Identification of two arginase isoenzyme activities along the nephron of Meriones shawi

 Conflicting theories on the existence of several renal arginase isoenzymes remain in debate. Because the activity of arginase is high in two embryologically different nephron segments of the Meriones shawi kidney, namely the cortical (CPST) and medullary (OSPST) proximal straight tubule and the outer medullary collecting duct (OMCD), we postulate that these nephron segments may contain different isoforms. Isolated nephron segments were dissected from collagenase-treated kidneys. Tubules were permeabilized with Triton X-100 (0.25%) and incubated with increasing Arg concentrations to characterize the arginase activity. The results were as follows: (1) in OMCD, one arginase isoform (E₁), characterized by a high Arg affinity (1.160 mM), was present; (2) in CPST, two arginase isoforms were discovered – one, E₁, had a similar K _m (1.407 mM) to that found in OMCD whereas the other (E₂) had a low affinity for Arg (K _m =18.8 mM); and (3) in OSPST, two isoenzymes were present – E₁ which had a K _m of 1.478 mM and the second isoform that we named E₂ which had a K _m of 9.07 mM. In addition, arginase located in CPST and OMCD was strongly inhibited by Orn and Lys. The K _i value for Lys varied between 1.635 and 2.288 mM. Therefore, this work demonstrates that two arginase isoforms are present in the kidney of Meriones shawi. Isoform E₁ is present in the proximal tubule and the collecting duct whereas isoform E₂ is restricted to the proximal tubule. Received: 13 August 1998 / Accepted: 25 September 1998     

Transport ofl-cystine in isolated perfused proximal straight tubules

Unidirectional fluxes ofl-³⁵S-cystine and intracellular³⁵S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux ofl-³⁵S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol·l^–1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration ofl-³⁵S-cystine and the rate of³⁵S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (J _max) of 1.45±0.27 (SEM) pmol min^–1 mm^–1, a Michaelis constant (K _m) of 0.20±0.07 mmol·l^–1, and an apparent permeability coefficient of 0.27±0.11 pmol min^–1 mm^–1 (mmol·l^–1)^–1 (approximately 0.06 m/s). The³⁵S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminall-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.Some of the work described here was presented as an abstract at the 8th International Congress of Nephrology, Athens, Greece, 1981     

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial bright portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7±19.1 fmol cAMP mm^–1 4 min^–1 (SE, N=13) whereas VIP-induced cAMP accumulation was less and more variable from one experiment to another (1 M, 97.2±17.8 fmol mm^–1 4 min^–1, SE, N=12). When used in combination, these two agonists were non-additive, indicating stimulation of a single pool of cAMP in DCTb. In DCTg, (granular) which consists of at least two cell types, PTH (100 nM) elicited a marked, reproducible accumulation of cAMP (154.3±27.0 fmol mm^–1 4 min^–1; SE, N=5). Isoprenaline (1 M) and VIP (1 M) induced much smaller increases in cAMP levels 20.9±2.7 and 29.4±4.1 fmol mm^–1 4 min^–1 (SE, N=5) respectively, and, when used in combination, were non-additive, demonstrating that VIP and isoprenaline are active on the same cell type. In DCTb, prostaglandin E₂ (PGE₂) inhibited both calcitoninand VIP-stimulated cAMP accumulation (calcitonin 57.8±2.7% inhibition, SE, N=16; VIP, 80.6±2.1% inhibition, SE, N=5). The EC₅₀ values for calcitonin were 1.21±0.33 ng/ml and 1.83±0.25 ng/ ml (SD, N=3) in the absence and presence of PGE₂ (300 nM) respectively with an IC₅₀ for PGE₂ of 26.3±6.3 nM (SE, N=4). In contrast, no effects of PGE₂ were seen in DCTg vis à vis PTH, isoprenaline or VIP. The percentage inhibition of calcitonin-stimulated cAMP accumulation by PGE₂ was of the same order in the presence of isobutylmethylxanthine (an inhibitor of all types of phosphodiesterase), Ro 20-1724 (inhibitor of low-K _m cAMP-specific phosphodiesterase) or in the absence of inhibitor. Preincubation of DCTb with pertussis toxin for up to 8 h in different experimental conditions did not relieve the inhibition by PGE₂. Protein kinase C activation by phorbol ester did not attenuate calcitonin responses. These data demonstrate that the inhibition by PGE₂ of cAMP production is restricted to the initial portion (DCTb) of the distal convoluted tubule and is effective on both calcitonin and VIP responses. When tested in the presence of Ro 20-1724, ionomycin, A₁-adenosine, ₂-adrenergic and muscarinic agonists were without effect on calcitoninand PTH-stimulated cAMP accumulation in DCTb and DCTg respectively.     

