The Flavone Luteolin, an Endocrine Disruptor, Relaxed Male Guinea Pig Gallbladder Strips.

BACKGROUND: Luteolin (3',4',5,7-tetrahydroxyflavone) is a flavone with a yellow crystalline appearance present in numerous plants such as broccoli, green chili, and carrot. Luteolin is considered to be an endocrine disruptor with potent estrogen agonist activity and potent progesterone antagonist activity. Luteolin has effects on smooth muscle. Luteolin relaxed guinea pig trachea smooth muscle as it inhibited both phosphodiesterase and reduced intracellular Ca2+. Luteolin also caused vasorelaxation in rat thoracic aorta smooth muscle by inhibiting intracellular Ca2+ release, inhibition of sarcolemmal Ca2+ channels, and activation of K+ channels. Luteolin or its glycosides from artichoke extracts may have an ameliorating effect on irritable bowel syndrome. The purpose of this study was to determine if luteolin had an effect on gallbladder motility. METHODS: An in vitro pharmacologic technique was utilized. Either cholecystokinin octapeptide (CCK) or KCl were used to induce tension in male guinea pig gallbladder strips maintained in Sawyer-Bartlestone chambers. Luteolin relaxed either the CCK- or KCl-induced tension in a concentration dependent manner. Various blockers were added to the chambers to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant. RESULTS: Treatment of the gallbladder strips with luteolin prior to either KCl or CCK significantly (P < 0.001) decreased the amount of either KCl- or cholecystokinin-induced tension. The 2-aminoethoxydiphenylborane was used to ascertain if the release of intracellular Ca2+ mediated the luteolin-induced relaxation. It significantly (P < 0.001) decreased the amount of luteolin-induced relaxation. To ascertain if PKA mediated the luteolin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < 0.01) reduced the amount of luteolin-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, genistein, tetraethylammonium, nor fulvestrant had a significant effect. To ascertain if PKC mediated the luteolin-induced relaxation, the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together. They had no significant effect. CONCLUSIONS: Luteolin relaxed cholecystokinin- or KCl-induced tension by blocking extracellular Ca2+ entry as well as intracellular Ca2+ release. In addition, the actions of PKA are also involved in mediating the luteolin effect.

Quercetin relaxes guinea pig gallbladder strips.

Quercetin, a phytoestrogen and flavonoid, relaxes intestinal and vascular smooth muscle. The purpose of this study was to determine if quercetin had an effect on gallbladder smooth muscle. An in vitro technique was used to determine the effects of quercetin on gallbladder strips and which system(s) mediated the relaxation. Paired t tests were used; differences between means of P < .05 were considered significant. Adding quercetin before cholecystokinin or KCl produced a significant (P < .001) decrease in the amount of tension (0.80 ± 0.04 vs 0.48 ± 0.04 g cholecystokinin octapeptide and 0.8 ± 0.06 vs 0.54 ± 0.05 g KCl, respectively). When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl- were simultaneously, a significant (P < .001) reduction in the quercetin-induced relaxation (45.7% ± 4.3% vs 27.6% ± 3.4%) was observed. To determine if protein kinase A (PKA) mediated the quercetin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < .05) decreased the amount (40.4% ± 3.7% vs 34.5% ± 3.3%) of quercetin-induced relaxation. The use of 2-APB also significantly (P < .001) reduced the amount of quercetin-induced relaxation (51.2% ± 3.5% vs 14.8% ± 3.6%). l-NG-methyl-l-arginine acetate salt, a nitric oxide synthase inhibitor, significantly (P < 001) decreased the quercetin-induced relaxation (45.7% ± 4.2% vs 35.2% ± 3.6%). KT5823, a PKC inhibitor, had no effect on the quercetin-induced relaxation. Quercetin blocked extracellular Ca2+ entry which affected downstream events such as activation of PKC, PKA, intracellular Ca2+ release, and activation of nitric oxide synthase. Quercetin relaxed cholecystokinin octapeptide and KCl-induced tension in a concentration dependent manner. Thus quercetin-induced relaxation was mediated by multiple signaling pathways.

Gallbladder motility and the sex of the guinea pig.

