Modelling of electrolyte transport in renal and intestinal epithelia

Summary Epithelia can be classified as leaky and tight epithelia due to their conductive properties and their modes of solute transport. Both the proximal segment of the nephron and the intestinal tract are leaky whereas the distal nephron and the colon are tight. Consequently, inborn errors and exogenous disorders of solute transport often involve both the proximal tubule and the small intestine. In addition, effects on ion and water transport in the distal nephron closely resemble those in the large intestine. Models of solute transport in leaky and tight epithelia are presented employing porter systems known in mammalian tissues. These porter systems are discussed as possible sites of transport defects and as targets for pharmacological agents.     

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption

In previous studies it has been demonstrated that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading.Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect.Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 mmol/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 min. After 18 min a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused tubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12% of the microinfused phosphate load within 24 min.In another series of experiments, the calcium specific ionophore A 23187 — a substance which is known to increase intracellular calcium — was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15% after 24 min. In contrast, when calcium chloride-free as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7%).From these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.This study was supported by Dr. Legerlotz StiftungParts of this study were presented at the fall meeting of the Nephrologische Gesellschaft in Bonn, 1977 and at the spring meeting of the Deutsche Physiologische Gesellschaft in Göttingen, 1978     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Renal tubular transport of glutathione in rat kidney

Filtered glutathione (-glutamyl-cysteinyl-glycine or GSH) is rapidly hydrolyzed by brush-border enzymes facing the tubular lumen and is reabsorbed in the form of the constituent amino acids. The first step of hydrolysis is catalyzed by -glutamyltransferase (-GT). We investigated localization and capacity of the rat renal glutathione degradation/reabsorption during elevation of the filtered load (intravenous infusion of 12 resp. 18 mol GSH/min). Fractional excretion went up from about 0.003 to 0.31±0.02 SEM during infusion of the lower and to 0.49±0.03 SEM during infusion of the higher glutathione dose. GSH degradation/reabsorption took place along the entire proximal tubule and was partially saturated by a 150–200-fold elevation of the normal filtered load. Net reabsorption of GSH up to the last accessible superficial loop was significantly lower during infusion of 18 mol GSH/min (0.3 mol/min) than during infusion of 12 mol GSH/min (1.6 mol/min). In further experiments, infusion of 18 mol GSH/min was preceded by the i.v. administration of acivicin (0.5 mmol/kg body wt.), an inhibitor of -GT. In these experiments, fractional glutathione deliveries to late proximal and early distal tubules did not significantly differ from 1, fractional excretion of GSH at the same time was 1.46±0.11 SEM, revealing net secretion of GSH with the final urine. Tubular secretion of GSH in the acivicin-treated animals occurred either in distal tubules and/or collecting ducts or in the proximal tubules of deep nephrons which are not accessible to micropuncture. The low net reabsorption of GSH up to the late proximal tubule during infusion of 18 mol GSH/min without prior administration of acivicin indicates tubular secretion of GSH along the proximal convolution of superficial nephrons. Net secretion of glutathione with the final urine in the acivicin-treated animals therefore probably originates from proximal tubules of deep nephrons.Supported by the Wilhelm-Sander-Stiftung. Parts of this work were presented at the 4th International Workshop on Ammoniagenesis, Cadarache, France, August 1987, and at the 65th meeting of the Deutsche Physiologische Gesellschaft, Würzburg, March 1988 (Pflügers Arch 411:R97)     

Activity of (Na+K+)-stimulated adenosintriphosphatase in the rat nephron

Summary In 17 male Wistar rats in antidiuresis 10 different nephron segments and arteries are identified with the aid of Lowry's technique, dissected and total-and (Mg⁺⁺)-adenosintriphosphatase (=ATPase) determined. (Na⁺K⁺)-activated ATPase in the distal tubule is four to five times (max. eight times) more active than in the proximal segment. This difference of activity may speak for a high pump mechanism mediated by the way of a (Na⁺K⁺)-activated enzyme system in the distal nephron and for a partially passive reabsorption of sodium from the proximal convolution.With the support of the Schweiz. Nationalfonds zur Förderung der wissenschaftlichen Forschung (Nr. 4256 and Nr. 4809.3)     

