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Although recent evidence suggests that reduced nitric oxide (NO) production may be involved in salt-induced hypertension, the specific NO synthase (NOS) responsible for the conveyance of salt sensitivity remains unknown. To determine the role of inducible NOS (NOS II) in salt-induced hypertension, we treated Dahl salt-resistant (DR) rats with the selective NOS II inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) for 12 days. Tail-cuff systolic blood pressures rose 29 +/- 6 and 42 +/- 8 mmHg in DR rats given 150 and 300 nmol AMT/h, respectively (P < 0.01, 2-way ANOVA) after 7 days of 8% NaCl diet. We observed similar results with two other potent selective NOS II inhibitors, S-ethylisourea (EIT) and N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W). Additionally, AMT effects were independent of alterations in endothelial function as assessed by diameter change of mesenteric arterioles in response to methacholine using videomicroscopy. We, therefore, conclude from these data that NOS II is important in salt-induced hypertension.

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Since thiols can undergo nitrosation and the cell membrane is rich in thiol-containing proteins, we considered the possibility that membrane surface thiols may regulate cellular entry of NO. Recently, protein disulfide isomerase (PDI), a protein that catalyzes thio-disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO transfer from the extracellular to intracellular compartment. We observed that PDI catalyzes the S-nitrosothiol-dependent oxidation of the heme group of myoglobin (15-fold increase in the rate of oxidation compared with control), and that NO reduces the activity of PDI by 73.1 +/- 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosphorothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxynucleotide decreased cell-surface PDI content by 74.1 +/- 9.3% and PDI folding activity by 46.6 +/- 3.5% compared with untreated or "scrambled" phosphorothioate oligodeoxynucleotide-treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guanosine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 +/- 26.7% reduction compared with control; P < 0.05), with no effect on cyclic adenosine monophosphate (cAMP) generation after prostaglandin E1 exposure. These data demonstrate that the cellular entry of NO involves a transnitrosation mechanism catalyzed by cell-surface PDI. These observations suggest a unique mechanism by which extracellular NO gains access to the intracellular environment.

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Doisynolic acids, D-ring seco-steroids derived from alkaline fusion of estrones, are hormonal curiosities: Their binding affinity for the estrogen receptor is low (ca. 1-2% that of estradiol), but their in vivo potency is high and they have a long duration of action. To study the in vivo behavior of the doisynolic acids, we prepared fluorine-substituted analogs of both trans-doisynolic acid (with the natural 14 alpha-hydrogen configuration, trans-FDA) and the more active cis-doisynolic acid (with the unnatural 14 beta-hydrogen configuration, cis-FDA) from estrone and 14 beta-estrone, respectively. Modification of the D-ring haloform cleavage approach of Meyers allowed us to introduce fluorine (or fluorine-18) on the carbon atom derived from C-16 in the estrones. Fluorine substitution had little effect on the estrogen receptor binding affinity of the doisynolic acids. Tissue distribution of the fluorodoisynolic acids (trans-[18F]FDA and cis-[18F]FDA) was unusual and very different from that of typical, high-affinity ligands for the estrogen receptor. At 1-3 h in immature female rats, trans-[18F]FDA shows low and rather nonselective uptake in the principal estrogen target tissue (uterus) and slow clearance. By contrast, cis-[18F]FDA shows high uptake in nearly all tissues, with significant uterine uptake that continues to increase over the 1-6-h period. The uterine uptake of this isomer was blocked at the later times by a sufficiently high dose of unlabeled cis-FDA. After administration of the trans-[18F]FDA, a more polar metabolite slowly accumulates in the blood. The cis-[18F]FDA, however, showed no apparent metabolism, with 84% of the blood activity at 5 h assigned as the unmetabolized radioligand. After 5 h, only limited clearance from blood, liver, and kidneys has occurred. No metabolite from this isomer accumulates in the uterus. Although fluorodoisynolic acids will not be useful breast-tumor imaging agents, their behavior was found to be interesting as it deviates from that of other F-18 estrogens. Further long-term studies of cis-doisynolic acid, labeled with tritium, may be needed to explicate fully its unusual distribution properties and high in vivo activity.

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In order to develop novel ligands for the estrogen receptor (ER) that might have high binding affinity and fluorescence properties suitable for assaying ER levels in cells, we have prepared a series of substituted 4'-hydroxyl-styrylpyridines and phenylethylpyridines and studied their optical spectroscopy and receptor binding properties. Several derivatives that contain alkyl substituents on the internal ethene or ethane carbons were prepared. While most of these compounds have only modest affinity for ER, one fluorescent analog, (E)-1-(4-hydroxyphenyl)-1-phenyl-2-(4-pyridinyl)ethene(13), has reasonably good binding affinity for ER and shows long wavelength fluorescence emission that is sensitive to solvent polarity and pH. This compound may prove to be a useful probe for detecting ER in cells.

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