Age-related variations in tissue angiotensin converting enzyme activities: comparison between spontaneously hypertensive and Wistar-Kyoto rats.

Angiotensin converting enzyme (ACE) activity was measured by fluorimetry in the plasma, lung, heart, aorta and kidney (cortex and medulla) of 3-, 5-, 8- and 11-week-old spontaneously hypertensive rats (SHR) and compared with that of age-matched Wistar-Kyoto rats (WKY). In the plasma, lung and kidney (cortex and medulla), ACE activity was lower in SHR than in WKY. This was evident as early as the age of 3 weeks. In contrast, there were no differences between SHR and WKY in the aorta and the heart. Age-related variations in ACE activities differed in each tissue and in both groups of rats, but no major modifications were correlated with the development of hypertension. A binding assay was performed with [3H]ramiprilat; affinity (KD) and the maximum number of binding sites (Bmax) were determined in plasma and tissues of 3-week-old SHR and WKY. The KD values were identical in the two groups but Bmax was lower in all SHR tissues except in the heart; these results might be related to the decrease in ACE activity. Our results probably reflect genetic differences in ACE activity between SHR and WKY, and suggest that ACE regulatory mechanisms act differently in each tissue.

Characterization of a high affinity piretanide receptor on kidney membranes.

The tritiated loop diuretic, piretanide, is a useful ligand for specific diuretic receptors which are present in the plasma membranes of renal medullary cells. Its high specific activity (30 Ci X mmol-1) made it possible to demonstrate the existence of a high affinity receptor (Kd approximately 5 nM) and a binding site with low affinity. High affinity binding is saturable, reversible and displaceable by a number of non-radioactive loop diuretics. Structural analogues, devoid of diuretic activity, do not displace piretanide binding. No specific binding occurs in liver or spleen membranes.

Coordinate expression of piretanide receptors and Na+,K+,Cl- cotransport activity in Madin-Darby canine kidney cell mutants.

Two receptor sites for [3H]piretanide, a sulfamoylbenzoic acid loop diuretic, have been identified in intact Madin-Darby canine kidney cells, an epithelial cell line derived from dog kidney. The two receptor sites differed in their affinity for piretanide (KD1 = 2.1 +/- 1.4 nM and KD2 = 264 +/- 88 nM) and the maximal number of sites (Bmax1 = 11 +/- 4 and Bmax2 = 120 +/- 80 fmol/mg of protein). Madin-Darby canine kidney cells are known to possess a tightly coupled and highly cooperative Na+,K+,Cl- cotransporter which is sensitive to loop diuretics. Under ionic conditions identical to those used to study piretanide binding (30 mM Na+, 30 mM K+, 30 mM Cl-), the Ki for inhibition of the initial rate of 86Rb+ uptake by piretanide was 333 +/- 92 nM, a value not significantly different from the KD of the low affinity receptor site. [3H]Piretanide binding to three low K+-resistant mutants derived from this cell line was also studied. These mutants had been previously characterized as being partially or completely defective in Na+,K+,Cl- cotransport activity (McRoberts, J. A., Tran, C. T., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 12320-12326). One of these mutants had undetectable levels of Na+,K+,Cl- cotransport activity and low to undetectable levels of specific piretanide binding. The second mutant had low but measurable levels of cotransport activity (11% of the wild-type levels) and displayed very low affinity (KD approximately 8000 nM) specific piretanide binding. In the third mutant, expression of Na+,K+,Cl- cotransport activity and both piretanide receptors was cell density-dependent. Subconfluent to just-confluent cultures of this mutant lacked detectable cotransport activity as well as specific piretanide binding, whereas very dense cultures displayed both piretanide receptors and had intermediate to nearly normal levels of cotransport activity. These results demonstrate that the Na+,K+,Cl- cotransporter is a receptor for loop diuretics, but they also raise questions about the functional significance of the two piretanide receptor sites.

Binding of (3H)-piretanide to a specific receptor of renal medulla.

We had previously demonstrated that (3H)-piretanide binds to a receptor located on medullary membranes of canine kidney. Here we show that binding is specific for a particular group of loop diuretics. Sulfonamide diuretics of the benzoic acid family, other substituted sulfonamides, and phenoxy-acetic acid derivates displace (3H)-piretanide from its receptor. Loop diuretics that do not act at the luminal tubular membrane do not displace piretanide, nor do diuretics with a different site and mode of action (thiazides; inhibitors of the Na+/H+ antiporter (amiloride), of Na+K+ ATPase (ouabain), or of carbonic anhydrase (acetazolamide). We demonstrate that no interference occurs between the piretanide receptor and membrane bound receptors of several neurotransmitters.

