• Characteristics and regulation of aspartate transport systems in rat ventral prostate epithelial cells

      Lao, Lixing; Franklin, Renty B.; Costello, Leslie (1992)
      A unique characteristic and function of rat ventral prostate, like human prostate, is the accumulation and secretion of high levels of citric acid. Aspartate is a proposed four-carbon precursor of citrate via transamination. Replenishment of endogenous aspartate requires continuous uptake of aspartate from circulation. This study was designed to identify aspartate transporters in isolated rat ventral prostate epithelial cells. The results indicated that two aspartate transporters, a high affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.01 mM) and a low affinity transporter (K{dollar}\sb{lcub}\rm m{rcub}{dollar} = 0.8 mM) exist in these cells. Both transporters are Na{dollar}\sp+{dollar}-dependent and pH sensitive. The optimal pH for the high affinity transporter is about 7.5, whereas for the low affinity transporter the optimum is between 6.5 and 7.0. The high affinity transporter is also temperature dependent. Competitive inhibitory studies indicate that L-aspartate uptake by the high affinity transporter is inhibited by L-glutamate and D-aspartate, but not by L-alanine and L-lysine. The low affinity system is inhibited by D-aspartate, but not by L-glutamate or L-alanine. These different characteristics suggest that the high affinity and the low affinity transporters are two distinct systems. The high affinity aspartate transporter is sensitive to the Na{dollar}\sp+{dollar}-K{dollar}\sp+{dollar} ATPase inhibitor vanadate but less sensitive to ouabain. This suggests that an ouabain-insensitive Na{dollar}\sp+{dollar}-ATPase exists on the cell membrane. High affinity aspartate uptake is not dependent on K{dollar}\sp+{dollar}. However, a Na{dollar}\sp+{dollar}-H{dollar}\sp+{dollar} antiport might be involved. Aspartate uptake is stimulated by testosterone in vivo and in vitro. The in vitro effect is rapid and is inhibited by cycloheximide and actinomycin D. Prolactin also stimulated aspartate uptake independent of testosterone and is inhibited by cycloheximide. The high affinity aspartate transporter is subject to transstimulation by aspartate and citrate.
    • Effects of experimental pulpitis on the expression of transient receptor potential channels on the rat trigeminal ganglia

      Duraes, Gabriela; Ro, Jin Y. (2010)
      Management of patients who present with tooth pain is one of the major challenges in dentistry. Further elucidation of mechanisms underlying tooth pain would lead to optimized management of these patients. Recent evidence suggests that Transient Receptor Potential (TRP) channels participate in pain sensation induced by chemical, thermal and mechanical stimuli. The aim of this study is to explore the molecular mechanisms underlying pulpitis by investigating the involvement of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels in tooth pain using an animal model. Experiments were performed on 42 male Sprague-Dawley rats. Pulpitis was induced by drilling the first maxillary molar of the animal and treating the cavity with either complete Freund's adjuvant (CFA) or saline. Naïve (untreated) rats were used as control. Trigeminal ganglia (TG) from both sides were extracted 1, 3 and 7 days after pulpitis induction and Western blot analysis was performed. The data was analyzed with one-way ANOVA or Kruskal-Wallis ANOVA on ranks depending on the outcome of normality test. TG from naïve and pulpitis-induced rats showed the expression of both TRPV1 and TRPA1. Increased expression of both receptors was observed in TG of rats treated with saline and CFA, compared to naïve (untreated) rats. However, results were not statistically significant due to a large variability. Potential factors that might have contributed to the variability, strengths and limitations of the pulpitis model employed in this study are discussed. Confirmation of our results with larger samples may provide a rationale for targeting these channels and reveal new therapeutic strategies for pulpitis.
    • Testosterone regulates mitochondrial aspartate aminotransferase gene expression in rat ventral prostate

