John D. Walters

Ganglioside modulation of cyclic AMP-dependent protein kinase and cyclic nucleotide phosphodiesterase in vitro.

Abstract: Purified cyclic AMP‐dependent protein kinase (cAK) catalytic subunit phosphorylated 180‐, 49‐, 31‐, 19‐, and 14‐kilodalton (kDa) proteins of rabbit sciatic nerve membranes. The ability of cAK to phosphorylate these membrane substrate proteins was inhibited by gangliosides GM1, GDIa, and GTlb with half‐maximal inhibitory concentration (I50) = 7–25 μM. Neutral glycolipids and lyso‐phosphatidylcholine were much less effective. Cyclic AMP (cAMP) kinase phosphorylation of histone IIA was inhibited by GM1, GDla, and GTlb (I50 = 115 μM, 75 μM, and 75 μM, respectively). Inhibition by GM1 was competitive with respect to histone (K‐, = 108 μM). Autophosphorylation of cAMP kinase was inhibited by GM1 (I50 = 15 μM). GTlb, GDla, and GM1 half‐maximally stimulated calmodulin‐de‐pendent cyclic nucleotide phosphodiesterase at 0.1 μM, 0.2 μM, and 0.3 μM, respectively. Although GTlb stimulated phosphodiesterase by increasing Vmax and decreasing Km (similar to calmodulin), GDla and GM1 produced only an increase in Vmax. These results suggest that ganglioside can modulate the activity of cAMP kinase by both direct inhibition of the enzyme and indirect reduction of cAMP levels through activation of phosphodiesterase. Through these mechanisms, gangliosides may alter cAMP‐dependent protein phosphorylation and cell function within the nervous system.

A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.

Azithromycin Decreases Crevicular Fluid Volume and Mediator Content

Azithromycin enhances the response to root planing and produces anti-inflammatory effects in treating chronic lung disease. This led us to hypothesize that azithromycin inhibits inflammatory mediator production in gingiva, leading to decreased gingival crevicular fluid (GCF) volume. To test this hypothesis, ten periodontally healthy volunteers received azithromycin every 24 hours for 48 hours. GCF samples were collected from 12 maxillary interproximal sites prior to azithromycin (baseline) and 2, 4, 7, and 14 days later. Samples were assayed for IL-1β, IL-8, TNF-α, VEGF, IL-6, and IL-10. With azithromycin treatment, GCF volume decreased significantly on days 2 through 7 (P < 0.05), but increased toward baseline levels on day 14. This was accompanied by a transient decrease in the content of IL-1β, IL-8, TNF-α, and VEGF (P < 0.05). IL-6 and IL-10 were not detected. Since plaque was absent throughout the study, the findings suggest that azithromycin produces anti-inflammatory effects in gingiva.

Transcriptional and post-transcriptional regulation of GM-CSF-induced IL-1β gene expression in PMN

Polymorphonuclear leukocytes (PMN) play an important role in inflammation, immune responses, and tissue repair by secreting interleukin‐1β (IL‐1β). We investigated the regulation of IL‐1β gene expression in human PMN treated with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced IL‐1β mRNA accumulation at 0.1 ng/ml and maximal induction was observed at 1 ng/ml. IL‐1β mRNA levels reached a maximum within 1–2 h after stimulation with GM‐CSF and returned to baseline levels by 4–6 h. The time course of IL‐1β mRNA induction by GM‐CSF was more protracted than previously reported for PMN stimulated with tumor necrosis factor‐α (TNF‐α, 10 ng/ml). Nuclear run‐on analysis indicated that GM‐CSF, like TNF, increases IL‐1β transcription. Kinetic studies with the RNA synthesis inhibitor, actinomycin D, showed that GM‐CSF induces stable IL‐1β mRNA. Cycloheximide enhanced the IL‐1β mRNA accumulation by GM‐CSF at the level of mRNA stabilization, but blocked IL‐1β mRNA expression by TNF. Thus, GM‐CSF increases IL‐1β message accumulation in PMN at both the transcriptional and post‐transcriptional levels by mechanisms that are different from TNF induction of IL‐1β gene expression.

Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.

Accumulation of Ciprofloxacin and Minocycline by Cultured Human Gingival Fibroblasts

Through a mechanism that is unclear, systemic fluoroquinolones and tetracyclines can attain higher levels in gingival fluid than in blood. We hypothesized that gingival fibroblasts take up and accumulate these agents, thereby enhancing their redistribution to the gingiva. Using fluorescence to monitor transport activity, we characterized the accumulation of fluoroquinolones and tetracyclines in cultured human gingival fibroblast monolayers. Both were transported in a concentrative, temperature-dependent, and saturable manner. Fibroblasts transported ciprofloxacin and minocycline with Km values of 200 and 108 μg/mL, respectively, at maximum velocities of 4.62 and 14.2 ng/min/μg cell protein, respectively. For both agents, transport was most efficient at pH 7.2 and less efficient at pH 6.2 and 8.2. At steady state, the cellular/extracellular concentration ratio was > 8 for ciprofloxacin and > 60 for minocycline. Thus, gingival fibroblasts possess active transporters that could potentially contribute to the relatively high levels these agents attain in gingival fluid.

