Gabriel A. Sánchez

Determination of salivary levels of mucin and amylase in chronic periodontitis patients.

BACKGROUND AND OBJECTIVE Patients with periodontal disease show differences in the profile of proteins in whole saliva. This profile reflects the nature and amplitude of the host response to a periodontal microbial challenge. Since periodontitis is a chronic inflammatory disease with different progression stages, the aim of the study was to evaluate the host response in these different clinical stages by assessing salivary flow rate, the concentrations of proteins and mucin and the amylase activity. MATERIAL AND METHODS Sixty adult subjects were clinically examined and distributed into four groups (n = 15) according to the periodontal status, namely, healthy, mild, moderate and severe periodontitis. Whole saliva was collected for 5 min, followed by a second 5 min sampling period with stimulation by chewing a paraffin block, and flow rate was determined. Salivary proteins, amylase and mucin were determined by colorimetric methods. RESULTS The concentrations of proteins, amylase and mucin increased in subjects with moderate and severe periodontal disease in unstimulated saliva, while flow rate decreased. A positive correlation was found between proteins and amylase or mucin concentrations among the different groups, indicating that the concentrations changed in the same way, being the response of salivary glands to the disease, possibly to enhance the protective potential of saliva. Mucin concentration was lower in the mild periodontitis group. Mechanical stimulation induced an increase in flow rate and output of proteins, amylase and mucin. CONCLUSION Periodontitis induces an increase in the output of proteins, including mucin and amylase, thereby enhancing the protective potential of saliva, but this is accompanied by a decrease in flow rate.

Relationship between salivary mucin or amylase and the periodontal status

OBJECTIVE Here we determine the relationship between salivary levels of mucin and amylase and the clinical parameters of periodontal disease before and after periodontal treatment. SUBJECTS Ninety two subjects were clinically examined and distributed into four groups namely clinically healthy, mild, moderate and severe periodontitis, according to the periodontal status, classified according the values of clinical attachment level (CAL) and probing pocket depth (PPD). Unstimulated saliva was collected for 5 min. Salivary proteins, amylase and mucin were determined by colorimetric methods. RESULTS A significant positive correlation (P < 0.0001) was observed between salivary mucin, amylase or protein and PPD or CAL before periodontal treatment while flow rate showed a negative correlation. Mucin and amylase output also showed a positive correlation with PPD or CAL. After treatment, the improvement of clinical parameters was accompanied by a diminution of salivary mucin, amylase or protein concentration and output in moderate and severe group. CONCLUSIONS The increment of mucin and amylase output in relation to periodontal status indicates that salivary glands respond to the disease by increasing the protective potential of saliva when necessary and return to the normal rate of secretion after the resolution of the inflammatory process.

Total salivary nitrates and nitrites in oral health and periodontal disease

It is well known that nitrites are increased in saliva from patients with periodontal disease. In the oral cavity, nitrites may derive partly from the reduction of nitrates by oral bacteria. Nitrates have been reported as a defence-related mechanism. Thus, the aim of the present study was to determine the salivary levels of total nitrate and nitrite and their relationship, in unstimulated and stimulated saliva from periodontal healthy subjects, and from patients with chronic periodontal disease. Nitrates and nitrites were determined in saliva from thirty healthy subjects and forty-four patients with periodontal disease. A significant increase in salivary nitrates and nitrites was observed. Nitrates and nitrites concentration was related to clinical attachment level (CAL). A positive and significant Pearsons correlation was found between salivary total nitrates and nitrites. Periodontal treatment induced clinical improvement and decreased nitrates and nitrites. It is concluded that salivary nitrates and nitrites increase, in patients with periodontal disease, could be related to defence mechanisms. The possibility that the salivary glands respond to oral infectious diseases by increasing nitrate secretion should be explored further.

Local Anesthetics Inhibit Ca-ATPase in Masticatory Muscles

Local anesthetics have myotoxic effects and inhibit Ca-ATPase activity and Ca transport in skeletal muscles. Such effects have not been fully elucidated in masticatory muscles. We tested the hypothesis that local anesthetics increase myoplasmic calcium in masticatory muscles by inhibiting Ca-ATPase at a concentration similar to that of dental cartridges. The effects of lidocaine and bupivacaine on Ca-ATPase from rabbit masseter and medial pterygoid muscles were tested with radioisotopic and colorimetric methods. Bupivacaine had an action similar to that of lidocaine on Ca-ATPase activity, but less effect on calcium transport. The pre-exposure of the membranes to the anesthetics enhanced the Ca-ATPase activity in the absence of calcium ionophore, supporting their permeabilizing effect. The results demonstrate that amide-type anesthetics do not inhibit calcium binding, but do reduce calcium transport and enzyme phosphorylation by ATP, and suggest that the myoplasmic calcium increase induced by lidocaine and bupivacaine might promote masticatory muscle contraction and eventual rigidity.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence

Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca2+ uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.