Expression of parathyroid hormone receptors in MDCK and LLC-PK1 cells

Parathyroid hormone (PTH) inhibits renal proximal tubular phosphate (P_i) and bicarbonate reabsorption by regulating the activity of apical Na/P_i cotransport and Na/H exchange. Two renal epithelial cell lines [proximal tubular, LLC-PK₁; distal tubular, Madin-Darby canine kidney, (MDCK) cells] were stably transfected with complementary deoxyribonucleic acids (cDNAs) encoding a cloned PTH receptor in order to examine the polarity of transfected receptor function and whether or not intrinsic P_i transport is regulated by the transfected PTH receptor. The receptors are functionally coupled to the stimulation of adenosine 35 cyclic monophosphate (cAMP) production at both cell surfaces in LLC-PK₁ cells, whereas this response is primarily limited to the basolateral surface in MDCK cells. Immunocytochemistry suggests an apical and basolateral localization of the transfected PTH receptor in LLC-PK₁ cells and only a basolateral localization in MDCK cells. PTH activation of the transfected receptors is not coupled to the regulation of intrinsic P_i transport in either LLC-PK₁ or MDCK cells.     

Unterschiedlich hoher Ureterdruck in Abhängigkeit von der Diureseform beim Hund

Zusammenfassung Bei Vorliegen einer normalen Diurese wird nach Ureterabklemmung der sog. hohe Ureterdruck, unter osmotischer Diurese der maximale erreicht. Die Differenz von Blutdruck und maximalem Ureterdruck war im Mittel der Versuche um 20 mm Hg kleiner als diejenige des hohen. Die Ursache dafür wird kurz diskutiert.Herrn Prof. Dr.S. Janssen zum 70. Geburtstag gewidmet.     

DNA-Antiviral Vaccines: New Developments and Approaches—A Review

Current vaccines can be divided into live, recombinant and killed vaccines. Live vaccines are traditionally composed of attenuated viruses or bacteria, selected for their reduced pathogenicity. Recombinant vaccines, driven by a viral or bacterial vector express foreign antigens, or only recombinant proteins injected as antigen. Killed vaccines consist of inactivated whole pathogens. But all these traditional vaccines have some disadvantages: Attenuated live vaccine are able to undergo mutation and as mutated viruses or bacteria can now provoke the diseases against which the vaccine should protect the organism. A further disadvantage of live vaccines is the possibility of shedding which is a real problem especially in veterinary medicine. Clearly, there is a need for better vaccines to protect against diseases without the disadvantages associated with vaccines presently in use. Modern vaccines might be characterized as safe, no risk of reversion to pathogenicity, and they should be stable without the necessity of a cold chain. Production should be simple, standardized and inexpensive. Vaccine development has now been improved by the ability to use direct inoculations of plasmid DNA encoding viral or bacterial proteins. One of the major benefits of DNA-vaccines, variously termed DNA-, genetic- or nucleic acid-immunization, is the endogenous synthesis of the encoded protein. Therefore DNA vaccines mimic natural infection and provoke both strong humoral and cellular immune response. This review summarizes new developments and approaches of DNA vaccination and explains the construction of expression plasmids as well as possible mechanisms of immune responses.     

Antikatatoxische Hormone

Zusammenfassung Nach einer kurzen Übersicht der Literatur über den Einfluß von Hormonen auf die Resistenz werden die grundlegenden Unterschiede zwischen syntoxisch und katatoxisch wirkenden Hormonen beschrieben. Die ersteren (z. B. die Glucocorticoide) greifen die schädigenden Stoffe selbst nicht an, unterdrücken jedoch die Reaktionen der Gewebe (z. B. Entzündung). Demgegenüber beeinflussen katatoxische Hormone die Gewebsreaktion nicht, sie beschleunigen jedoch den Abbau pathogener Stoffe. Gewöhnlich sind die Abbauprodukte weniger toxisch als der ursprüngliche Giftstoff, und in diesen Fällen üben katatoxische Hormone eine Schutzwirkung aus.Die neuesten, hier ausführlich beschriebenen Versuche zeigen jedoch, daß die Schutzwirkung katatoxischer Hormone, z. B. des Pregnenolon-16-carbonitrils (PCN) und des Spironolactons (SNL) sowohl gegen Digitoxin als auch gegen Indomethazin durch Triamcinolon und Thyroxin unterdrückt werden können. Oestradiol ist in dieser Beziehung nur gegen Digitoxin wirksam. Eine derartige echte antikatatoxische Wirkung hängt nicht schlechtweg von der Steroidstruktur der Wirkstoffe ab, da Desoxycorticosteron (DOC), Progesteron und Hydroxydion unter gleichen Versuchsbedingungen weder gegen Digitoxin noch gegen Indomethazin die Schutzwirkung von PCN oder SNL unterdrücken, während Thyroxin diesbezüglich hoch wirksam ist.Die im Text erwähnten Arbeiten wurden vom Medical Research Council of Canada (Block Term Grant MT-1829), dem Ministère des Affaires Sociales, Québec, der Quebec Heart Foundation und der Succession J. A. DeSève unterstützt. — Die folgenden Substanzen wurden in großzügiger Weise von den Firmen Upjohn (PCN), Searle (SNL), Squibb (Triamcinolon), Organon (DOC-Acetat), Roussel (Oestradiol, Progesteron) und Pfizer (Hydroxydion) zur Verfügung gestellt.     

