Synergy of nitric oxide and azoles against Candida species in vitro.

The candidacidal activity of nitric oxide (NO) as delivered by a class of compounds termed diazeniumdiolates has been investigated. Diazeniumdiolates are stable agents capable of releasing NO in a biologically usable form at a predicted rate, and three such compounds were examined for activity. One compound, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA-NO), proved to be most suitable for examining NO activity due to its relatively long half-life (20 h) and because of limited candidacidal activity of the uncomplexed DETA nucleophile. DETA-NO was active against six species of Candida for which the MICs necessary to inhibit 50% growth (MIC50s) ranged from 0.25 to 1.0 mg/ml. C. parapsilosis and C. krusei were the most susceptible to the compound. In addition to a determination of NO effects alone, the complex was utilized to investigate the synergistic potential of released NO in combination with ketoconazole, fluconazole, and miconazole. Activity was investigated in vitro against representative strains of Candida albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, and C. dubliniensis. Determination of MIC50, MIC80 and MICs indicated that DETA-NO inhibits all strains tested, with strains of C. parapsilosis and C. krusei being consistently the most sensitive. The combination of DETA-NO with each azole was synergistic against all strains tested as measured by fractional inhibitory concentration indices that ranged from 0.1222 to 0.4583. The data suggest that DETA-NO or compounds with similar properties may be useful in the development of new therapeutic strategies for treatment of Candida infections.

Phencyclidine inhibits epinephrine-stimulated platelet aggregation independently of high affinity N-methyl-D-aspartate (NMDA)-type glutamatereceptors.

The psychotomimetic analgesic phencyclidine (PCP), which binds to a high affinity site on the neuronal N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor, has previously been found to bind to platelets with high affinity and to specifically delay the onset of epinephrine-stimulated platelet aggregation (Jamieson et al. (1992) Biochem. J. 285, 35-39). We have now shown that the rank order of binding affinities of 14 synthetic PCP analogs at the high affinity binding site on platelets does not parallel the rank order of their affinities in binding to rat brain membranes, indicating that the high affinity PCP binding sites in platelets is distinct from the neuronal NMDA receptor. The order of potency of six of these analogs in delaying the onset of epinephrine-stimulated platelet aggregation also did not parallel the rank order of their binding affinities for platelet or brain binding sites. These data indicate that the ability of PCP analogs to inhibit epinephrine-stimulated aggregation is not related to their ability to bind to the high affinity platelet PCP binding site. Furthermore, (+)MK-801, which binds to the same high affinity binding site in neurons as does PCP, failed to inhibit epinephrine-stimulated platelet aggregation, further suggesting that the site at which PCP acts in platelets is not related to the NMDA-type glutamate receptor. Further studies showed that 5-HT2 receptors and effects on platelet secretion are not involved in PCP-mediated inhibition of epinephrine-induced platelet aggregation.

Differences in imidazoline and phenylethylamine alpha-adrenergic agonists: comparison of binding affinity and phosphoinositide response.

The imidazoline class of compounds, reported to be partial agonists at alpha 1 adrenoceptors, were compared with phenylethylamines for their ability to displace the binding of [3H]prazosin and to stimulate hydrolysis of phosphoinositides in the cerebral cortex of the rat. Both classes of alpha adrenoceptor compounds exhibited two sites of interaction with binding sites for [3H]prazosin in 30 mM Tris buffer. In a Na+ containing ionic buffer, the competition by phenylethylamines for [3H]prazosin sites shifted to a one-site best-fit, while imidazolines retained their two-site best-fit. Phenylethylamines stimulated hydrolysis of phosphoinositides in a dose-dependent manner, with ED50 values that correlated with Kd values from competition curves. In contrast, imidazolines were not potent or efficacious at stimulating hydrolysis of phosphoinositides and the binding affinities did not correlate with the ED50 values. The alpha 1 adrenoceptor antagonist, prazosin potently inhibited phenylethylamine, but not imidazoline-stimulated hydrolysis of phosphoinositides. Dose-response curves to the imidazoline, oxymetazoline, in the presence and absence of maximally stimulating concentrations of norepinephrine, indicated that oxymetazoline caused a dose-dependent inhibition of norepinephrine-stimulated hydrolysis of phosphoinositide. The inhibition of norepinephrine-stimulated hydrolysis of phosphoinositides was evident up to 100 microM, at which point oxymetazoline elicited hydrolysis of phosphoinositides through a non-alpha 1 adrenoceptor-mediated mechanism. These data indicate that imidazolines act primarily as antagonists at the alpha 1 adrenoceptor, coupled to hydrolysis of phosphoinositide and stimulate the hydrolysis of phosphoinositide through a non-alpha 1 adrenoceptor mechanism.

Effects of nBM lesions on muscarinic-stimulation of phosphoinositide hydrolysis.

The nucleus basalis magnocellularis (nBM) is believed to be the major path of cholinergic innervation to the frontal cortex. The cerebral cortex is known to contain muscarinic receptors that are coupled to the hydrolysis of phosphoinositides (PI) (9,14). Adult male Sprague-Dawley rats were unilaterally and bilaterally lesioned at the nBM with the excitotoxin ibotenic acid and killed at 7 or 21 to 23 days postsurgery. In rats unilaterally lesioned 7 days previously, the carbachol dose-response curves in lesioned fronto-parietal cortex were identical to control fronto-parietal cortices. In rats studied 21 to 23 days postsurgery, carbachol dose-response curves were again identical in control vs. lesioned fronto-parietal cortices. Similar results are obtained when bilaterally lesioned rats are compared to sham-operated controls. For each group, the hydrolysis is linear with respect to time until 15 minutes with a maximum reached at approximately 40 minutes. Receptor density, as measured by [3H]-QNB binding or agonist competition for [3H]-QNB binding, was not changed by any of the lesions studied. These results suggest that the loss of cholinergic innervation from the nBM does not result in compensatory denervation supersensitivity in cerebral fronto-parietal cortical muscarinic receptors.