Chronic treatment with sulfhydryl angiotensin-converting enzyme inhibitors reduce susceptibility of plasma LDL to in vitro oxidation, formation of oxidation-specific epitopes in the arterial wall, and atherogenesis in apolipoprotein E knockout mice.

The effects of chronic treatment with the new sulfhydryl angiotensin-converting enzyme (ACE)-inhibitor, zofenopril, in comparison with the classical sulfhydryl ACE-inhibitor captopril or enalapril or placebo on the development of atherosclerosis were determined in apolipoprotein-E knockout (apoE(-/-)) mice. Groups of 2-month-old male mice received either placebo (N=10), 0.05 mg/kg/day of zofenopril (N=10), 1 mg/kg/day of zofenopril (N=10), 5 mg/kg/day of captopril (N=10) or 0.5 mg/kg/day of enalapril (N=8). After 29 weeks of treatment, computer-assisted imaging analysis revealed that zofenopril reduced the aortic cumulative lesion area by 78% at 0.05 mg/kg/day and by 89% at 1 mg/ml/day of zofenopril compared to that of the placebo (P<0.0001). Captopril reduced by 52% aortic lesions compared to placebo (P<0.01 vs. placebo; P<0.05 vs. zofenopril at both doses). Enalapril did not reduce aortic lesions. Furthermore, 0.05 mg/kg/day of zofenopril reduced susceptibility of plasma LDL to in vitro oxidation compared to captopril, enalapril or placebo, as shown by significant reduction of malondialdehyde content (P<0.001 vs. placebo or enalapril; P<0.05 vs. captopril), as well as by the prolongation of lag-time (P<0.01 vs. placebo or enalapril P<0.05 vs. captopril). More importantly, mice treated with 1 mg/ml/day of zofenopril had a significant decrease in the intimal immunohistochemical presence of oxidation-specific epitopes on oxLDL (NA59 monoclonal antibody, P<0.01), macrophages derived foam cells (F4/80 monoclonal antibody, P<0.05) and native LDL (NP monoclonal antibody, P<0.01) compared to placebo, captopril or enalapril. Thus, chronic treatment with the new sulfhydryl ACE-inhibitor zofenopril has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic apoE(-/-) mice. This protection was significantly higher than that reached with captopril and at lower doses of the drug. Treatment with 0.5 mg/kg/day of enalapril did not provide any protective effect.

Loop diuretics enhance the secretion of prostacyclin in vitro, in healthy persons, and in patients with chronic heart failure.

BACKGROUND: Previous studies suggest that the acute haemodynamic effects of loop diuretics are due to a direct dilation of blood vessels and are not related to diuretic properties, but possibly to prostaglandin secretion. OBJECTIVES: We investigated whether in vitro human endothelial and renal epithelial cells responded to torasemide or furosemide with enhanced secretion of the vasodilator prostaglandin prostacyclin (PGI2). We also investigated the effects of loop diuretics on plasma concentrations of PGI2 and its physiological antagonist thromboxane after 25 min of administration of drugs in 44 patients with congestive heart failure (CHF) and 44 healthy volunteers. METHODS: The PGI2 levels were measured after extraction in ethyl acetate by RIA as levels of 6-KetoPGF1alpha, a stable metabolite from a non-enzymatic degradation. TxB2 concentration, the stable hydrolysis product of TxA2, was also measured by RIA. RESULTS: In human endothelial and renal epithelial cells, both loop diuretics induced an increase of 6-KetoPGF1alpha secretion that reached a peak after about 5 min and remained stable for 30 min of exposure to the drugs. The magnitude of the phenomenon was lesser in epithelial than in endothelial cells. Moreover, in both cell lines, there was a significantly higher secretion of 6-KetoPGF1alpha to torasemide than furosemide (P < 0.05). Concentrations of 6-KetoPGF1alpha at baseline were similar between the groups of CHF patients receiving the two different drugs. After 25 min of both drugs, 6-Keto-PGF1alpha significantly increased (P < 0.01), and this was significantly higher in patients treated with 10 mg of torasemide (P < 0.05 vs furosemide). Levels of PGI2 at baseline were lower in healthy controls than those reached by CHF patients and similar between groups. After 25 min of both drugs, PGI2 plasma levels were significantly increased (P < 0.01). Baseline values of TxB2 were significantly higher in CHF patients compared with controls (P < 0.01 vs respective groups). and, more importantly, furosemide but not torasemide increased TxB2 levels in patients and controls (P < 0.05 vs baseline). CONCLUSIONS: Our study is the first demonstration in human tissue of increased secretion of PGI2 both in vitro and in vivo, after torasemide or furosemide administration. This phenomenon, which may explain in part the vasodilatory effects of these drugs, was more evident with torasemide and was reached at lower concentrations of the drug. Accordingly, we also found that furosemide but not torasemide stimulated the release of the PGI2 physiological antagonist thromboxane in CHF patients and healthy controls.

Olfactory receptors in aquatic and terrestrial vertebrates.

In species representing different levels of vertebrate evolution, olfactory receptor genes have been identified by molecular cloning techniques. Comparing the deduced amino-acid sequences revealed that the olfactory receptor gene family of Rana esculenta resembles that of Xenopus laevis, indicating that amphibians in general may comprise two classes of olfactory receptors. Whereas teleost fish, including the goldfish Carassius auratus, possess only class I receptors, the 'living fossil' Latimeria chalumnae is endowed with both receptor classes; interestingly, most of the class II genes turned out to be pseudogenes. Exploring receptor genes in aquatic mammals led to the discovery of a large array of only class II receptor genes in the dolphin Stenella Coeruleoalba; however, all of these genes were found to be non-functional pseudogenes. These results support the notion that class I receptors may be specialized for detecting water-soluble odorants and class II receptors for recognizing volatile odorants. Comparing the structural features of both receptor classes from various species revealed that they differ mainly in their extracellular loop 3, which may contribute to ligand specificity. Comparing the number and diversity of olfactory receptor genes in different species provides insight into the origin and the evolution of this unique gene family.