Neuron Activity in the Prefrontal Cortex of the Brain in Rats with Different Typological Characteristics in Conditions of Emotional Stimulation

Male Wistar rats were separated according to the emotional resonance method (groups of animals avoiding (altruists) and not avoiding (egotists) the pain cries of partner rats) and neuron activity in the prefrontal areas of the cortex was studied in the right and left hemispheres. Assessments were made of changes in the frequency of nerve cell spike activity (in relation to the baseline activity of neurons in sated animals) in rats subjected to one day of food deprivation and after electrical stimulation of emotionally positive (lateral hypothalamus) and negative (tegmentum of the midbrain) brain structures and after exposure to the pain cries of partner rats. The results of these experiments revealed a series of differences in the cell activities of the two groups of rats. In conditions of hunger, the discharge frequency in the altruists was higher than that in egotists. Cortical neuron responses to positive stimulation were greater than those to negative stimulation in rats of both groups. Intracerebral stimulation produced significantly greater increases in discharge frequency in neurons of both prefrontal areas of the cortex in altruists than in egotists. In both groups of rats, neurons in the right hemisphere responded to emotionally negative stimulation with significantly greater activation than cells in the left hemisphere, while activity in the left hemisphere was greater in conditions of emotionally positive stimulation. Altruists showed significantly greater neuron responses during exposure to pain cries from victim rats in both the right and left hemispheres. The responses of egotists to victim cries were not significantly different from baseline activity levels.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.     

Presence of two isoforms of Na,K-ATPase with different pharmacological and immunological properties in the rat kidney

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the ₁ isoform of the catalytic subunit, whereas the collecting duct expresses an ₃-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC₅₀ 5.10^–6 M) which is recognized by an anti- ₃ antibody and another moiety of lower affinity for ouabain (IC₅₀ 5.10^–4 M) which is recognized by an anti- ₁ antibody. Whether these two subpopulations correspond to different isoforms of the subunit of Na,K-ATPase ( ₁ and ₃-like) remains to be determined.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Untersuchungen zur prognostischen Bedeutung der „Stromareaktion“ beim malignen Melanom

Zusammenfassung Es ist für den Histopathologen äußerst schwierig, beim malignen Melanom über die globale Malignität hinaus eine prognostische Aussage zu treffen. Wir griffen daher dieses Problem am Beispiel der Stromaveränderungen des malignen Melanoms erneut auf. Dabei untersuchten wir zunächst die prognostische Bedeutung einiger möglichst genau definierter histologischer Kriterien der Vascularisation anhand von 100 mindestens 5 Jahre klinisch nachbeobachteten Melanom-Fällen. Unter diesen Patienten aus 6 in der Arbeitsgemeinschaft Malignes Melanom kooperierenden Dermatologischen Universitätskliniken hatten 55 im gleichen Zeitraum eine schlechte und 42 eine gute Prognose (3 waren prognostisch uninformativ).Anstelle einer globalen histologischen Beurteilung wurden die Tumorschnittfläche und ihre Umgebung in mehrere gedachte Kompartimente unterteilt. Dabei fanden wir oft Vasodilatation und/oder quantitativ verstärkte Gefäßproliferation innerhalb oder unterhalb des Melanoms. Diese Kriterien der Vascularisation, die sich nur teilweise auf Lymphgefäße bezogen, waren besonders bei Auftreten am oberen Tumorpol, weniger deutlich auch im Zentrum oder außerhalb des Melanoms, mit ungünstiger Prognose korreliert. Damit wird der fördernde Einfluß bereits oberflächlicher äußerer Irritationen des malignen Melanoms auf dessen Frühmetastasierung unterstrichen. Des weiteren deuten intravasale Tumorzell-Einbrüche auf eine hohe, wenn auch nicht generelle Koinzidenz mit ungünstigem Verlauf hin. Ähnliche, prognostisch schwächere Tendenzen ergaben sich bezüglich des Vorkommens von partiellen Tumor-Nekrosen oder/und von hämorrhagischen Extravasaten.

