Pitfalls of using lucigenin in endothelial cells: implications for NAD(P)H dependent superoxide formation.

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (approximately 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.

Dose-dependent reduction of myocardial infarct mass in rabbits by the NHE-1 inhibitor cariporide (HOE 642).

The aim of this study was to investigate the dose-dependent effect of pretreatment with the selective sodium-hydrogen exchange NHE-subtype 1 inhibitor cariporide on myocardial infarct mass in a rabbit model of coronary ligation and reperfusion. Furthermore, in a second part of the study, we tested the effect of cariporide in the rabbits when given prior to reperfusion. Rabbits (n=49) were randomized in 7 groups: saline vehicle, cariporide: 0.01, 0.03, 0.1 and 0.3 mg/kg, and subjected to a 30 min occlusion of a branch of the left coronary artery followed by 2 h reperfusion. Cariporide was given as a bolus intravenously 10 min before occlusion or 5 min before reperfusion. After reperfusion, myocardial infarct mass was determined by triphenyl tetrazolium chloride staining and expressed as a percent of area at risk. Cariporide given intravenously 10 min before occlusion in doses of 0.01, 0.03, 0.1, 0.3 mg/kg, led to a dose-dependent reduction in infarct mass from 58+/-6% in controls to 48+/-4% (-17%, NS), 36+/-5% (-38%, p<0.05), 26+/-6% (-55%, p<0.05), 11+/-4% (-81%, p<0.05) respectively, whereas area at risk did not differ in between the groups. The effect of the lowest dose of 0.01 mg/kg did not reach significance. Plasma levels at different doses of cariporide were correlated to the respective infarct mass. After coronary occlusion left ventricular end-diastolic pressure (LVEDP) significantly increased throughout occlusion and reperfusion. Cariporide in the doses of 0.3, 0.1 and 0.03 mg/kg normalized LVEDP when measured after 2 h reperfusion. In controls hemodynamic parameters such as mean arterial blood pressure (MAP), heart rate (HR), left ventricular pressure (LVP) and LV dP/dt(max) were not significantly changed by ischemia/reperfusion with the exception of MAP, LVP and LV dP/dt(max) which were significantly decreased after 120 min reperfusion. Cariporide at doses of 0.1, 0.03 and 0.01 mg/kg did not significantly influence these parameters, whereas the highest dose of 0.3 mg/kg prevented the decrease of MAP and LVP. Cariporide (0.3 mg/kg i.v.) administered 5 min before reperfusion significantly reduced infarct mass by 31%. Under these conditions the increase of LVEDP after coronary occlusion was not influenced by cariporide. As in the pretreatment experiments, the decrease of MAP and LVP was prevented when measured 2 h after reperfusion. The results show that pretreatment with the NHE-subtype 1 inhibitor cariporide is cardioprotective by reducing infarct mass in rabbits in a dose-dependent manner. While the cardioprotective effect of pretreatment could be demonstrated over a broad range of doses, the efficacy of the compound when given only on reperfusion was significant but more limited.

Arginine-vasopressin analogues with high antidiuretic/vasopressor selectivity. Synthesis, biological activity, and receptor binding affinity of arginine-vasopressin analogues with substitutions in positions 1, 2, 4, 7, and 8.

In a search for more selective agonists of arginine-vasopressin (AVP), 10 analogues of [Sar7]- and [MeLa7]AVP with additional substitutions in positions 1 (beta-mercaptopropionic acid), 2 (phenylalanine), 4 (valine), or 8 (D-arginine) were synthesized and tested for antidiuretic and vasopressor activities. All analogues are characterized by a relatively high antidiuretic activity and by a sharp decrease in pressor activity. Their antidiuretic/vasopressor (A/P) selectivities were 2-3 orders higher (except for peptides 2 and 3) than that of the parent hormone. The additivity of the effects of changes in positions 1, 2, 4, and 8 combined with the sarcosine or N-methylalanine substitutions in position 7 on the biological activity is observed. Binding affinities of AVP analogues to plasma membranes from bovine kidney inner medulla and from rat liver containing specific vasopressin receptors were also determined. Generally, these analogues retained high binding affinities to renal vasopressin receptors, and on the other hand they are characterized by a large decrease in binding affinities to hepatic vasopressin receptors, which share characteristics with vasopressor receptors.