Progesterone (P), 17ß-estradiol (E2), and dihydrotestosterone (DHT) affect gallbladder motility. When gallbladders were taken from women and men, women had more estrogen and P receptors than men. Both P and E2 had an inhibitory effect upon gallbladder contractility in men and premenopausal and postmenopausal women. Similar findings have been reported in gallbladder strips from male and female guinea pigs. In the present study, there was no significant difference in the amount of E2-, P-, or DHT-induced relaxation of CCK-induced tension when the responses in gallbladder strips from male and female guinea pigs were compared. Three metabolites of P were used: 17-hydroxyprogesterone (17-P), 20α-hydroxyprogesterone (20-P), and 21-hydroxyprogesterone (21-P). There was no significant difference in the responses from strips from male and female guinea pigs. In order to determine if the effects of E2 and P were additive, strips from male animals were exposed to either E2 or P and the amount of relaxation recorded. After recovery, the strips were exposed to E2 or P in reverse order to ensure the order of treatment had no effect. Then, the strips were treated with both E2 and P simultaneously and the relaxation recorded. This procedure was repeated with strips from female guinea pigs. The effect of E2 and P was found to be additive; however, the response of the strips from each sex were not significantly different. It is concluded that the sex of the guinea pig has no significant effect on the response to the sex hormones used.

Curcumin Relaxes Precontracted Guinea Pig Gallbladder Strips via Multiple Signaling Pathways.

BACKGROUND: Curcumin (diferuloymethane) is the active ingredient of the dietary spice turmeric. Curcumin modulates various signalling molecules, including inflammatory agents, transcription factors, protein kinases and cell cycle regulatory proteins. The purpose of this study was to determine if curcumin had an effect on gallbladder motility. METHODS: A pharmacologic in vitro technique was used. Since curcumin relaxed both cholecystokinin octapeptide- (CCK) and KCl-induced tension of guinea pig gallbladder strips in a concentration dependent manner, an in vitro technique was used to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests, t-tests and analysis of variance were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant. RESULTS: To determine if protein kinase A (PKA) mediated the curcumin-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no significant effect on the amount of curcumin-induced relaxation. When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, a significant (P < 0.01) reduction in the curcumin-induced relaxation was observed. The use of tetraethylammonium chloride (TEA) caused a significant (P < 0.01) decrease in the amount of curcumin-induced relaxation. Adding curcumin prior to the KCl caused a significant (P < 0.001) decrease in the amount of KCl-induced tension. CONCLUSIONS: The results suggested that the curcumin-induced relaxation is mediated by multiple signaling pathways including the PKC second messenger system, inhibiting extracellular Ca2+ entry and K+ channels.

The Resveratrol-induced Relaxation of Cholecystokinin Octapeptide- or KCl-induced Tension in Male Guinea Pig Gallbladder Strips Is Mediated Through L-type Ca2+Channels.

BACKGROUND/AIMS: Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic compound (stilbene) and a phytoalexin. The purpose of this study was to determine the mechanism which mediated the resveratrol-induced relaxation of cholecystokinin octapeptide- or KCl-induced tension in male guinea pig gallbladder strips. METHODS: Gallbladder strips were prepared and suspended in in vitro chambers filled with Krebs-Henseleit solution. The strips were attached to force displacement transducers, and the changes in tension were recorded on a polygraph. All reagents were added directly into the chambers. RESULTS: To determine if intracellular Ca(2+) release mediated the resveratrol-induced relaxation of cholecystokinin octapeptide-induced tension, 2-aminoethoxydiphenylborane (2-APB) was used. 2-APB significantly (P < 0.01) decreased the amount of RSVL-induced relaxation. To determine if protein kinase A (PKA) mediated the resveratrol-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no effect on resveratrol-induced relaxation. Neither KT5823, N(G)-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor, nor fulvestrant had a significant effect on the amount of resveratrol-induced relaxation. Genistein, a protein tyrosine kinase inhibitor, significantly (P < 0.01) increased the RSVL-induced relaxation. To determine if protein kinase C mediated the RSVL-induced relaxation, the protein kinase C inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, and a significant (P < 0.05) increase in resveratrol-induced relaxation was observed. The pretreatment of the strips with resveratrol significantly (P < 0.001) decreased the amount of KCl- and cholecystokinin octapeptide- induced tension. CONCLUSIONS: Resveratrol-induced relaxation is mediated by its effects on L-type Ca(2+) channels and intracellular Ca(2+) release.