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Investigations on the prognostic significance of the stroma reactivity to malignant melanomaI. Vascularization and prognosis

Summary In histopathology there are many difficulties in the evaluation of the histological features of malignant melanoma and of the surrounding tissue reactivity with regard to prognosis.As a contribution to this problem we studied the influence of stroma variations on the prognosis of malignant melanoma. We selected some defined histological criteria of vascularization and investigated their incidence in 100 melanoma patients with reference to either 5 years' survival free of symptoms or death or relapse, within this period.Among 97 prognostically informative cases of malignant melanoma from 6 Dermatological Departments in W. Germany which co-operate as a special Arbeitsgemeinschaft for some years, the prognosis was poor in 55 and good in 42. The microscopic examination was carried out in several virtually defined compartments of the tumor section and thus yielded more differentiated results than a general survey on the melanoma as a whole.In many cases we found vasodilatation and/or increased proliferation of vessels within the melanoma or surrounding it, only a varying proportion of them representing lymphatics. These criteria of vascularization were more often combined with a marked tendency to poor prognosis when localized in the superficial tumor area than when they were in the center or in the tumor's surrounding tissue. This may explain why even superficial irritations promote early metastasization of malignant melanoma cells. Furthermore tumor cells within blood vessels or lymphatics were observed being accompanied by a rather high incidence of poor prognosis. With regard to necrosis of tumor cells and/or hemorrhage into the tumor parenchyma similar but less distinct relations to impaired prognosis were detected.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Herrn Professor Erich Müller, Erlangen, zum 70. Geburtstag gewidmet.     

Microtubule stability in severed axons

Summary We examined severed axons of cat sympathetic nerves and severed neurites of cultured chick sensory neurons for evidence of extensive microtubule depolymerization. Cat sympathetic fibres fixed at various times after severing were cross-sectioned for electron microscopy from both cut ends. The number density of microtubules was determined at various times after severing for matched proximal and distal regions equidistant from cut ends. These data show that the number density of microtubules was nearly identical in proximal and distal fragments at 10, 20 and 60 min and at distances between 10 and 50 m from the cut ends. In chick sensory neurites the microtubule array was examined in longitudinal sections. In order to define objectively a normal microtubule array, the distance between 100 microtubule pairs was measured in seven control neurites, giving a mean distance (±S.D.) of 33nm±19nm. A normal array of microtubules was defined as having microtubules within 52nm of their nearest lateral neighbour. Among 11 neurites at times from 1 to 15 min after transection, the mean distance from the cut tip to the first microtubule was 1.3 m in proximal fragments and 0.5 m in distal fragments. The mean distance to the normal microtubule array was 2.8 m in proximal fragments and 2.1 m in distal fragments. There was no trend or pattern with respect to the times after severing that the neurite was fixed and the amount of microtubule depolymerization. Our results show no evidence for stabilization of axonal/neurite microtubules by capping structures at their ends. We conclude that microtubule instability is unlikely to play a role in the response of axons to axotomy.     

Effect of muscular work on the motor function of the stomach and duodenum

Summary The effect of muscle work on the gastric and duodenal motor function was studied in dogs.It was observed that the static stress inhibited the hunger contractions of the stomach and duodenum; the inhibitory effect disappeared as the stress ceased. The static muscle work effect was more pronounced in hunger contractions of the stomach than in those of the duodenum. Under the influence of static stress, the periodic hunger contractions of the stomach varied more extensively than its acid and digestive contractions.Presented by Active Member Acad. Med. Sci. USSR V. N. Chernigovsky     

Ribonuclease activity of the “myofibrillary” fraction of normal and autolyzed muscle tissue

The separation in a sucrose gradient of the myofibrillary fraction of normal and autolyzed muscle tissue gave 4 components. During post-mortem destruction of the tissue there was observed a slight decrease of the myofibrillary fraction yield and also certain changes in the distribution of protein between different components. Under the selected conditions RNase activity was found in all 4 components. During the course of autolysis enzymatic activity increased in the whole myofibrillary fraction, as well as in the lysosomal-mitochondrial components of myofibrils.Research Laboratory, Ministry of Health of the USSR, Moscow. Translated from Buylleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 533–537, May, 1978.