Effect of angiotensin converting enzyme inhibitors on the vasoconstrictor action of angiotensin I on isolated rat kidney.

On the isolated perfused rat kidney, the angiotensin converting enzyme (ACE) activity was evaluated with two approaches: one, pharmacological, through the vasoconstrictor response to angiotensin I (ANG I), and the other, biochemical, through the measurements of the enzymatic activity on renal homogenate. ANG I and angiotensin II (ANG II) induced a concentration-dependent renal vasoconstriction (EC50 = 10.5 +/- 1.8 X 10(-9) and 1.1 +/- 0.5 X 10(-9) M, respectively). Both responses were competitively antagonized by an ANG II receptor antagonist, saralasin (pA2 = 8.65 +/- 0.63 and 8.94 +/- 0.28, respectively). The effects of ACE inhibitors were studied in vitro after addition of captopril and ramiprilat (10(-5) M) directly to the perfusion medium, and ex vivo, after pretreatment of the rats with ramipril (50 mg/kg, i.p. the day before or 10 mg/kg/day, per os, over 3 weeks). In spite of the high doses of ACE inhibitors used, the ANG I concentration-response curve was only shifted to the right by a factor of 4, although renal tissue ACE activity was completely inhibited. Saralasin (10(-5) M) totally abolished the ANG I-induced vasoconstriction elicited in the presence of ACE inhibitors, this response being therefore linked to a generation of ANG II from ANG I. Our results suggest that, on the isolated perfused rat kidney, ANG II may be formed from ANG I by a peptidyl dipeptidase different from the ACE.

Vascular effects of selective dopamine receptor agonists and antagonists in the rat kidney.

The renal vascular effects of dopaminomimetics and dopaminolytics were studied in the isolated perfused rat kidney after pretreatment with phenoxybenzamine (10(-5) M) and sotalol (10(-5) M) and after contraction of the vascular bed with prostaglandin F2 alpha. The DA1- and D1-selective antagonist, SCH 23390, antagonized competitively the relaxation induced by dopamine (pA2 = 9.7 +/- 0.08, m +/- S.D.). On the other hand, (+/-)-DO 710, a D2-preferential benzamide, only antagonized the renal vascular response to dopamine at a concentration 30 times higher than that active on D2 receptors. The ergot derivative, quinpirole, a selective agonist for DA2 and D2 receptors had no renal vascular dopaminomimetic activity, whereas (-)-EOE, a D2-selective ergoline, seemed to be a partial agonist, but 10 times less potent than dopamine. These results confirm the existence of DA1 receptors on the vascular bed of isolated rat kidney but rule out the presence of DA2 receptors. They also reinforce the analogy between DA1 and D1 dopamine receptors.

Tissue converting enzyme activity is low in the kidney of spontaneously hypertensive rats.

Angiotensin-converting enzyme (ACE) activity was measured in the plasma, the kidney, and other organs of 5-, 8-, and 11-week-old spontaneously hypertensive male rats (SHR) of the Okamoto-Aoki strain and compared with that of age-matched Wistar-Kyoto (WKY, bred by Iffa-Credo) or normotensive Wistar rats. ACE activity was measured spectrofluorometrically, using the artificial substrate N-CBZ-L-phe-L-his-L-leu. ACE activity was constantly lower in the plasma and renal cortex of SHR from 5 weeks on than in WKY rats. This difference in the renal cortex ACE activity persisted after 1 h of open circuit perfusion of the isolated kidney with Krebs-Henseleit medium. On the other hand, there lung, the brain occipital cortex, or the abdominal aorta of hypertensive or normotensive rats. The percentage inhibition of ACE activity provoked by 7 days of oral administration of ramipril (Hoe 498, 1 mg/kg/day) was analogous in the kidneys and lungs of WKY rats and SHR. Enalapril (MK 421, 30 mg/kg/day) was equipotent to ramipril in the kidney but had lower inhibitory effects on the pulmonary ACE activities of WKY rats and SHR.

Effects of SK&F 82526 and SK&F 83742 on the renal vascular dopamine receptor.

The renal vascular effects of benzazepine derivatives were studied on the isolated perfused rat kidney in the presence of phenoxybenzamine and sotalol after contraction of the vascular bed with prostaglandin F2 alpha. SK&F 82526 was a very potent dopaminomimetic drug (ED50 = 7.6 +/- 0.8 X 10(-9) M) on the renal vascular dopamine receptor. It displayed partial agonist activity (similar to SK&F 38393) and was devoid of alpha-adrenomimetic effects. SK&F 83742 was a potent dopaminolytic drug. It antagonized dopamine-induced relaxation of the renal vascular bed in a competitive way, with an apparent pA2 of 7.47 +/- 0.23.