The flavonoid chrysin, an endocrine disrupter, relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips through multiple signaling pathways.

The bioflavonoids have effects on vascular smooth muscle and gastrointestinal smooth muscle. The flavone and phytoestrogen, chrysin, has been shown to have a vasorelaxant effect on resistance blood vessels. This effect was mediated by nitric oxide (NO). Chrysin inhibited aromatase/estrogen biosynthesis in postmenopausal women. The purpose of this study was to determine if chrysin had an effect on cholecystokinin- or KCl-induced tension in male guinea pig gallbladder strips. In addition, the second messenger(s) system(s) that mediated the effect were to be determined. A pharmacologic approach was used. Male guinea pig gallbladder strips were placed in in vitro chambers filled with Krebs solution, maintained at 37 °C, and gassed with 95% O2-5% CO2. Changes in tension were recorded using a polygraph. It was shown that the PKA/cAMP second messenger system mediated part of the observed chrysin-induced relaxation of cholecystokinin-induced tension, the PKC system also mediated part of the relaxation, and the inhibition of both extracellular Ca(2+) entry and intracellular Ca(2+) release also mediated the chrysin-induced relaxation. This is the first report of chrysin having an effect on gallbladder smooth muscle contraction.

Osteocalcin expression in pulp inflammation.

INTRODUCTION: Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation. METHODS: Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay. RESULTS: OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial growth factor, fibroblast growth factor, macrophage inflammatory protein-1ß, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-17, and soluble IL-2 receptor α and negatively correlated to that of IL-1α, IL-1ß, IL-8, granulocyte macrophage colony-stimulating factor, and macrophage inflammatory protein-1α. CONCLUSIONS: Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment.

A novel experimental model for studying transverse orthodontic tooth movement in the rat mandible.

OBJECTIVES: To establish a rat model of a one-piece mandible using the principles of gingivoperiosteoplasty and guided bone regeneration to fuse the midline symphyseal area. MATERIAL AND METHODS: Twenty-four Sprague-Dawley female rats were divided into two groups: 12 experimental and 12 control. Both groups were imaged using in vivo micro-computed tomography at baseline and at end point (5 months). The experimental group received regenerative surgery at the symphysis area; the control group received no treatment. Outcomes were evaluated by radiographic examination of gross and volumetric bony changes in the symphyseal region of interest marked between the mental foramina bilaterally and the two central incisors near the most coronal margin of the alveolar crests. These landmarks were chosen as they can be reproduced on the computed tomography images at baseline and end point. Histologic examination was performed on all samples at a level 5 mm apical to the alveolar bone crest. RESULTS: Radiologic and histologic examinations of the experimental group revealed complete bony fusion of the symphyseal area in three subjects, partial fusion in five subjects, and thickening of the alveolar bony socket in three subjects; one rat died of anesthesia-related complications. No evidence of fusion or alveolar bone thickening was found in any of the controls. CONCLUSIONS: This surgical animal model demonstrates that a rat mandible can be surgically manipulated to mimic the one-piece human mandible. This novel model may prove useful in studying mandibular bone remodeling and orthodontic mandibular tooth movement.

A comparison of the effects of various sex steroids on cholecystokinin- and KCl-induced tension in female guinea pig gallbladder strips.