Relaxation of chemically skinned guinea pig taenia coli smooth muscle from rigor by photolytic release of adenosine-5′-triphosphate

Summary The mechanical events following release of ATP from P³-1-(2-nitro)phenylethyladenosine-5-triphosphate (caged-ATP) in skinned guinea pig taenia coli smooth muscle in rigor were investigated. A rigor force of about 25–35% of the maximal active force was obtained by removing ATP at the plateau of a maximal active contraction. In the rigor solution free-Mg²⁺ was 2mm, ionic strength 90mm and pH 7.0. When caged-ATP (12.5mm) was diffused into the preparation there was no change in the rigor force. Photolytic production of about 2mm ATP was achieved with a xenon flash lamp. Following illumination, force decreased with an approximate initial rate constant of 0.7 s^–1. The rate of relaxation was increased in the presence of inorganic phosphate (at 3mm: 1.3 s^–1; 10mm: 2.2 s^–1). At higher Mg²⁺ concentrations the rate of relaxation was slower (5mm: 0.2 s^–1) and at lower concentrations the rate was faster (0.5mm: 1.2 s^–1). An increased rate of relaxation was observed when ionic strength was increased to 150mm (2.2 s^–1). Phosphate increased the rate of relaxation at the different levels of Mg²⁺ (0.5–10mm) and ionic strength (90, 150mm). In preparations shortened (by 1–3%) to give reduced rigor force, a small transient increase in tension was recorded after ATP release. In comparison to the rates of ATP-induced dissociation of actomyosin in solution, reported in the literature, the rate of relaxation from rigor is slower. This may reflect a slow rigor cross-bridge dissociation or mechanical interactions not associated with cross-bridges in the muscle fibre. However, the results may also be interpreted on the basis of a model proposed for striated muscle by Goldmanet al. (1984) where the relaxation from rigor in the absence of Ca²⁺ involves a phase of reattaching cross-bridges whose lifetime in a tension-producing state is influenced by phosphate.     

L-ornithine transaminase synthesis in Saccharomyces cerevisiae: Induction by allophanate,intermediate and inducer of the urea degradative pathway adds to arginine induction

Summary Yeast ornithine transaminase is known to be induced by arginine and ornithine, through the action of regulatory elements common to arginase induction. We show here that it is subject to a second induction circuit, that which is responsible for urea amidolyase and urea permease induction by allophanate and defined by the regulatory mutants durL ^– and durM ^–     

Thermodynamic analysis of calcium binding to frog parvalbumin

Summary The enthalpy of calcium binding to frog parvalbumin (Rana temporaria isoenzyme IVb, pI 4.75) has been measured by microcalorimetry. The reaction is exothermic; the heat of the reaction in 100mm KCl, 50mm Tris, pH 8.0 at 12° C is –19 kJ (mol site)^–1 and –33 kJ (mol site)^–1 in the presence of 1mm magnesium. The shape of the titration curve indicates that the properties of the two calcium binding sites are different. The thermodynamic parameters measured for frog parvalbumin are compared with those of related parvalbumins from carp and whiting.     