Association between Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque and clinical parameters, in Argentine patients with aggressive periodontitis

BACKGROUND Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) have been associated with aggressive (AgP) and chronic periodontitis. OBJECTIVE The aim of this study was to evaluate the levels of Aa and Pg in gingival crevicular fluid (GCF) of patients with AgP and its relation with clinical parameters. DESIGN Sixteen females and fourteen males with clinical diagnosis of AgP aged 17-23 years and their matchs controls, were included in this study. Clinical recording concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 30 and 60 days after baseline. After clinical examination GCF samples were analyzed for Aa and Pg with a real-time polymerase chain reaction technique. Patients group was treated with a combined of mechanical and oral antibiotic therapy (doxycycline 100 mg/day, during 21 days). A multivariate analysis was used to determine the relationship between Aa and Pg counts with clinical parameters. RESULTS GCF from all subjects was positive for Aa and PG. In controls Pg concentration was higher than Aa (Pg: 42,420 ± 3,034 copies/ml; Aa: 66.6 ± 5.4 copies/ml p < 0.001) while in patients both microbes showed the same concentration (Aa: 559,878 ± 39,698 Pg: 572,321 ± 58,752). A significant and positive correlation was observed between counts of Aa and Pg (R square: 0.7965, p < 0.0001). Female showed more counts/ml. Aa might be closely associated with clinical parameters while Pg did not. At 30 and 60 days Aa counts in patients were similar to controls while Pg counts were equal to baseline. However, in spite of Pg presence a clinical improvement was observed in all patients. CONCLUSIONS In our population the presence of Aa may be associated with AgP while Pg may be in GCF as an opportunistic pathogen which might caused disease when the ecological balance was favorable.

Characteristics of the Sarcoplasmic Reticulum Ca2+-dependent ATPase from Masticatory Muscles

We compared the sarcoplasmic reticulum (SR) Ca-ATPase from masseter (M) and medial pterygoid (MP) muscles with that from fast muscles (FM) to examine whether its calcium transport capability and enzymatic activity are different. SR vesicles from FM, M, and MP muscles were obtained according to Champeil et al.(1985). Assays for characterization of the enzyme properties were performed. The results showed similar optimal conditions for the Ca-ATPase activity and calcium transport in M, MP, and FM. However, the maximal values of calcium transport, Ca-ATPase activity, and Ki for thapsigargin were significantly lower in the masticatory muscles. These findings are likely related to different Ca-ATPase isoforms. Since the local anesthetics used in dentistry inhibit Ca-ATPase and calcium transport in FM, it will be important for the effects of these drugs on the Ca-ATPase of masticatory muscles to be assessed.

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.

Parameters of oxidative stress in saliva from patients with aggressive and chronic periodontitis

Objectives: Free radicals play an important role in the onset and progression of many diseases. The aim of this study was to investigate the contribution of oxidative stress in the pathology of aggressive (AgP) and chronic (CP) periodontitis and its relation with the clinical periodontal status. Methods: Eighty subjects were divided into two groups: 20 patients with AgP and 20 patients with CP with their 20 corresponding matched controls, based on clinical attachment loss (CAL), probing pocket depth (PPD), and bleeding on probing (BOP). Saliva reactive oxygen species (ROS), lipid peroxidation, and non-enzymatic antioxidant defences were measured by luminol-dependent chemiluminescence assay, as thiobarbituric acid-reactive substances (TBARs) and total radical-trapping antioxidant potential (TRAP), respectively. Pearsons correlation and multivariate analysis were used to determine the relationship between ROS and TBARs and the clinical parameters. Results: ROS and TBARs were increased in AgP while TRAP was decreased, comparing with CP. In AgP, a strong and positive correlation was observed between ROS and TBARs and they were closely associated with CAL and PPD. Discussion: In AgP, but not in CP, oxidative stress is a high contributor to periodontal pathology and it is closely associated with the clinical periodontal status.

Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis

OBJECTIVE In a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE(2) production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol. DESIGN Experimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate. RESULTS The effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10(-6)M carbachol was inhibited by 4-DAMP (10(-6)M), U-73122 (5 × 10(-6)M) and trifluoperazine (5 × 10(-6)M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration-response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE(2). CONCLUSIONS Parotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca(2+) and cAMP in the fusion/exocytosis step of secretory vesicles.