Estrogen (E) has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. During pregnancy E and progesterone (P) levels are elevated. A biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17ß-estradiol (E2), dihydrotestosterone (DHT), P, 17-hydroxyprogesterone (17-P), and a P metabolite, 20α-hydroxyprogesterone (20-P) on contraction in female guinea pig gallbladder strips. DHT, P, 17-P, 20-P, and E2 each induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK) induced tension. DHT, E2, and P also induced a concentration-dependent relaxation of KCl-induced tension. When the response to E2 was compared to strips from young female guinea pigs with those taken from guinea pigs in late pregnancy, there was no significant difference in the response to either 50 or 100 µM E2; however, 10 µM E2 caused a significant increase (p<0.05) in the amount of relaxation in strips from pregnant guinea pigs. Treatment of the strips from young guinea pigs with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Treatment of the strips with 2-APB, an inhibitor of IP3 induced Ca(2+) release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used. Neither KT5823, a PKG inhibitor, nor L-NMMA, a nitric oxide (NO) synthase inhibitor, had a significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl(-), PKC blockers, were used in combination with no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) increased the amount of E2-induced relaxation. When either E2 or P were added to the chambers 3 min prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca(2+) entry mediates both P-induced and E2-induced relaxation of CCK- and KCl-induced tension in female guinea pig gallbladder strips.

Dentin matrix protein-1 activates dental pulp fibroblasts.

INTRODUCTION: Dentin matrix protein-1 (DMP-1) is involved in the mineralization of hard dental tissues. DMP-1 is localized in several soft tissues, but its role is unclear. METHODS: Human inflamed dental pulps were collected from the endodontic clinic and human normal pulps from impacted teeth. Dental pulp cells from 8 subjects were explanted to test the effect of DMP-1 on interleukin-6 (IL-6) and IL-8 production by using enzyme-linked immunosorbent assay. RESULTS: DMP-1 was localized in pulp inflammation by using immunohistochemistry but was not present in impacted root pulps. Wherever found, areas of calcification were positively stained against DMP-1, suggesting its possible involvement in pulp inflammation and in pathologic calcification. To test this hypothesis, primary human pulp fibroblasts were cultured. The fibroblasts were identified on the basis of their morphology, immunoreactivity against vimentin and collagen 1a1 by immunofluorescence and negative staining to CD45, CD34, and cytokeratin by flow cytometry. DMP-1 (10 ng/mL) stimulated the production of IL-6 and IL-8 from pulp fibroblasts. DMP-1 showed an additive effect with lipopolysaccharide in IL-6 and IL-8 production. Inhibition of the p38 mitogen-activated protein kinase pathway blocked the proinflammatory effect of DMP-1 on pulp fibroblasts. CONCLUSIONS: Our data indicate that DMP-1 might participate in the development of inflammatory changes in the dental pulp. DMP-1 inhibition might be a new therapeutic strategy to target pulp inflammation and pathologic calcification.

17ß-Estradiol relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips.

Estrogen has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since estrogen and progesterone levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17ß-estradiol (E2) on contraction in male guinea pig gallbladder strips. E2 induced a concentration-dependent relaxation of either CCK-induced tension or KCl-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the E2-induced relaxation. Pretreatment of strips with 2-APB, and inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used to induce tension. KT5823, an inhibitor of PKG, also significantly (p<0.001) increased the amount of E2-induced relaxation. Genistein, an inhibitor of protein tyrosine kinase, had no significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl- when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.001) increased the amount of E2-induced relaxation. When E2 was added to the chambers prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca(2+) entry mediates the E2-induced relaxation of CCK- and KCl-induced tension in male guinea pig gallbladder strips.

Effects of calcitonin, calcitonin gene-related peptide, human recombinant bone morphogenetic protein-2, and parathyroid hormone-related protein on endodontically treated ferret canines.