Regulation of ciprofloxacin uptake in human promyelocytic leukemia cells and polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) actively internalize ciprofloxacin, a capability that can enhance killing of intracellular bacteria and facilitate delivery of the antimicrobial agent to infection sites by migrating PMNs. In this study we investigated mechanisms for upregulation of this process. Activation with N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP; 100 nM) enhanced PMN ciprofloxacin uptake by 50% (P < 0.05). Phorbol myristate acetate (PMA; 10 nM) enhanced uptake by at least 36‐fold, mainly by stimulating an increase in the V max of the ciprofloxacin transporter. This effect of PMA was inhibited by antagonists of protein kinase C (H7 and chelerythrine) and the mitogen‐activated protein kinase cascade downstream (PD 098059). Under resting and PMA‐activated conditions, ciprofloxacin uptake by immature human promyelocytic leukemia (HL‐60) cells was much lower than in PMNs. However, when HL‐60 cells were induced to mature into PMN‐like cells, their ciprofloxacin uptake activity increased markedly. These findings implicate a role for protein kinase C in upregulation of the ciprofloxacin transporter and suggest that myeloid cells acquire an enhanced ability to take up ciprofloxacin as they mature to end‐stage PMNs. J. Leukoc. Biol. 61: 619–623; 1997.

Azithromycin concentrations in blood and gingival crevicular fluid after systemic administration.

BACKGROUND Azithromycin is a macrolide antibiotic that is active against several periodontal pathogens. Macrolides are taken up and concentrated inside gingival fibroblasts, which could influence their pharmacokinetics. This study tests the hypothesis that steady-state levels of azithromycin are higher and more sustained in gingival crevicular fluid (GCF) than in serum. METHODS Four healthy patients received an initial dose of 500-mg azithromycin followed by 250-mg doses on each of the next 2 days. Serum and GCF samples were obtained 2 hours after the last dose on day 2, and on days 4 and 7. GCF samples were collected from maxillary posterior sites with paper strips. The strips were pooled and eluted with high-purity water. After extraction, the azithromycin content of the serum samples and GCF eluates was determined with an agar diffusion bioassay. RESULTS On days 2, 4, and 7, the concentrations of azithromycin in blood serum were 0.22 ± 0.02, 0.08 ± 0.02, and 0.04 ± 0.01 μg/mL, respectively. The concentrations in GCF were 8.82 ± 1.25, 7.90 ± 1.72, and 7.38 ± 1.15 μg/mL, respectively. Mean GCF levels were significantly higher than mean serum levels (P ≤0.02; paired t test). CONCLUSIONS The findings demonstrate that the pharmacokinetic profiles of azithromycin are different in GCF and serum. At steady state, azithromycin concentrations in GCF were higher and more sustained than those in serum. Based on previous studies, the levels observed in GCF were above the minimal inhibitory concentration for Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and Prevotella intermedia.

Clarithromycin Transport by Gingival Fibroblasts and Epithelial Cells

Macrolide antibiotics penetrate cells, but the mechanism by which this occurs is unclear. The objective of this study was to characterize the mechanisms of clarithromycin uptake by gingival fibroblasts and oral epithelium. Cultured human gingival fibroblasts and SCC-25 cells were incubated with [3H]-clarithromycin. We assayed clarithromycin transport by measuring cell-associated radioactivity over time. Fibroblasts and epithelial cells rapidly accumulated clarithromycin, attaining steady-state intracellular concentrations within 15 minutes. Incubation in medium containing 2 μg/mL clarithromycin yielded steady-state intracellular concentrations of 75.8 μg/mL in fibroblasts and 6.6 μg/mL in SCC-25 cells. Clarithromycin transport exhibited Michaelis-Menten kinetics and was inhibited below 37°C. The Michaelis constants for fibro-blasts and SCC-25 cells were 78.4 and 227 μg/mL, respectively, while the maximum transport velocities were 264 and 381 ng/min/106 cells, respectively. Thus, both types of cells take up clarithromycin via a concentrative active transport system. By increasing intracellular clarithromycin levels, this system may enhance the effectiveness of clarithromycin against invasive periodontal pathogens.

Effect of Ciprofloxacin on Killing of Actinobacillus actinomycetemcomitans by Polymorphonuclear Leukocytes

ABSTRACT Actinobacillus actinomycetemcomitans, a pathogen associated with aggressive periodontitis, resists phagocytic killing by polymorphonuclear leukocytes (PMNs). It is susceptible to ciprofloxacin, which PMNs actively accumulate. This study tested the hypothesis that ciprofloxacin-loaded PMNs are more effective at killing A. actinomycetemcomitans than control PMNs. Isolated human PMNs were loaded by brief incubation with 0.5 μg of ciprofloxacin/ml. Opsonized bacteria (ATCC 43718) were incubated at 37°C with control and ciprofloxacin-loaded PMNs and in the presence and absence of 0.5 μg of ciprofloxacin/ml. When assayed at bacteria-to-PMN ratios of 30:1 and 90:1, ciprofloxacin-loaded PMNs killed significantly more bacteria and achieved significantly shorter half times for killing than control PMNs (P < 0.05; Tukeys test). At ratios of 3:1 and 10:1, these differences were not significant.