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake

Summary The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a K_M of about 2 × 10^–4 M for both media and a V of about 5 nmol min^–1mg^–1 and 2 nmol min^–1mg^–1 for derepressed and repressed cells respectively. The pho 1–118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1–118 mutation.     

Role of vasopressin V2 receptors in modulation of the renal cortico-papillary NaCl gradient

In order to examine if modulation by vasopressin of NaCl transport in Henle's loops (via V2 receptors) can significantly modify medullary ionic hypertonicity, the effects of stimulation or inhibition of these receptors were studied in anaesthetized Wistar rats. Total electrolyte concentration in the medullary interstitium was continuously measured as tissue admittance (reciprocal impedance), using needle electrodes recording from the inner and outer medulla of the in situ kidney. Deamino-[Cys¹,D-Arg⁸]vasopressin (dDAVP], a V2 agonist, infused i.v. at 7.5 ng · min^–1 · kg^–1, significantly increased admittance by 9% and 8% in the inner and outer medulla, respectively. A slightly pressor i.v. infusion of natural arginine vasopressin (AVP) induced pressure natriuresis and did not affect medullary electrolyte concentration. Inhibition of V2 receptors with [d(CH₂)⁵,D-Phe², Ile⁴]-AVP, infused i.v. at 133 g · h^–1 kg^–1 in indomethacin-treated rats, decreased admittance (significant in the inner medulla). Neither of the three agents used caused significant changes in the renal blood flow (RBF) or clearance of inulin (C _in). The demonstration that changing activity of V2 receptors affects the corticopapillary NaCl gradient indicates that, at least in rodents, stimulation of loop salt transport by AVP may represent an additional mechanism enhancing urine concentration.     

Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules

Binding of [¹²⁵I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensititive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16–27×10^–18 mol mm^–1) were found in the thick ascending limb of the Henle's loop and in the distal convoluted tubule and lower binding levels (2–5×10^–18 mol mm^–) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henle's loop. In the medullary thick ascending limb, Scatchard analysis of specific [¹²⁵I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation.Abbreviations PCT proximal convoluted tubule - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT medullary collecting tubule     

Generation of tension by glycerol-extracted vertebrate skeletal muscle fibres in the absence of calcium

Summary When a small bundle of glycerol-extracted fibres from either frog, tortoise or rabbit skeletal muscle was first exposed to high MgATP (5mm) in the absence of Ca²⁺ (<1nm) and at low ionic strength (<0.11) at 20° C, it produced a single sharp transient contraction followed by a lower maintained isometric tension. The maintained tension was investigated further in rabbit psoas fibres. Ca²⁺-free tension was dependent on the ionic strength in the range 0.04–0.10, on the temperature in the range 6–20° C and the free Mg²⁺ in the range 0–6mm. It was promoted by low ionic strength, low Mg²⁺ and high temperature, and was unaffected by varying the MgATP^2– in the range 0.4–4mm and by adding ATP regenerating components. A separate regime of tension generation was detected at MgATP^2– concentrations of less than 0.1mm, in which MgATP^2– concentration was critical. The results are interpreted on the assumption that binding of Mg²⁺ to some component of the regulatory system is necessary to maintain its inhibitory effect in the absence of Ca²⁺. Ionic strength and temperature, on the other hand, may affect actomyosin directly.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Aquaporin 6 is permeable to glycerol and urea