Photoaffinity labelling of the renal V2 vasopressin receptor. Identification and enrichment of a vasopressin-binding subunit.

To identify renal vasopressin receptor proteins, analogues of 1-deamino-vasopressin i.e. ([1-(2-mercapto)propionic acid]vasopressin, [Mpa1]VP) with photoreactive aryl-azido groups in position 4 and 8 of the vasopressin sequence were prepared. In the absence of ultraviolet light, these ligands exhibit a high binding affinity for the V2 vasopressin receptor in plasma membranes from bovine and rat kidney medulla (apparent dissociation constants 1.8 X 10(-9) M to 1.7 X 10(-8)M); the photoreactive analogues stimulate the renal vasopressin-sensitive adenylate cyclase. In photoaffinity labelling experiments with tritium-labelled ligands (34-50 Ci/mmol), a membrane protein from bovine kidney or rat kidney medulla with an apparent relative molecular mass (Mr) of 30 000 was preferentially and specifically labelled. The labelling of the 30 000-Mr protein was completely inhibited by a 10-100-fold molar excess of vasopressin; in contrast, angiotensin II, bradykinin or low-affinity analogues of vasopressin did not suppress the incorporation of the reactive ligands into this protein. The highest specific labelling yield and only a low amount of unspecific labelling was obtained with the analogue [Mpa1,Lys(N6-4-azidobenzoyl)8]VP. Preparative sodium dodecyl sulfate gel electrophoresis of bovine kidney membranes photolabelled with this analogue resulted in a 20-30-fold enrichment of the 30 000-Mr vasopressin-binding protein. Our results suggest that this photoreactive analogue of [1-deamino, 8-lysine]vasopressin is a suitable tool for further purification of the renal V2 vasopressin receptor binding subunit.

Determination of the functional molecular size of vasopressin isoreceptors.

The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.

[1,6-alpha-aminosuberic acid,3-(p-azidophenylalanine), 8-arginine] vasopressin as a photoaffinity label for renal vasopressin receptors: an evaluation.

The photoreactive analog of vasopressin [1,6-alpha-aminosuberic acid,3-(p-azidophenylalanine), 8-arginine]vasopressin was labeled with tritium (specific radioactivity: 39 Ci/mmole). In the absence of UV-light, the tritium-labeled ligand retained a high binding affinity to the vasopressin receptor in plasma membranes from bovine kidney inner medulla (apparent dissociation constant KD: 1.4 X 10(-8) M). Renal plasma membranes were irradiated under conditions of a high ratio of specific versus non-specific binding of the reactive ligand. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction demonstrated the preferential labeling of a polypeptide with an apparent molecular weight of Mr = 32 000. A comparison with stained gels revealed that this protein is a minor constituent of the bovine kidney membrane. Our results suggest the 32 000-dalton polypeptide is either a component of the vasopressin receptor or that it is located in the immediate vicinity of the vasopressin binding protein.

Interactions of vasopressin agonists and antagonists with membrane receptors.

