Measurements of GnE/GnM from the 2H(e-->,en-->)1H Reaction to Q2=1.45 (GeV/c)2.

We report new measurements of the ratio of the electric form factor to the magnetic form factor of the neutron, G(n)(E)/G(n)(M), obtained via recoil polarimetry from the quasielastic 2H(e-->,e(')n-->)1H reaction at Q2 values of 0.45, 1.13, and 1.45 (GeV/c)(2) with relative statistical uncertainties of 7.6% and 8.4% at the two higher Q2 points, which points have never been achieved in polarization measurements.

Phenotypic instability of Salmonella strain YG1024 during mutagenicity assays of arylamine promutagens.

During spot tests using Salmonella TA98 derivatives (YG1021, YG1024) and TA100 derivatives (YG1026, YG1029), a unique response of O-acetyltransferase (OAT)-enhanced strains YG1024 and YG1029 to arylamines was observed. On plates containing rat-liver S9, these strains yielded revertant colonies induced in two separate concentric rings around the site of application, while the parent (TA98, TA100) and nitroreductase-enhanced strains (YG1021, YG1026) did not exhibit this response. The inner ring of revertants was accompanied by cytotoxicity and microcolony formation, with the outer ring in a region without background lawn toxicity. Addition of tetracycline to the top agar eliminated formation of the inner ring of YG1024 revertants in spot tests and reduced the revertant count in preincubation assays at cytotoxic dose levels of 2-aminoanthracene, 2-aminofluorene, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline. Tetracycline sensitivity indicates that mutant colonies developing at high concentration/toxicity arose, in effect, from TA98 regenerated by functional loss of the tetracycline-resistance plasmid (pYG219) from YG1024. Mutant colonies found at low concentration/toxicity arose from normal plasmid-bearing YG1024. These results indicate the need to consider coincidental toxicity-induced instability in YG1024 during quantitative mutagenicity assays of arylamines and uncharacterized complex mixtures.

The antagonist properties of S 11978 on 5HT3 receptors in N1E-115 neuroblastoma cells.

The effects of the novel antagonist S 11978 (Endo-7-[(8-methyl-8-azabicyclo[3,2,1]-3-octyl)oxycarbonyl] benzo[b] thiophene) on 5HT3 receptors were examined in N1E-115 mouse neuroblastoma x rat glioma hybrid cells, with radioligand binding and whole cell patch clamp techniques. The 5HT3 receptor ligand [3H] quipazine was displaced by ICS 205-930, GR 38032F and S 11978 with KI values of 2.25 nM, 36.5 nM and 1.75 nM respectively. Electrophysiological studies showed that S 11978 is a potent 5HT3 antagonist: IC50 values for inhibition of 5HT-induced inward current by ICS 205-930, GR 38032F and S 11978 were 0.22 nM, 0.63 nM and 0.43 nM respectively at a holding potential of -65 mV. It is concluded that S 11978 is a potent, high affinity 5HT3 receptor antagonist.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca2+ channel inhibition in a rat osteoblast-like cell line, UMR 106, by a new dihydropyridine derivative, S11568.

UMR 106 rat osteogenic sarcoma cells were studied with the whole cell patch clamp technique to investigate the presence of voltage-gated inward currents. In barium (Ba2+)-containing medium, depolarizing jumps revealed both transient (T-type) and sustained (L-type) Ba2+ currents. The L-type component was dihydropyridine-sensitive: the agonist Bay K 8644 increased the amplitude of the L-type Ba2+ current. A new dihydropyridine calcium channel blocker, S 11568 ((+/-)-2(2-[2-(aminoethoxy)ethoxyl]methyl)4-(2',3'- dichlorophenyl)3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4- dihydropyridine, and its enantiomers, S 12967 ((+)-S 11568) and S 12968 ((-)-S 11568), inhibited the L-type Ba2+ current. IC50 values at a holding potential (VH) of -50 mV were 90 nM for S 11568, 800 nM for S 12967 and 45 nM for S 12968. At VH = -80 mV, S 12968 was less potent (IC50 near 500 nM). In contrast, S 12968 was without appreciable effect on the T-type component of the inward current through Ca2+ channels. Our results indicate that UMR 106 cells express both T-type and L-type Ca2+ channels and could be used to study the modulation by Ca2+ channel blocking agents, such as S 12968, of the hormonal regulation of Ca2+ fluxes across the osteoblast membrane.

