Cultured renal collecting duct cells from neonatal rabbit kidney were used to examine the influence of aldosterone on enyzmatic activity of citrate synthase during increase in Na⁺ transport. Control epithelia showed citrate synthase activity of 71 ± 3 mU/mg protein (n=28), while after aldosterone treatment citrate synthase activity was significantly increased to 79 ± 6 mU/mgat 1 h(n=5), to 88 ± 6 mU/mg at 2 h(n=6) and to 93 ± 8 mU/mg protein at 3 h (n=5). Citrate synthase activity subsequently decreased to basal values. Spironolactone fully blocked the aldosterone-induced increase in citrate synthase activity. The time course of enzyme stimulation after aldosterone administration indicates that the hormone activates citrate synthase during the physiological early response phase.

The effects of aldosterone on protein synthesis in the latent period were investigated on cultured renal collecting duct cells from neonatal rabbit kidneys. Tissue was incubated with radioactively labelled uridine and amino acids and then precipitated with trichloroacetic acid in order to determine the intracellular precursor pool and identify new synthesis of RNA and protein. During the latent period, aldosterone increased the intracellular radioactive uridine pool and total radioactive RNA content already 20 and 60 min after its application; conversely 40 min after aldosterone introduction, no stimulation was found. Further experiments revealed that the intracellular radioactive amino acid pool was generally increased by aldosterone after 20, 40 and 60 min, while a distinct increased radioactive protein content was found to be induced by aldosterone only after 40 min. This indicates that aldosterone increases the uptake of RNA and protein precursors and the new synthesis of RNA and proteins. These events seem to to be regulated not continously but intermittently. The induced proteins possibly take part in the mediation of the early hormone response. Experiments with the aldosterone antagonist, spironolactone, provide evidence for the specificity of the described hormone effects. The results after application of the Na⁺ channel blocker, amiloride, and the Na⁺/K⁺-ATPase inhibitor, G-strophanthin, indicate that the aldosterone effects are controlled by Na⁺ channels and Na⁺ pumps and therefore by the intracellular Na⁺ content. The inhibitory effect of cycloheximide on the aldosterone-induced protein synthesis indicates the role of these proteins on the hormone-stimulated Na⁺ transport.

To investigate whether ‘aldosterone-induced proteins’ could be detected in mammalian species, cultured renal collecting duct epithelia from neonatal rabbit kidneys were labelled under aldosterone administration with radioactive methionine and subsequently fractionated into cytosolic and coarse membrane protein fractions. Newly synthesized proteins were then analyzed by SDS-PAGE, isoelectric focussing and two-dimensional electrophoresis. Quantitative estimates of individual newly synthesized proteins were performed utilizing gel slicing, scintillation counting and autoradiography. The labelling experiments demonstrated that, in comparison to controls, aldosterone (1× 10^-6 M) generally increased the amount of radioactive protein. No qualitative changes in the pattern of newly synthesized proteins and, therefore, no classical aldosterone-induced proteins were observed. The increase of radioactive protein was already seen after 1, 6, and 18 h of hormone treatment. The effect could be blocked partially by spironolactone (1.5×10^-4M), and totally by amiloride (1×10^-6M), g-strophantin (5×10^-4M), and cycloheximide (1 × 10^-6M). Thus, the interference of aldosterone action at the receptor level, the Na⁺ channels and the Na⁺/K⁺-ATPase pump demonstrate that the expression of proteins in cultured renal collecting duct cells is a sensitive system and seems to be controlled by aldosterone at the receptor level, but also counter-controlled by specific plasma membrane sites.

Die pH-Abhängigkeit der biologischen Inaktivierung von ot-L-AspartyP-Angiotensin II (A) und von a-L-AsparaginyP-Angiotensin II (H) durch menschliches Plasma sowie die pH-Abhängigkeit der Hemmbarkeit von "Angiotensinasen" durch EDTA und ihre Reaktivierung durch zweiwertige Metallionen wurden vergleichend untersucht. Beide Substrate werden durch mindestens drei in verschiedenen pHBereichen optimal wirksame Enzyme gespalten. In allen pH-Bereichen fanden sich unterschiedliche Eigenschaften der A-bzw. H-iniktivierenden Enzyme. Zusätzlich wurde die pH-Abhängigkeit der biologischen Inaktivierung von H und der Abspaltung von Asparagin i lus H (Aminopeptidase) verglichen. Die Annahme, daß die bei pH 5,6 optimal wirksamen Enzyme Endopeptidasen und die im Neutral>ereich und im Alkalischen optimal wirksamen Enzyme Aminopeptidasen sind, ist mit unseren Ergebnissen vereinbar. Studies on the pH-dependence y Inhibition and reactivation of angiotensin II and angiotensin II-amlde splitting enzymes In human plasmaWe compared the pH-dependance of the inactivation of a-L-aspartyP-angiotensin II (A) and of a-L-asparaginyP-angiotensin II (H) by human plasma. In addition, the pH-dependance of EDTA-inhibition of "angiotensinases" and its reversal by divalent cations were investigated. Both Substrats are split by at least three enzymes with different pH-optima. There are differences between the A-and H-splitting enzymes in the acid, neutral and alkaline range. We further investigated the pH-dependance of the hydrolysis of the N-terminal peptide bond in H. Our results are compatible with the assumption that the enzymes most active at pH 5.6 are endopeptidases, while the en-: zymes with pH-optima in the neutral and alkaline range are aminopeptidases.£ REGOLI und Mitarbeiter (1) äußerten als erste die Vermutung, daß AsparaginyP-Angiotensin ( ) und AspartyP-Angiotensin ( ) im Blutplasma durch unterschiedliche Enzyme inaktiviert werden. NAGATSU und Mitarbeiter (2) konnten diese Annahme weitgehend bestätigen. Sie fanden, daß die Hydrolyse von ÖC-LAspartyl 1 -bzw. -L-Glutamyl-jff-Naphthykmid durch A, nicht aber durch H kompetitiv gehemmt wird. Die Hydrolyse von L-Leucyl-j9-Naphthylamid wird andererseits nur durch H kompetitiv gehemmt. Während die Hydrolyse der N-terminalen Peptidbindung von A in unverdünntem Serum bei pH 7,0 durch 12mMCa' i

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