Cloning and expression of odorant-binding proteins Ia and Ib from mouse nasal tissue.

We had previously reported the purification and partial characterisation of four distinct odorant-binding proteins from male mouse nasal epithelium. One of these, named OBP-I appeared to be a heterodimer, whose subunits, Ia and Ib showed significant similarity in their N-terminal amino acid sequences with hamster aphrodisin. In this paper, we report the complete amino acid sequences of these two polypeptide chains, as deduced from nucleotide sequences of their relative cDNA. These data confirm the high similarity of both proteins with hamster aphrodisin. A comparison with the sequences of other known OBPs indicate that they are more closely related to members of class I, including bovine OBP, rat OBP-I and pig OBP-I. A putative odorant-binding site is indicated by the presence of amino acid residues conserved with respect to the bovine protein, whose three-dimensional structure has been recently resolved. In-situ hybridisation has revealed identical expression patterns for the two proteins, further supporting the heterodimeric structure of these proteins in the nasal mucus.

Subfamily of olfactory receptors characterized by unique structural features and expression patterns.

The complex chemospecificity of the olfactory system is probably due to the large family of short-looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.

The calcium-binding protein VILIP in olfactory neurons: regulation of second messenger signaling.

Visinin-like-protein (VILIP), a member of the neuronal subfamily of EF-hand calcium-sensor proteins is shown to be expressed in olfactory sensory cells of the rat nasal epithelium. Its prominent localization in cilia and dendritic knobs-the chemosensory compartments of olfactory neurons-suggests that the calcium-binding protein could be involved in olfactory signal transduction. Consistent with this assumption, it was found that recombinant VILIP attenuates in a calcium-dependent manner odorant-induced cAMP formation in olfactory cilia preparations. Kinetic data indicate that VILIP does not interfere with odorant-induced receptor desensitization. Since VILIP inhibits the forskolin-induced formation of cAMP, it is conceivable that VILIP may directly affect the olfactory adenylyl cyclase. Thus, VILIP may play a role in adaptation of olfactory neurons.

G-protein(S), G alpha q/G alpha 11, in the olfactory neuroepithelium of the channel catfish (Ictalurus punctatus) is altered by the herbicide, dichlobenil.

The immunohistochemical localization of G alpha q/G alpha 11 was studied in the olfactory neuroepithelium of the channel catfish. Antigenicity in the rosette was found at the apical surfaces of cells, within the apical neck regions of some cells, and within the area of the basal nerve tracts. Specific labeling was eliminated by preincubation of the G alpha q/G alpha 11 antibodies with the cognate peptide. Catfish, exposed to a 20 ppm dose of the herbicide, dichlobenil, displayed a reduction in G alpha q/G alpha 11 antigenicity. Proteins were extracted from olfactory tissues and solubilized. Using SDS-PAGE and Western blotting, bands corresponding in apparent molecular weight to a 38 kD protein were found. These data demonstrate that the herbicide may be a potent nasal olfactory toxicant in aquatic situations.

G alpha 9/G alpha 11: immunolocalization in the olfactory epithelium of the rat (Rattus rattus) and the channel catfish (Ictalurus punctatus).

The immunohistochemical localization of G alpha 9/G alpha 11 was studied in the olfactory and respiratory epithelium of two representative vertebrates, the rat and the channel catfish. Localization in the rat was found at the apical surface of cells in the epithelium and within nerve tracts in the lamina propria. Immunostaining of neuronal cilia and supporting cell microvilli was confirmed by electron microscopy. Immunoreactivity on the ipsilateral neuroepithelium was abolished five weeks following unilateral bulbectomy. An emergence of patchy immunoreactivity was found, however, after fifteen weeks. In catfish, G alpha 9/G alpha 11 antigenicity was found at the apical surface of cells within the olfactory epithelium, at supranuclear regions within some cell bodies and in basal nerve tracts of the olfactory rosette. Immunoreactivity was removed with unilateral bulbectomy. Specific labelling in both rat and catfish was eliminated by preincubation of the G alpha 9/G alpha 11 antibodies with the cognate peptide. Proteins were extracted from olfactory tissues of both species and solubilized. Using western blotting, bands corresponding in apparent molecular weight to a 38,000 mol. wt protein were found. These data demonstrate the presence of G alpha 9/G alpha 11 in the olfactory tissues of these vertebrates and suggest a role in olfaction for this class of G-protein.

Purification and characterization of two odorant-binding proteins from nasal tissue of rabbit and pig.

1. Soluble proteins showing binding activity to 2-isobutyl-3-methoxypyrazine have been purified to homogeneity from rabbit and pig nasal tissue; their characteristics are similar to the bovine odorant-binding protein and are to be considered members of the same family. 2. The rabbit protein is a homodimer with subunits of Mr 19k and an isoelectric point of 4.7, whereas the pig protein appears to consist of a single polypeptide chain of Mr 22k and an isoelectric point of 4.2. 3. Both proteins bind 2-isobutyl-3-methoxypyrazine with dissociation constants in the micromolar range. 4. Antibodies against the bovine OBP react well with the rabbit protein, and slightly with the porcine one.