Loren W. Kline

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species

SummaryCalcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.

Progesterone inhibits gallbladder motility through multiple signaling pathways.

Progesterone (P) has an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since P 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 P and two metabolites on contraction in guinea pig gallbladder strips. P induced a concentration-dependent relaxation in guinea pig gallbladder strips precontracted with cholecystokinin octapeptide (CCK). Pretreatment of gallbladder strips with P (50 microM) also reduced the amount of CCK-induced tension. Nifedipine (1 microM) produced a similar effect. Pretreatment of the strips with PKA inhibitor 14--22 amide myristolated (180 nM) or the PKG inhibitor KT5823 (1.2 microM) either separately or in combination significantly reduced the amount of P-induced relaxation. Rp-cAMPs (0.1mM) or H-89 (10 microM) separately or in combination significantly reduced the P-effect; however, the combination of agents produced the largest reduction. Genistein (1 microM), an inhibitor of protein tyrosine kinases, significantly (p<0.01) reduced the amount of P-induced relaxation. The use of strontium in the Krebs solution as a substitute for Ca(2+) significantly (p<0.01) reduced the amount of CCK-induced tension. Pretreatment of the strips with 2-APB (26 microM), an inhibitor of IP(3,) induced Ca(2+) release, produced a significant (p<0.01) reduction in P-induced relaxation. We conclude that P inhibits gallbladder motility rapidly by nongenomic actions of the hormone. Several pathways that include tyrosine kinase and PKA/cAMP activity may mediate this effect.

Calcitonin gene-related peptide in the bullfrog, Rana catesbeiana: localization and vascular actions

Calcitonin gene-related peptide (CGRP) was found using radioimmunoassay in the brain and spinal cord of the bullfrog, Rana catesbeiana. The brain extracts were found to contain 241.7 +/- 68.1 pg/mg tissue, and the spinal cord contained 1753.0 +/- 96.8 pg/mg tissue. An intense immunocytochemical reaction was observed in the dorsal spinal cord. Vascular studies using helical strips of the dorsal aorta, iliac artery, and femoral artery showed CGRP to exert a vasorelaxant effect which was most pronounced in the femoral artery and minimal in the aorta. As in the rat, CGRP was shown to exert its vasorelaxant effect by inhibiting the mobilization of intracellular calcium.

Calcitonin gene-related peptide relaxed rat tail artery helical strips in vitro in an intracellular calcium-dependent manner.

The vasoactive properties of calcitonin gene-related peptide (CGRP) were examined using the helical strip of the rat tail artery as a model. CGRP was found to inhibit norepinephrine-induced contraction but not KCl- or vasopressin-induced contraction. No significant inhibition occurred in either KCl- or norepinephrine-stimulated strips when Ca2+ was added incrementally to otherwise Ca2+-free bathing solution. However, a dose-related inhibition to norepinephrine-stimulated contraction in Ca2+-free bathing solution was observed. This strongly suggests that CGRP exerts its effect by inhibiting the mobilization of intracellular Ca2+.

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.

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.

Nitric oxide modulates the calcitonin gene-related peptide-induced relaxation in guinea pig gallbladder strips in vitro

Calcitonin gene-related peptide (CGRP) induces a dose-dependent relaxation in cholecystokinin-induced tension in guinea pig gallbladder strips in vitro. This effect seems to be modulated, in part, via activation of the nonadrenergic noncholinergic nervous system. Blockers of nitric oxide synthesis significantly reduced the CGRP effect. Increases in nitric oxide (NO) after treatment with CGRP suggested the involvement of NO in the observed relaxation.

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-1β, 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-1β, 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.

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.

Calcitonin gene-related peptide in neural tissues: a phylogenetic study.

A heterologous radioimmunoassay using a rabbit antiserum raised against human calcitonin gene-related peptide (CGRP) was used to measure levels of immunoreactive CGRP (IR-CGRP) in the brain, pituitary, and spinal cord in species representing all classes of vertebrates from cyclostomes to mammals, except amphibians. All the brain extracts except those from the trout, goldfish, and iguana demonstrated the presence of IR-CGRP. Pituitary extracts from all animals, except the ratfish, goldfish and trout, contained IR-CGRP. CGRP was present in all classes of animals tested and seems to be highly conserved in the nervous system, where it may act as a neurotransmitter or neuromodulator.