Plasma membranes containing one class of non-cooperative binding sites for tritium-labelled [8-arginine]vasopressin were isolated from bovine kidney inner medulla and from rat liver. By using a weighted, non-linear least squares fit to logistic curves, the binding parameters of eight vasopressin agonists and antagonists were determined in competition experiments. Vasopressin analogues with sarcosine or N-methyl-L-alanine in position 7 instead of proline showed a high ratio of antidiuretic to vasopressor activity. These analogues retained a high binding affinity to the renal vasopressin receptor with apparent dissociation constants KD in the order proline less than sarcosine less than methylalanine . In contrast, the affinity to the hepatic vasopressin receptor, which shares characteristics with vasopressor receptors, was drastically reduced with KD values being in the order proline much less than N- methylalanine less than sarcosine. By combining the substitutions at position 7 with substitutions of cysteine in position 1 by either deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid, inhibitors of the oxytocoic and vasopressor responses were obtained. These additional substitutions at position 1 led to a drastic decrease in the binding affinity to the vasopressin receptor in bovine kidney. The intrinsic activity of these analogues to stimulate the renal vasopressin sensitive adenylate cyclase was strongly reduced or completely lost. In the rat liver system, however, these vasopressin antagonists showed a remarkably increased affinity to vasopressin receptors as compared to analogues substituted only at position 7. GTP reduced the binding affinity of all analogues to the hepatic receptor. The results show that these structural modifications which influence both the conformational properties of the vasopressin molecule and the biological activities of the hormone had strikingly different effects on the interactions of the resulting analogues with physiologically important receptors in the kidney and the liver. These studies may lead to the development of more specific vasopressin agonists and antagonists.

[1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin: a new photoaffinity label for hydroosmotic hormone receptors. Characterization of the ligand and irreversible stimulation of hydroosmotic water flow in toad bladder by photoaffinity labeling.

The photoreactive analog of vasopressin [1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin [( Asu1,6, Phe (p-N3)3]AVP) has been synthesized. This analog retains a high binding affinity for the vasopressin receptor in plasma membranes from bovine kidney inner medulla (apparent dissociation constant, KD = 8.5 X 10(-9) M). [Asu1,6, Phe (p-N3)3] AVP was found to be biologically active in triggering the characteristic increase in toad bladder permeability to water. Photolysis of the analog in the presence of the toad bladder results in a hydroosmotic response which persists, in spite of repeated washings, for more than 18 h. The irreversible stimulation of the bladder is inhibited when photolysis is carried out in the presence of vasopressin. Our findings indicate that with photoactivation [Asu1,6, Phe(p-N3)3]AVP binds covalently to hormonal receptors and forms an active hormone-receptor complex. This analog, therefore, is a suitable tool for studies of hydroosmotic receptor function and for receptor isolation.

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver.

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.

Synthesis and biological activities of arginine-vasopressin analogues with reactive groups.

The synthesis and biological activities of arginine-vasopressin analogues are described, where p-azido-L-phenylalanine [Phe(pN3)] or p-(bromoacetylamino)-L-phenylalanine [Phe-(pNHCOCH2Br)] replace Tyr2 or Phe3. The hormone analogues are prepared via precursors containing p-aminophenylalanine [Phe(pNH2)] in position 2 or 3. During peptide synthesis the p-amino group of [Phe(pNH2)] is protected by the tert-butyloxycarbonyl or the benzyloxycarbonyl group, the side chains of cysteine and arginine by the acetamidomethyl residue and the tosyl group, respectively. The amino and guanidino protecting groups are removed from the nonapeptides by trifluoromethanesulfonic acid yielding the S-protected derivatives which are cyclized by means of iodine. The ring closure by disulfide formation is confirmed by Edman degradation, CD and 1H-NMR spectroscopy. Modification at the p- and alpha-amino groups result in [Phe(pN3)2]-vasopressin, [Phe(pNHCOCH2Br)2]vasopressin, Nalpha-dansyl-[Phe(pN3)2]vasopressin, [Phe2,Phe-(pN3)3]vasopressin and [Phe2,Phe(pNHCOCH2-Br)3]vasopressin. The analogues modified only in position 2, [Phe(pN3)2]vasopressin stimulate the adenylate cyclase derived from bovine kidney inner medulla to similar maximal velocities as arginine vasopressin and show high apparent affinities for enzyme activation. The Nalpha-dansyl derivative and the analogues with reactive groups in position 3 have reduced maximal velocities and apparent affinities for vasopressin-sensitive adenylate cyclase. These results suggest that especially the derivatives with reactive groups in position 2 are useful for the labelling of vasopressin receptors in plasma membranes and for studies of covalent hormone-receptor complexes.