Klaus Thurau

Perivascular potassium and pH as determinants of local pial arterial diameter in cats. A microapplication study.

The effects of and the interaction between local perivascular variation in potassium and bicarbonate concentration on the diameter of pial arteries were studied in cats by the microapplication technique. At 11 mEq/liter of bicarbonate, a direct correlation existed between vascular diameter and K+ concentrations between 0 and 10 mEq/liter. At K+ concentrations between 10 and 20 mEq/liter, no further increase in vascular diameter was observed. At a K+ concentration of 5 mEq/liter, an inverse relationship existed between pial arterial diameter and perivascular bicarbonate concentrations between 0 and 22 mEq/liter. At K+ concentrations of 0 and 10 mEq/liter, the pial arterial diameter was determined strongly by the K+ concentration and was only slightly, if at all, influenced by bicarbonate concentrations between 5 and 22 mEq/liter. At lower bicarbonate concentrations the local acidity induced a marked vasodilation. The results indicate that the diameter of pial arterioles in cats is affected by periarteriolar concentrations of K+ and H+; the degree of the vasoreaction induced by H+ is modulated by K+.

Micropuncture studies on the filtration rate of single superficial and juxtamedullary glomeruli in the rat kidney

SummarySingle nephron filtration rates of superficial and juxtamedullary nephrons were determined in high and low sodium rats. Single nephron GFR was calculated from TF/P inulin and tubular flow rate in superficial nephrons and single juxtamedullary GFR from corresponding data in long loops of Henle. In low sodium rats superficial nephron GFR was 23.5±6.4 (SD)×10−6 ml/min×g KW, juxtamedullary nephron GFR was 58.2±13.6 and total kidney GFR (CIn) was 0.94±0.16 ml/min×g KW. Using these single nephron values, total kidney GFR and a total number of 30,000 glomeruli per kidney, the number of superficial and juxtamedullary glomeruli was calculated to be 23,267 and 6,733, respectively. During high sodium diet superficial nephron GFR increased to 38.1±11.3 and single juxtamedullary GFR decreased to 16.5±6.6, total kidney GFR increasing to 1.01±0.24. Calculation again revealed the same distribution of the two nephron types. End-proximal TF/P inulin in superficial nephrons was 2.36±0.36 in low sodium and 2.31±0.28 in high sodium rats. Loops of Henle TF/P inulin and intratubular flow rate were inversely related: the highest TF/P inulin values and lowest intratubular flow rates were found in the descending limb. These data quantify the distribution of superficial and juxtamedullary nephrons on a functional basis and suggest a mechanism by which the kidney adjusts sodium excretion by altering the contribution of each nephron type to total kidney GFR.

Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

SummaryFor elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-μm thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard.The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringers solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringers solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10−4m) produced an increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10−4m). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride.The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing meium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be, prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain.The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the frog skin epithelium can be regarded as a functional syncytium for Na.

Micropuncture evaluation of the importance of perivascular pH for the arteriolar diameter on the brain surface

SummaryA micropipette technique was used to induce local changes of the bicarbonate concentration of the cerebro-spinal fluid surrounding arterioles on the exposed cerebral cortex of anaesthetized rats and cats. Injection volumes of a few nanoliters caused circumscribed and pronounced changes of the diameter of the arterioles under study: mock spinal fluid without bicarbonate dilated, while a solution containing 25 meq/l of bicarbonate constricted the vessels. In such experiments the localpCO2 of the arteriolar wall remains practically constant, since it is set by thepCO2 of the arterial blood and of the cerebral tissue. Hence the microinjections essentially consisted in a local change of the pH of the fluid surrounding a small segment of a cerebral arteriole. Since metabolic changes of the nervous tissue changes the periarteriolar pH, it is probable that local pH induced vasomotor changes of the type reported here participate in the so called metabolic regulation of the cerebral blood flow which underlies the local adaptation of the cerebral blood flow to changing functional demands.

Preparation of freeze-dried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissues.