The passive water permeability (L_p) of AQP6 is activated by Hg²⁺. Our aim was to test if L_p was associated with a permeability to small hydrophilic molecules such as glycerol and urea. AQP6 was expressed in Xenopus laevis oocytes and activated by 0.3 mM of HgCl₂ in the bathing solution. HgCl₂ caused the oocytes to swell at a rate of about 0.3% min^–1. The L_p of AQP6 was determined from brief additions or removals of mannitol from the bathing solution, and compared to the L_p obtained from adding glycerol or urea. In paired experiments, L_{p (mannitol)} was 2.4±0.1, L_{p (glycerol)} 1.5±0.2, and L_{p (urea)} 0.7±0.1 (units: 10^–5 cm s^–1 osmol^–1; 14 oocytes); the latter was not different from L_ps obtained for native oocytes. The L_ps were independent of the Hg²⁺-induced swelling and of the magnitude of the osmotic challenge (–75 to +100 mosmol). Hg²⁺ stimulated the uptake of [¹⁴C]glycerol fivefold and [¹⁴C]urea twofold in AQP6-expressing oocytes. There were no significant uptakes in native oocytes nor in AQP1-expressing oocytes whether these were treated with HgCl₂ or not. We conclude that water, glycerol and urea share an aqueous pathway in AQP6.     

Lack of effect of intraluminal pressure on renin release from isolated afferent arterioles

To evaluate the role of the proposed baroreceptor mechanism in the afferent arteriole in regulating renin release, we modified the isolated perfused tubule technique to perfuse afferent arterioles. Arterioles with attached glomeruli were isolated from rabbit kidneys and perfused using standard methods. To stop the arteriolar flow and allow perfusion pressure, as set by a mercury manometer, to be built up in the lumen of the vessel, the glomerulus was sucked into a constriction pipette. The preparation was continuously superfused with Krebs-Ringer solution in the first series of experiment, and a cell culture medium in the second series of experiment. The superfusate droplets were collected under mineral oil with 10-min collection intervals. The renin content of the samples was assayed by radioimmunoassay of the angiotensin I generated. In the two series of experiments we tested the effects of sequential changes in intraluminal pressure on renin release. In the first series of experiments (n=6) the renin release was 56.3 nGU arteriole^–1 min^–1 in the first 10 min of sampling. The renin release was then constant for 80 min with an average of 21.6 nGU arteriole^–1 min^–1. In the last 30 min the renin release was 96.5 nGU arteriole^–1 min^–1. In the second series of experiments (n=8) the renin release was 26.5 nGU arteriole^–1 min^–1 throughout the course of the experiment. These results indicate that under these conditions there is no relation between renin release and intraluminal pressure in afferent arterioles.     

Lifelong variations in heart rates in SPF Sprague Dawley rats of both sexes

In unanesthetized Sprague Dawleys, SPF, of both sexes, housed indl _12:12 (110 lux), at a temperature of 18–23°C and a hygrometry of 60–75%, the measurements of heart frequencies during most part of their life point out a continuous decrease with age and an always higher (40–20 c. min^–1) heart rate in females than in males. Significant correlations between rate and body weight can account for these heart which corresponds to the growth period, a linear relationship was established between Log heart rate (y;c. min^–1) and Log body weight (x; gram); for males: Logy=–0.122 Logx+Log 938 and for females: Logy=–0.166 Logx+Log 1217. After the age of 600 days, which corresponds to senescence, decreases in heart rate as well as in body weight were observed in both sexes.We acknowledge the technical assistance of Serge Hazout, Christian Lemercerre and Micheline Duriez     

Messungen des tubulären Harnstromes,seine Beziehungen zum Blutdruck und zur Inulin-Clearance