Akuammine and dihydroakuammine, two indolomonoterpene alkaloids displaying affinity for opioid receptors.

Akuammine [1], an indolomonoterpene alkaloid, which is the major component of the seeds of Picralima nitida, was reduced to dihydroakuammine [4]. This compound has structural analogy with eseroline [7], for which affinity for opiate receptors was reported. The present investigation showed that 1 and 4 also bind (with lower affinity however) to mu and kappa opiate receptors. 1H- and 13C-nmr spectra of 1 and 4 have been fully assigned by 2D nmr experiments.

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.

Binding of loop diuretics to their renal receptors: use as a screening model for potential diuretic activity.

Loop diuretics of the benzoic acid and aryloxyacetic acid families inhibit Na+K+Cl- cotransport. The ranking order of potencies measured in the thick ascending limb of Henle's loop and the ranking order of affinities for [3H]piretanide receptors on renal plasma membranes are the same. Potencies and affinities correlate well (correlation coefficient r = 0.959 for the medulla and r = 0.951 for the cortex). Therefore, measurement of [3H]piretanide binding is proposed to facilitate screening for loop diuretic action.

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.

[Effects of conversion enzyme inhibitors on the synthesis of angiotensin II by the isolated rat kidney]. / Effets des inhibiteurs de l'enzyme de conversion sur la synthèse d'angiotensine II par le rein de rat isolé.

On the isolated perfused rat kidney, angiotensin-converting-enzyme activity was evaluated by two approaches: one, biochemical, through the measurements of the enzymatic activity on renal homogenate, the other, pharmacological, through the vasoconstrictor response to angiotensin I. Renal tissue angiotensin-converting-enzyme activity was not modified by setting the kidney under perfusion with a modified Krebs-Henseleit solution but was inhibited after addition of captopril into the perfusion medium (10(-5) M, 100 p. 100 inhibition) or after pretreatment of the animals with ramipril (10 mg/kg/day over 3 weeks, per os, 60 p. 100 inhibition). On the isolated perfused rat kidney, angiotensin I and angiotensin II induced a concentration dependent renal vasoconstriction (EC50 = 1.05 +/- 0.18 X 10(-8) and 0.11 +/- 0.05 X 10(-8) M) which was competitively antagonized by saralasin, an angiotensin II receptor antagonist. Addition of angiotensin-converting-enzyme inhibitors to the perfusion medium (captopril or ramiprilat, 10(-5) M) or pretreatment of the animals with ramipril (50 mg/kg, i.p. the day before or 10 mg/kg/day over 3 weeks, per os) only shifted the angiotensin I concentration-response curve to the right by a factor 3 to 4. The residual vasoconstrictor effect of angiotensin I was abolished by 10(-5) M saralasin and remains linked to a local generation of angiotensin II. Our results suggest that, on the isolated perfused rat kidney, besides the angiotensin-converting-enzyme, an iso-enzyme may also be able to generate angiotensin II.

Renal and iliac vascular effects of dopamine in the anaesthetized rat.

The renal and iliac vascular effects of dopamine were compared in pentobarbital anaesthetized rats. Local vascular resistances were calculated from simultaneous measurement of blood pressure, renal and iliac blood flow. Without pretreatment, dopamine increased renal and iliac vascular resistance. After pretreatment with prazosin, dopamine decreased the renal vascular resistance while the iliac vascular resistance was still increased. After a combination of yohimbine and prazosin pretreatment, dopamine lowered both the renal and iliac vascular resistance by 30%. These responses were not modified by the beta-adrenoceptor antagonist, sotalol, or by pretreating the rats with reserpine. The renal but not the iliac vascular response to dopamine was abolished by (+)-butaclamol, a stereoselective dopamine receptor antagonist, and by SCH 23390, the DA1-selective dopamine receptor antagonist. The decrease in iliac vascular resistance was not modified by indomethacin or the non-selective 5-HT receptor antagonist, metitepin. These results show that after blockade of alpha 1 and alpha 2-adrenoceptors, dopamine induces iliac vasodilation by a postsynaptic mechanism independent of an interaction with beta-adrenoceptors, dopamine or serotonin receptors. They also confirm in the rat in vivo the existence of renal vasodilation mediated by DA1 dopamine receptors.

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.

Characterisation of the alpha-adrenoceptors of the rat renal vascular bed.