SummaryA procedure is described which allows the evaluation of wet weight concentrations of diffusible substances in biological soft tissue on a cellular level by the use of energy dispersive X-ray microanalysis. Epithelia of frog skin and toad urinary bladder were used to prepare freeze-dried cryosections without the use of chemical fixatives, cryoprotectants, floating solutions or coating materials.The specimens were shock-frozen inl-propane (−180°C), cryosectioned (−80°C), sandwiched between collodion films and freeze-dried (−80°C). The analysis was performed in a scanning electron microscope at an acceleration voltage of 15 kV, probe current of 0.5 nA, using scanning areas of 1–2 μm2. The spatial resolution power using 1–2 μm thick sections was about 0.7 μm. In a superficial layer of about 30 μm the analysis was found not to be influenced by tissue damage due to ice crystal formation. The mass loss during electron bombardement was restricted to constituents of the organic matrix (30%). No changes of characteristic radiation were observed for Na, K and Cl. Albumin standards, containing electrolyte concentrations in the range of biological interest, revealed linear calibration curves. To obtain reliable wet weight concentrations, the characteristic X-rays of the tissue were compared to those of an internal standard which was applied to the specimen prior to freezing and analysed simultaneously.

Quantitative analysis of electrolytes in frozen dried sections

During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock‐freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 μm thick serial sections at 170 K and subsequent freeze‐drying at 190 K and 10−4 Pa. The analysis of the frozen dried cryosections is performed in a scanning electron microscope which is equipped with an energy dispersive X‐ray detector. The measuring conditions selected are 17–20 kV acceleration voltage and 0·1–0·5 nA probe current. For quantification, the cellular X‐ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X‐ray intensities is performed using a computer program. Some critical points of this technique will be discussed.

Dependency of Pial Arterial and Arteriolar Diameter on Perivascular Osmolarity in the Cat: A Microapplication Study

The effect of perivascular osmolarity on the diameter of pial arteries was studied in cats by the microapplication technique. Between 251 and 360 mosmoles/liter, concentration-response curves were obtained for single vessels. Constriction occurred when perivascular osmolarity was decreased below 317 mosmoles/liter, and dilation occurred at osmolarities above this value. The effect was the same whether the osmolarity was changed by addition of mannitol or NaCl. Reduction of sodium concentration from 156 to 133 mEq/liter at constant osmolarity did not affect arteriolar diameter, but greater reductions in sodium concentration induced vasoconstriction. The results indicate that the resistance of pial arteries can be influenced by local changes in perivascular osmolarity.

Die frühdistale Natriumkonzentration in Rattennieren nach renaler Ischämie und hämorrhagischer Hypotension

SummaryIn micropuncture experiments on rat kidneys early distal sodium concentration, approximating that of the macula densa site, together with glomerular filtration rate and renal excretory function were determined before and after renal ischemia or hemorrhagic hypotension. In the ischemia experiments micro-puncture samples were obtained using the same early distal puncture site of distal convolutions before and after ischemia.Early distal [Na+] was found to be increased after ischemia or hypotension, in some experiments approaching plasma [Na+]. The degree of increased [Na+] was inversely related to GFR. In order to eliminate the influence of reduced tubular flow rate which occurs after renal ischemia, single loops of Henle were perfused at a constant rate and the increase in early distal [Na+] was found to be in the same range of magnitude. U/P inulin was decreased after renal ischemia. UNa·V and urine volume were increased in most experiments.The results indicate that the ability of the ascending limb of Henles loop to establish a [Na+] gradient across the tubular wall is reduced after renal ischemia or hemorrhagic hypotension. According to the hypothesis that GFR is regulated by [Na+] at the macula densa cells, it is suggested that persistent renal vasoconstriction and fall in GFR following renal damage is caused by the elevated [Na+] of the tubular fluid in the macula densa segment. It is concluded that reduction of RBF and GFR after renal damage is the response to decreased tubular Na+ reabsorption, and that the renin angiotensin system participates in this reaction at the level of the juxtaglomerular apparatus. The reaction is considered as a Na+-conserving mechanism.

HEAT SHOCK PROTEINS HSP25, HSP60, HSP72, HSP73 IN ISOOSMOTIC CORTEX AND HYPEROSMOTIC MEDULLA OF RAT KIDNEY

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.

Electron microprobe analysis of the different epithelial cells of toad urinary bladder. Electrolyte concentrations at different functional states of transepithelial sodium transport.

SummaryThe electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemi-bladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at −180°C. From the frozen material 1–2μm thick cryosections were cut and promptly freeze-dried for 12 hr at −80°C and 10−6 Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110mm/kg wet wt) and low in Na (about 13mm/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringers as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10−2m. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis that the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.