Zusammenfassung An Nembutal-narkotisierten Ratten wurde eine kontinuierliche Zunahme aller tubulären Passagezeiten von Lissamingrün (LG) bei arterieller Blutdrucksenkung (Entblutungshypotonie) gefunden. Bei einem Mitteldruck von 60 mm Hg hat die proximale Passagezeit um den dreifachen, die distale Passagezeit um den zehnfachen Betrag zugenommen.An jungen Katzen beträgt die Passagezeit von LG für den an der Nierenoberfläche sichtbaren Anteil des proximalen Konvolutes im Mittel 7,5 sec, die Schleifen-Passagezeit 35 sec und die distale Passagezeit 41 sec. Im Mitteldruckbereich unter 80 mm Hg sind bei Katzen wiederum alle Passagezeiten verlängert (distal > proximal), während die Passagezeit oberhalb dieses Wertes vom Druck unabhängig erscheint. Bei Blutdrucksenkungen sind neben der bekannten Einschränkung der Inulin-Clearance und der Diurese auch Abnahmen der proximalen Tubulusdurchmesser zu beobachten. Eine Abhängigkeit zwischen proximaler Stromstärke und arteriellem Mitteldruck findet sich nur im Druckbereich unter 80 mm Hg. Zwischen proximaler Stromstärke und Inulinclearance besteht eine fast lineare Korrelation. Aus direkten Strömungsgeschwindigkeits-Messungen und der proximalen Passagezeit wird die mittlere Länge des oberflächlich sichtbaren Anteiles eines proximalen Konvolutes abgeleitet, sowie der Nettowasserfluß für die innere Tubulusoberfläche berechnet.In der Diskussion wird das Problem der glomerulär-tubulären Balance erörtert. Unsere Ergebnisse scheinen die Hypothese von Gertz zu bestätigen. Ferner werden aus der Schleifen-Passagezeit und morphologischen Daten Strömungsgeschwindigkeiten in dem für die Harnkonzentrierung wichtigsten Bereich des Nierenmarkes berechnet und die Bedeutung von Strömungsbeschleunigungen für die Harnkonzentrierung diskutiert.

---

Summary When the blood-pressure of rats is reduced by bleeding, we found a continous increase of all the tubular passage-times of Lissamingreen measured on the renal surface by incident-light microscopy. With a blood-pressure of 60 mm Hg the proximal passage-time of Lissamingreen was three-times the normal and the distal passage-time ten-times the normal.On young cats with normal blood-pressure Lissamingreen took an average of 7,5 sec to pass through the visible part of the proximal convolution; the average times taken to pass through the Henle-loops and the distal convolution were 35 sec and 41 sec respectively.As in the case of rats all passage-times increased when the mean blood-pressure was less than 80 mm Hg (the distal passage-time increased more than the proximal passage-time), whereas the proximal passage-time on higher values was not affected by pressure. This corresponds to the so-called autoregulation of the kidney. When the blood-pressure is reduced, a decrease of the diameter of the proximal tubules can be seen in addition to the known reduction in the inulin-clearance and diuresis. The correlation between the inulin-clearance and the diameter of the tubules is then at its most obvious. There is only a connection between the proximal tubular volume-flow and the mean blood-pressure when the pressure is below 80 mm Hg. However there is almost a linear correlation between the proximal tubular volume-flow and the inulin-clearance.The average length of the part of a proximal convolution visible on the surface is calculated from direct measurements of the flow-velocity and the passage-time at 2,6 mm. With those length an average proximal tubular volume-flow of 1.2±0.07 · 10^–4 mm³ · sec^–1 is to be found withCin>3.0 ml · min^–1 · kg^–1. OnCin 2.1 to 2.9 ml · min^–1 · kg^–1 the volume-flow is 8.9±0.7 · 10^–5 mm³ · sec^–1 and onCin<2.0 the volume-flow is 3.9±0.9 · 10^–5 mm³ · sec^–1.A net water reabsorption for internal tubular surface of approximately 5.0·10^–4 mm³ · mm^–2 is calculated from the decrease of the flow-velocity in the proximal convolution.The flow-velocity in the descending and ascending limb of the Henle-loop was calculated from the passage-times for the loops and from morphological data.The question of the glomerular-tubular balance was discussed and the conclusions tended to support the Gertz-theory.

---

---

Mit 8 Textabbildungen

---

Mit Unterstützung der Deutschen Forschungsgemeinschaft.

---

Auszugsweise vorgetragen auf der 29. Tagung der Deutschen Physiologischen Gesellschaft, Tübingen 1964²⁹.

---

Kir. klin. Sahlgrenska Sjukhuset, Universität Göteborg/Schweden.     