      Qian, Kaifeng; Franklin, Renty B.; Costello, Leslie (1991)
      One of the physiological functions of the rat ventral prostate, like human prostate, is to secrete an extraordinarily high quantity of citrate. This unique characteristic is under the influence of testicular androgen. The continuous secretion of citrate from prostate results in loss of a 6-carbon compound from the metabolic pool which must be replenished. Citrate is synthesized from the concentration of acetyl-CoA and oxaloacetate. Acetyl-CoA may come from various sources. However, recent studies indicated that the possible available source for oxaloacetate is the transamination of aspartate. Mitochondrial aspartate amino-transferase (mAAT) is an important enzyme which regulates citrate production in rat ventral prostate. Testosterone, as a major testicular androgen, stimulates citrate production and mAAT activity in a similar pattern. The hypothesis in this dissertation is that the ability of testosterone to increase citrate production and mAAT activity is the result of stimulation of mAAT gene expression. To verify this hypothesis, numerous in vivo and in vitro studies were utilized to analyze the steady-state level of mAAT mRNA in response to testosterone depletion and repletion. The rats were castrated, followed by testosterone or oil vehicle injection. Twenty-four (or forty-eight) hours later all animals were killed, prostates removed, RNA and nuclei isolated. In vitro studies were performed using primary cultured pig prostate cells. The quantity of mAAT mRNA was measured using northern hybridization. The rates of transcription and degradation of mAAT mRNA were determined by in vitro transcription assay and pulse chase labeling assay, respectively. Results show that castration caused a significant decrease in the content of mAAT mRNA and the transcription rate of the mAAT gene. However, testosterone administration reversed these hormonal depletion effects. Moreover, testosterone administration also prolonged the half life of mAAT mRNA. These results support the hypothesis that one of the major physiological functions of testosterone is to stimulate mAAT mRNA gene expression, which in turn enhances citrate production in rat ventral prostate. Furthermore, these results confirm that the increase in the steady-state level of mAAT mRNA in response to testosterone administration results from stimulation of mAAT gene transcription and inhibition of mAAT mRNA degradation.
    • Therapeutic Evaluation of a Novel Topical Antimicrobial Formulation against Candida-Associated Denture Stomatitis in an Experimental Rat Model

      Sultan, Ahmed; Jabra-Rizk, Mary Ann; 0000-0001-5286-4562 (2019)
      Candida-associated denture stomatitis (DS), caused by the fungal species Candida albicans, is the most common manifestation of oral candidiasis and is prevalent in up to 70% of denture wearers. DS tends to be a persistent and recurrent oral condition as a consequence of the ability of C. albicans to adhere to denture material and invade associated palatal tissue. There are currently no effective therapeutic strategies targeting DS, and despite antifungal therapy, infection is often re-established after treatment ceases. Therefore, it has become crucial to identify novel therapeutic approaches. Antimicrobial peptides have attracted significant attention as candidates for drug development due to their potent antimicrobial and anti-inflammatory properties, lack of toxicity and lack of development of drug resistance. Specifically, histatin-5 (Hst-5), naturally produced and secreted by host salivary glands, has demonstrated potent antifungal activity, including against strains resistant to traditional antifungals. However, our laboratory has previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans secreted proteolytic enzymes at specific amino acid residues. Therefore, to generate a resistant derivative of Hst-5, we engineered a variant (K11R-K17R) with substitutions in the amino acid residues at the cleavage sites. The new peptide proved to be more stable, and unlike the native Hst-5, resistant to proteolysis by C. albicans proteases. Importantly, for clinical application, we designed a polymer-based bioadhesive hydrogel as a delivery system for the peptide and developed a therapeutic formulation specifically designed for oral topical application. The potency of the new formulation in inhibiting C. albicans adherence and biofilm formation on denture acrylic material was demonstrated in vitro indicating a potential clinical applicability against DS. To that end, using 3D digital design and printing technology, we engineered and fabricated a universal intraoral device that was successfully used in the animals to develop clinical disease mimicking DS as in humans. Using the novel animal model, we established the clinical utility of the formulation for the prevention of biofilm formation on denture device and DS development. Importantly, in addition to DS, the formulation can also be used for treatment of other forms of candidiasis as well as serve in augmenting host natural immune defenses.