Juan José Segura

CHARACTERIZATION OF FUNCTIONAL RECEPTORS FOR VASOACTIVE INTESTINAL PEPTIDE(VIP) IN RAT PERITONEAL MACROPHAGES

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.

Talon cusp affecting permanent maxillary lateral incisors in 2 family members

The term talon cusp refers to a relatively rare dental anomaly in which an accessory cusplike structure projects from the cingulum area or cement-enamel junction. The condition can occur in either maxillary or mandibular anterior teeth in both the primary and permanent dentitions. This article reports 2 cases of talon cusp affecting consanguineous first cousins: a case of bilateral talon cusps on the permanent maxillary lateral incisors of a 16-year-old girl, and a case of talon cusp on the maxillary permanent lateral incisor of an 11-year-old boy. The talon cusps caused clinical problems that were related to caries or occlusal interferences. The presence of the dental anomaly in 2 members of the same family suggests that genetic inheritance may be a causative (related) factor. Examination of relatives could facilitate early diagnosis of the talon cusp and aid in preventing carious and occlusal problems.

Expression of VIP receptors in mouse peritoneal macrophages: Functional and molecular characterization

Receptors for VIP in mouse peritoneal macrophages (MPM) were examined using [125I]labeled VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, pH, temperature and cell concentration. At 15 degrees C, the stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 1.05 +/- 0.2 and 66.4 +/- 11.0 nM and binding capacities of 19.2 +/- 2.8 and 706.6 +/- 172.0 fmol VIP/10(6) cells. The interaction showed a high degree of specificity, as suggested by competition experiments with various peptides structurally related to VIP as follows: VIP > helodermin > rGRF > PHI >> secretin. Glucagon, pancreastatin, somatostatin, insulin, and octapeptide of cholecystokinin (CCK 26-33) were ineffective at concentrations as high as 1 microM. VIP was a potent and efficient stimulator of cyclic AMP production in MPM. The stimulation was observed at a concentration as low as 0.01 nM VIP. Half-maximal stimulation (ED50) was observed at 1.0 +/- 0.2 nM VIP, and maximal stimulation (three-fold above basal levels) was obtained between 0.1-1 microM. The cyclic AMP system of mouse peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP > helodermin > rGRF > PHI >> secretin. Glucagon, insulin, pancreastatin, somatostatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

EDTA inhibits in vitro substrate adherence capacity of macrophages: Endodontic implications

The disodium salt of ethylenediamine tetraacetate (EDTA) is a calcium ion chelator used in endodontics to enlarge root canals. This study investigated the effect of EDTA on substrate adherence capacity of rat inflammatory macrophages to determine if EDTA leakage to periapical tissues during root canal therapy can alter macrophage function. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index (AI) was calculated. Results showed that EDTA decreased substrate adherence capacity of inflammatory macrophages in a time and dose-dependent manner. The lowest EDTA concentration that caused a significant inhibition of AI was 50 mM (p < 0.05), and the EDTA concentration that caused half-maximal inhibition (IC50) was 194 +/- 20 mM (p < 0.01). Calcium chloride (10 mM) increased the adherence index of macrophages by 17.1% (p < 0.05) and decreased the EDTA inhibitory effect on AI by 49.5% (p < 0.05). We conclude that an EDTA concentration lower than that used in endodontics decreased the substrate adherence capacity of macrophages significantly. Adhesion is the first step in the phagocytic process and in antigen presentation, but leakage of EDTA to periapical tissues during root canals preparation may inhibit macrophage function and reduce periapical inflammatory reactions.

The disodium salt of EDTA inhibits the binding of vasoactive intestinal peptide to macrophage membranes: Endodontic implications

The purpose of this study was to investigate the effect of the disodium salt of ethylenediamine tetraacetate (EDTA), a calcium ion chelator used in the root canal therapy, on vasoactive intestinal peptide (VIP) binding to macrophage membranes (MMs). Binding assays were conducted at 15 degrees C in 0.5 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 1.6% (w/v) bovine serum albumin, 1.2 mg/ml of bacitracin, and different EDTA concentrations, using 45 pM of [125I]VIP as tracer. Results showed that EDTA inhibits VIP binding to MMs in a dose-dependent manner, with an IC50 value of 5.4 mM (p < 0.01). EDTA concentrations equal or higher than 100 mM of abolished VIP-MM interaction. Taking into account that the macrophage plays an essential role in inflammatory reactions and the immune response, we conclude that the apical extrusion of EDTA during root canal therapy could modify VIP-macrophage interaction modulating the inflammatory mechanisms involved in periapical lesions.

Calcium Hydroxide Inhibits Substrate Adherence Capacity of Macrophages

The purpose of this study was to investigate the effect of calcium hydroxide on substrate adherence capacity of rat inflammatory macrophages to determine if calcium hydroxide can alter macrophage function. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index (AI) was calculated. Results showed that calcium hydroxide decreased substrate adherence capacity of inflammatory macrophages in a time and dose-dependent manner. The lowest calcium hydroxide concentration that caused a significant inhibition of AI was 1 mM (p < 0.05), and the concentration of calcium hydroxide that caused half-maximal inhibition (IC50) was 1.54 mM (p < 0.01). We conclude that calcium hydroxide decreased substrate adherence capacity of macrophages. When adhesion as the first step in the phagocytic process and in antigen presentation is taken into account, calcium hydroxide could inhibit macrophage function and reduce inflammatory reactions in periapical tissues or in dental pulp when it is used in root-canals therapy or in direct pulp capping and pulpotomy, respectively. Moreover, this effect could explain, at least in part, the mineralized tissue-inducing property of calcium hydroxide.

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.

Characteristics of Receptors for VIP in Rat Peritoneal Macrophage Membranes

Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with Kd values of 0.60 +/- 0.08 and 275 +/- 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC50 = 1 nM) > rGRF (IC50 = 13 nM) > PHI (IC50 = 421 nM) >> secretin. Glucagon, somatostatin, insulin octapeptide of cholecystokinin [CCK(26-33)], and pancreastatin were ineffective at concentrations up to 1 microM. Binding of [125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [125I]VIP-protein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.

Concomitant hypohyperdontia: Simultaneous occurrence of a mesiodens and agenesis of a maxillary lateral incisor

A 13-year-old boy appeared for evaluation with a missing maxillary left lateral incisor. He also had an abnormally shaped tooth in the midline between his maxillary central incisors. This mesiodens had an incompletely developed root. The unusual association of these 2 anomalies is discussed as a possible transposition of the lateral incisor to the mesiodens position.

The effect of the bleaching agent sodium perborate on macrophage adhesion in vitro: implications in external cervical root resorption.

The purpose of this study was to investigate the in vitro effect of sodium perborate, which is used as a bleaching agent in the treatment of discolored pulpless teeth, on substrate adherence capacity of macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. As a test of macrophage adhesion, the adherence capacity of macrophages to a plastic surface was determined. Assays were conducted in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that sodium perborate decreased in a dose-dependent manner and decreased significantly (p < 0.05) the adherence index of rat peritoneal macrophages. Sodium perborate was less potent than sodium hypochlorite and eugenol in inhibiting macrophage adhesion. The inhibitory effect of sodium perborate on macrophage adhesion further supports the concept that this agent is not implicated in external cervical root resorption associated with intracoronal bleaching.