Evidence for sltA1 as a salt-sensitive allele of the arginase gene (agaA) in the ascomycete Aspergillus nidulans

Strains ofAspergillus nidulans carrying thesltA1 mutation, conferring sensitivity to KCl and NaCl, also showed an arginine-sensitive phenotype whereby concentrations of thel-amino acid at or above 10 mM were toxic to growth. Sexual progeny of a cross between asltA1 mutant and a wild-type strain showed a co-segregation of salt and arginine sensitivity. Similarly, revertants to salt tolerance showed a loss of arginine sensitivity as didsltA1 strains that were transformed with a cosmid carrying the putativesltA1 ⁺ wild-type allele. In addition, arginine sensitivity could be relieved byl-ornithine. It is suggested thatsltA1 is a salt-sensitive allele of the arginase gene (agaA).     

Reduced exercise hyperaemia in calf muscles working at high contraction frequencies

Summary The effects of muscle contraction frequency on blood flow to the calf muscle (Q _calf) were studied in six female subjects, who performed dynamic plantar flexions at frequencies of 20, 40, 60, 80 and 100 contractions · min^–1, in a supine position. TheQ _calf measured by a mercury-in-rubber strain gauge plethysmograph, increased as contraction frequency increased and reached a peak at 60–80 contractions · min^–1. After 100 plantar flexions at 60 contractions · min^–1, the meanQ _calf was 30.95 (SEM 4.52) ml · 100 ml^–1 · min^–1. At 100 contractions · min^–1, however, it decreased significantly compared with that at 60 contractions · min^–1 at a specified time (2 min or exhaustion) or after a fixed amount of work (100 contractions). The contraction frequency at whichQ _calf reached a peak depended on the duration of exercise. The heart rate showed its highest mean value at 60 contractions · min^–1 and decreased significantly at 100 contractions · min^–1. The mean blood pressure was lower at 100 contractions · min^–1 than at 60 contractions · min^–1. The relaxation period between contractions, measured by recording the electromyogram from the gastrocnemius muscles, shortened markedly as the frequency increased; the mean value at 100 contractions · min^–1 was 0.14 (SEM 0.02) s, which corresponded to 35.7% of the contraction time. This shortened relaxation period between contractions should have led to the inhibition of exercise hyperaemia at the higher contraction frequencies.     

Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10^–11 m Ca²⁺ and 2.7×10^–9 m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10^–5 m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10^–4 m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10^–4 m Ca²⁺ buffered with 10mm EGTA. At 1.3×10^–7 m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10^–4 m.     

Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

Summary We have investigated the conditions for isolation of Ca²⁺-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml^–1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g^–1 artery wet weight) were made up of actin, tropomyosin and a 120 000M _r protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca²⁺. Up to 80% inhibition was observed in the absence of Ca²⁺. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca²⁺ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000M _r protein.     

Differenzierung der Wirkungen von CO2-Druck und Wasserstoffionenkonzentration im Blut auf die Atmung beim Menschen

Summary Data published in the preceding paper on effects of augmented arterial CO₂ tensions on lung ventilation before and after inducing acidosis by NH₄Cl or CaCl₂ or acetazolamide (diamox) were used to differentiate the partial effects of p _H changes at constant CO₂ tension and of CO₂ tension changes at constant p _H in arterial blood on lung ventilation.The total increase of lung ventilation caused by elevation of CO₂ tension was found to be 3.2 l min^–1 Torr^–1. The partial p _H effect was 1.4 l min^–1 Torr^–1 or 43% per cent of the total effect and the partial CO₂ effect was 1.8 l min^–1 Torr^–1 or 57 per cent of the total effect. This is in agreement with the prediction of Gray and the experimental results of Lambertsen and Semple.

---

---

Mit 1 Textabbildung

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

---

Die Ergebnisse wurden teilweise mitgeteilt in Vorträgen vor der Gesellschaft für Morphologie und Physiologie in München und in Oxford im Juli 1959.

---

Stipendiat der A. v. Humboldt-Stiftung.