INTRODUCTION: The purpose of this study was to determine whether human recombinant bone morphogenetic protein-2 (rhBMP-2), calcitonin gene-related peptide (CGRP), calcitonin (CT), or parathyroid hormone-related protein (PTHrP) promoted reparative tertiary dentin or osteodentin formation in ferret canines. METHODS: Ferrets had up to 4 pulpotomies performed under anesthesia. All pulps had sterile absorbable sponge of a standard size placed in contact with the pulp. The sponge contained sterile saline, rhBMP-2, CGRP, CT, or PTHrP. The opening was filled with an intermediate restorative material. After 6 weeks, the ferrets were anesthetized, and the pulpotomized teeth were extracted. The canines were fixed, decalcified, sectioned, and stained with hematoxylin-eosin. Sections were selected from the area of the opening, and the amount of reparative tertiary dentin and osteodentin was measured by using a digitizer. RESULTS: Analysis of the photomicrographs showed that rhBMP-2 induced 0.58 +/- 0.19 mm(2) osteodentin and 0.56 +/- 0.18 mm(2) tertiary dentin. CGRP induced 0.46 +/- 0.05 mm(2) osteodentin and 0.38 +/- 0.04 mm(2) tertiary dentin. The amount of rhBMP-2-induced and CGRP-induced osteodentin and tertiary dentin was significantly (P < .001) more than that found in the sterile saline-treated teeth (0.29 +/- 0.03 mm(2) osteodentin and 0.14 +/- 0.03 mm(2) tertiary dentin) or CT (0.2 +/- 0.06 mm(2) osteodentin and 0.16 +/- 0.05 mm(2) tertiary dentin; P < .01). PTHrP significantly (P < .05) reduced the amount of osteodentin (0.17 +/- 0.02 mm(2)) observed in the saline-treated teeth but was not significantly different in the amount of tertiary dentin observed. CONCLUSIONS: RhBMP-2 and CGRP promoted more pulpal healing than either CT or PTHrP.

Testosterone and dihydrotestosterone inhibit gallbladder motility through multiple signalling pathways.

Testosterone (T) has been shown to cause vasodilation in rabbit coronary arteries through a nongenomic pathway. Part of this T-induced relaxation was shown to be mediated by opening voltage dependent K(+) channels. T infusion also reduces peripheral resistance in human males with heart failure. The effects of T or its active metabolite 5-alpha dihydrotestosterone (DHT) are not well studied. This study investigates the effect of T and DHT on contraction in guinea pig gallbladder strips. T or DHT induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK)-induced tension. Pretreatment of the strips with PKA inhibitor 14-22 amide myristolated had no significant effect on the relaxation induced by either T or DHT. Pretreatment of strips with 2-APB, an inhibitor of IP(3) induced Ca(2+) release, produced a significant (p<0.001) reduction in the T- or DHT-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl(-) when used in combination had no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) decreased the amount of T- or DHT-induced relaxation. The flavone chrysin, an aromatase inhibitor, and genistein, an isoflavone, each produced a significant (p<0.01) reduction in CCK-induced tension. Chrysin significantly (p<0.05) increased T-induced relaxation; however, genistein had no effect on T-induced relaxation. It is concluded that T and DHT inhibits gallbladder motility rapidly by nongenomic actions of the hormones. Multiple pathways that include inhibition of intracellular Ca(2+) release, inhibition of extracellular Ca(2+) entry, and the actions of PKC may mediate this effect.

Protein kinase C masks nitric oxide synthase activity in vascular smooth muscle under basal conditions.

Under basal conditions there is no observable nitric oxide synthase (NOS) activity in vascular smooth muscle (VSM). Pretreatment of endothelium-denuded aortic rings from Sprague-Dawley rats with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), (0.1 micromol/L) significantly attenuated phenylephrine (PE)-induced contractile responses in a dose-dependent manner. In the presence of 10 micromol/L Nomega-nitro-L-arginine (L-NNA) or 0.1 mmol/L aminoguanidine (AG), the inhibition of contractions at 10 nmol/L PE by H-7 was blocked by 88% or 52%, respectively. The blockade by antagonists was completely reversed by l-arginine but not by d-arginine, and alone they did not significantly alter PE-induced contraction of endothelium-denuded aorta. Methylene blue (MB, 50 micromol/L) also inhibited the action of H-7. The inhibitory effect of H-7 occurred after 5 minutes and was reversible. PE-induced contraction was also inhibited by the selective protein kinase C inhibitors calphostin C (10 micromol/L), and bisindolylmaleimide IV (Bis-IV, 10 micromol/L), but not by the selective protein kinase A inhibitor H-89 (0.1 micromol/L). These results indicate protein kinase C inhibits NOS activity in VSM under basal conditions. Incubation of tissues with either H-7 or calphostin C stimulates NO production, and immunocytochemical studies reveal the presence of NOS in VSM under basal conditions.