic anhydrase independent bicarbonate reabsorption.

Absorption↗
PubMed1982

Membrane transport in the proximal tubule and thick ascending limb of Henle's loop: mechanisms and their alterations.

Over the past few years, our knowledge on renal tubular transport mechanisms has increased considerably. Due to new technical developments, it is now possible to understand in part transepithelial transport and its pathological and pharmacological alterations at the level of the cell membranes. Different membrane transport mechanisms are discussed in this article, whereby sodium coupled solute transport in the proximal tubule and sodium chloride transport in the thick ascending limb of Henle's loop are taken as examples. It is indicated that an altered function of the kidney can often be equated with an alteration of the membrane transport.

Animals↗
PubMed1982

A cytotoxin of Pseudomonas aeruginosa acts directly on the cortical thick ascending limb of Henle's loop of rabbit kidney.

The present study examines directly the effect of a cytotoxin of Pseudomonas aeruginosa on the in vitro perfused rabbit cortical thick ascending limb of the loop of Henle (cTAL). 25 cTAL segments were perfused at high rate. The open circuit transepithelial electrical PD (PDte) and the specific electrical transepithelial resistance (Rt) were recorded continuously. From PDte/Rt the equivalent short circuit current (Isc) was calculated. The Isc was 214 +/- 30 mu A.cm-2 under control conditions, and decreased significantly to 74 +/- 34 mu A.cm-2 60 s after the addition of toxin (2 mg.1(-1)) to the lumen perfusate. Microscopic observation and photographs taken at that time clearly indicated swelling of the cTAL cells. Thereafter inhibition of active transport proceeded further, Rt fell progressively, and cells started to desquamate from the basement membrane. This effect of the toxin was dose dependent, and was half maximal at approximately 1.2 mg.1(-1). From the bath side the effect was less marked and higher doses of toxin had to be used (half maximal effect at 5 mg.1(-1)). We conclude that this toxin of Pseudomonas aeruginosa exerts its toxic effect on the cTAL segment by increasing primarily the permeability of the lumen membrane.

Animals↗
PubMed1982

The effect of respiratory and metabolic acid-base changes on ionized calcium concentration: in vivo and in vitro experiments in man and rat.

Correlation of ionized calcium concentration, [Ca2+] and blood pH has long been recognized. So far no distinction of the acid-base changes, i.e. respiratory changes or metabolic changes seemed necessary. The present study, with the use of a recently developed system for in vivo analysis of [Ca2+], and with in vitro experiments reinvestigates this question. In a first series respiratory and metabolic changes were induced in rats. Changes of [Ca2+] (delta [Ca2+]) and of plasma pH (delta pH) were recorded continuously in vivo, plasma bicarbonate, [HCO-3] was measured in vitro. In a second series respiratory and metabolic changes were induced in sixteen volunteers and, separately, in vitro in plasma and modified Ringer solution, and the same parameters were determined. In all experiments delta [Ca2+] correlates negatively with delta pH. However, the correlation in respiratory changes was significantly less as compared to that in metabolic changes. As expected, delta [HCO-3] correlates positively with pH in metabolic and negatively in respiratory changes. We conclude from these experiments that in metabolic changes the effects of calcium-albumin interaction and calcium complexation with bicarbonate are additive, whereas both effects oppose each other in respiratory changes. This might explain the blunted effect of pH changes on [Ca2+] in respiratory changes.

Acidosis↗
PubMed1981

A modified system for in vitro perfusion of isolated renal tubules.

A modified system for the in vitro perfusion of isolated tubule segments is presented. The system consists of four holders each of which carries an acrylic cylinder. The acrylic cylinders are used to fix the glass pipettes in a concentric position. The four holders are mounted onto a support consisting of two holding pieces and three steel rods. Three of the holders contain ball-races so that they can slide on the rods with high accuracy and little friction. The holders to which the sylgard pipette and the perfusion pipette are attached are moved by electric motors. Compared with the classical V-track system this modification has the advantage of higher precision. Once the different pipettes are centered, concentricity is maintained even when the pipettes are moved forward or backward. Thus, this equipment facilitates the cannulation of tubules and increases the number of successful experiments.

Animals↗