Postjunctional renal alpha-adrenoceptors were studied (1) in vivo, on the renal vasculature of the anaesthesized rat and compared with those in the femoral vasculature, and (2) in vitro, on the renal vascular bed of isolated perfused rat kidney. In vivo, renal and iliac blood flows were measured with an electromagnetic flow meter. The i.v. injection of (-)-phenylephrine (1-16 micrograms/kg) and B-HT 920 (0.6-600 micrograms/kg) induced an increase in both renal and iliac vascular resistance, inhibited respectively with prazosin (300 micrograms/kg) or yohimbine (300 micrograms/kg). In the kidney, maximum response to B-HT 920 was equivalent to 64% of that to (-)-phenylephrine; on the iliac vasculature, vasoconstrictor responses to both drugs were identical, but only corresponded to 50% of the maximum renal response to (-)-phenylephrine. This indicates the predominance of alpha 1- over alpha 2-adrenoceptors in the renal vascular bed. In vitro, on the isolated perfused rat kidney, vasoconstriction was induced by the preferential alpha 1-adrenoceptor agonists [(-)-phenylephrine, cirazoline and methoxamine] and the preferential alpha 2-adrenoceptor agonists (alpha-methylnoradrenaline, dopamine and clonidine) at concentrations at which they lose their selectivity for the alpha 2-adrenoceptors; all responses were antagonised by prazosin but not by yohimbine. B-HT 920, the selective alpha 2-adrenoceptor agonist, only induced renal vasoconstriction in vitro under concomitant infusion of rabbit plasma.

[Comparison of the vasomotor effects of dopamine on the renal and iliac arterial beds of the anesthetized rat]. / Comparaison des effets vasomoteurs de la dopamine sur les lits artériels rénal et iliaque chez le rat anesthésié.

Dopamine remains the reference in the study of the mechanisms involved in the antihypertensive effects of dopaminomimetics. Its renal vasodilator effects are well characterized but relaxation of other vascular beds is less known. The iliac vascular response to dopamine was studied in the anesthetized rat (pentobarbital) and compared to the renal response. Simultaneous measurements of arterial pressure, iliac and renal blood flows (electromagnetic flowmeter probes, Skalar, Delft) allowed iliac and renal vascular resistance (IVR, RVR) to be calculated. Their variations were studied after intravenous injections of increasing doses of dopamine (1.5 to 200 micrograms/kg) in unpretreated animals and in animals receiving various pretreatments. Without pretreatment, dopamine induced a biphasic renal response, vasodilation partially masked by subsequent vasoconstriction for doses above 12.5 micrograms/kg of dopamine. Simultaneously, IVR was increased. After alpha-adrenolytic pretreatment (prazosin 2.5 mg/kg, i.v.), dopamine decreased the RVR while iliac vasoconstriction persisted. The association of yohimbine (5 mg/kg, i.v.) to prazosin completely abolished the vasoconstrictive effects: dopamine lowered then by about 30% both IVR and RVR. Dopamine-induced iliac and renal decrease in vascular resistance persisted in the presence of a beta-adrenoceptor antagonist [+/-)-sotalol 30 mg/kg, i.v.), after depletion of catecholamines from sympathetic terminals (reserpine 10 mg/kg, i.p. 20 h before the experiment) or inhibition of cyclo-oxygenase (indomethacin 2.5 mg/kg, i.p. 20 h and 1 h before dopamine). On the contrary, a specific antagonist of dopamine receptors, (+)-butaclamol (60 micrograms/kg/min, i.v.) stereoselectivity inhibited the dopamine-induced renal vasodilation but did not modify the iliac response.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Ochratoxin A on enzyme activities and macromolecules synthesis in MDCK cells.

Protein, RNA, and DNA synthesis, and enzyme (gamma GT, LDH, LAP, A1-P, NAG) activities were assayed in Madin Darby Canine Kidney cells (MDCK) treated by ochratoxin A, a mycotoxin known to be nephrotoxic in animal and man. Both cellular macromolecules syntheses and enzymatic activities are inhibited by OTA in a dose-dependent manner after 24 h incubation. The protection by 100 microM additional phenylalanine is optimal when MDCK cells were pretreated 4 h before the poisoning by OTA. When OTA 25 microM and phenylalanine 100 microM were added simultaneously the prevention of toxic effects is evident, but not complete in spite of the respective intracellular concentrations of OTA 2.22 microM and phenylalanine 2.4 microM which are normally not in favor of a strong inhibition by OTA, except if one admits that MDCK cells are really very sensitive to OTA.