Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.

Dynamics of prolactin secretion from diethylstilbestrol-induced rat prolactinoma tissue in vitro.

Experiments were performed to determine whether PRL secretion in the rat diethylstilbestrol (DES)-induced prolactinoma model is affected by the addition of thyrotropin-releasing hormone (TRH) and/or immunoneutralization of intrapituitary vasoactive intestinal polypeptide (VIP) in vitro. Male Fischer 344 rats were implanted with either a 10 mg DES or placebo pellet 30 days prior to obtaining the anterior pituitary glands for perifusion. The anterior pituitaries were quartered and used in three different perifusion experiments. In Experiment I, placebo-treated tissue channels were perifused for 2 baseline hr followed consecutively by a 30-min exposure to 1:100 nonimmune rabbit serum (NRS), a 30-min wash, and a final 30-min exposure to 10(-5) M TRH. Additional placebo channels were run as above except 1:100 VIP antiserum (AVIP) was substituted for NRS and AVIP was added to the TRH. In Experiment II, the same perifusion protocol was used as in Experiment I, except DES-induced tumor tissue was used instead of placebo tissue. Results from Experiment I and II reveal that AVIP significantly decreased PRL secretory rate in both DES and placebo groups. In the tumor group, both TRH alone and in the presence of AVIP significantly increased the PRL secretory rate. In Experiment III DES-induced tumor tissue channels were perifused with a similar protocol, except the concentrations of NRS and AVIP were increased to 1:10. Both NRS and AVIP significantly decreased PRL secretory rate; however, AVIP had a significantly greater effect than NRS. In this experiment, 1:10 AVIP overcame the stimulatory effect of TRH. In conclusion, AVIP decreases and TRH increases, even in the presence of AVIP, PRL release in DES-induced prolactinoma tissue in vitro. Increasing the AVIP concentration 10-fold diminished the PRL-releasing action of TRH in the tumor tissue. These data suggested that PRL secretion is not autonomous in these prolactinomas and can be affected by exogenous TRH and partial immunoneutralization of endogenous VIP.

Vasoactive intestinal polypeptide and thyrotropin-releasing hormone stimulate newly synthesized, not stored, prolactin.

Experiments were designed to determine whether vasoactive intestinal polypeptide (VIP), reported to stimulate basal PRL secretion, affects PRL processing by lactotrophs. Initially, rat anterior pituitary quarters were incubated for 2 h with [3H]leucine, with and without 10(-5) M VIP, and immunoreactive and immunoprecipitable rPRL were measured during 56 mM KCl perifusion to determine total and 3H-labeled PRL, respectively. Inclusion of VIP increased immunoreactive PRL (P less than 0.05), decreased immunoprecipitable PRL (P less than 0.01), and, therefore, decreased the specific activity of labeled PRL (P less than 0.001). These results suggested an enhanced release of newly synthesized PRL before KCl depolarization, thus decreasing the release of labeled PRL. To discriminate between the two PRL pools, newly synthesized and storage, pituitary quarters were incubated with and without 10(-5) M VIP for 4 h with [14C]leucine, 2 h in cold medium and 2 h with [3H]leucine. Immunoprecipitable PRL was measured during perifusion with 56 mM KCl. Data were depicted as the 3H/14C disintegrations per min ratio of PRL released/3H/14C disintegrations per min of total tissue to account for any differences in tissue labeling. This ratio was greater for tissue labeled in the presence of VIP (P less than 0.002). To determine whether VIP, as a secretagogue, differentiates between the newly synthesized and storage pools, VIP was added after pulse chase, as previously described. No preferential release was observed between the two groups. Finally, using the same [3H]- and [14C]leucine-labeling protocol with and without 10(-5) M VIP, tissue was perifused with medium 199 for 1 h, with 10(-5) M TRH for 30 min, with medium 199 for 30 min, and with 56 mM KCl for 1 h. Inclusion of VIP increased the 3H/14C released/3H/14C total tissue ratio during basal perifusion (P less than 0.04) and TRH exposure (P less than 0.05). Within the control group, the TRH ratio was greater than basal (P less than 0.003). These experiments suggest that newly synthesized PRL is preferentially secreted over stored PRL from tissue incubated with VIP during pulse-chase labeling; however, addition of VIP as a secretagogue did not affect either PRL pool preferentially.