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 Kreb's 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.

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) relax cholecystokinin-induced tension in guinea pig gallbladder strips.

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have been shown to relax various types of smooth muscle, e.g. vascular, uterine and gastric. This study demonstrates that PTH and PTHrP both relaxed cholecystokinin octapeptide (CCK)-induced tension in guinea pig gallbladder strips. This relaxation was concentration-dependent. The use of PTHrP (7-34) blocked the relaxant effect of both agents. This suggested PTH and PTHrP were acting through the same receptor. The use of Rp-cAMPs, an inhibitor of cAMP activation of protein kinase A, and H-89, a selective inhibitor of protein kinase A, suggested that cAMP mediated the relaxant action of PTH and PTHrP. The use of iberiotoxin indicated that the high conductance Ca(2+)-activated potassium channels also mediated the actions of PTH/PTHrP. The use of KT5823, a selective blocker of protein kinase G, also decreased the amount of relaxation induced by PTH/PTHrP. This suggested that crosstalk between the two second messenger (cAMP and cGMP) systems occurred.

Protein kinase C mediates the contractile actions of pituitary adenylate cyclase activating polypeptide in guinea pig gallbladder strips.

Pituitary adenylate cyclase activating polypeptide (PACAP) was shown to relax guinea pig gallbladder strips contracted with cholecystokinin. This relaxation was mediated by PACAP interacting with VIP/PACAP receptors. PACAP was also shown to cause contraction in guinea pig gallbladder strips. The present study demonstrated that calphostin C and bisindolylmaleimide IV, both blockers of protein kinase C, significantly reduced tension, Rp-adenosine 3', 5'-cyclic monophosphatase triethylamine, a blocker of protein kinase A, had no effect on PACAP-induced tension. Nifedipine also significantly reduced the PACAP effect. The contractile effects of PACAP are mediated by protein kinase C.

Cyclic AMP modulates part of the relaxant action of calcitonin gene-related peptide in guinea pig gallbladder strips.

Calcitonin gene-related peptide (CGRP) has been shown to relax cholecystokinin-induced tension in guinea pig gallbladder strips. This relaxation is dependent on the concentration of CGRP, and is primarily due to the opening of ATP sensitive K+ channels; however, other mechanisms may also be involved. Studies using forskolin, 8-bromoadenosine 3', 5' cyclic monophosphate, dibutyryl cAMP, cholera toxin, and Rp-adenosine 3', 5'-cyclic monophosphothioate triethylamine, which measured changes in tension suggest that cAMP may be involved in mediating the actions of CGRP. Radioimmunoassay of strips precontracted with cholecystokinin octapeptide (CCK) and either treated with CGRP or its solvent demonstrated that cAMP concentrations increased with CGRP treatment. The results of these studies demonstrate that CGRP acts through multiple mechanisms to induce relaxation of guinea pig gallbladder strips precontracted with CCK.

Cyclic AMP induces a relaxation response in the bullfrog Rana catesbeiana, but nitric oxide does not.

Cholecystokinin octapeptide (CCK), acetylcholine (ACh) and ceruletide have been shown to produce contraction in bullfrog (Rana catesbeiana) gallbladder strips. Agents capable of relaxing the bullfrog gallbladder are less numerous. Calcitonin gene-related peptide reduced the amount of both CCK- and ACh-induced tension in bullfrog gallbladder strips. The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), nitric oxide (NO) and the second messengers cyclic GMP or cyclic AMP had any effect on gallbladder motility in the bullfrog. In vitro tension studies using l-NG-nitro-arginine methyl ester, Methylene Blue, sodium nitroprusside and N2,2'-O-dibutyryl guanosine 3',5'-cyclic monophosphate suggested that nitric oxide did not modulate gallbladder motility in the bullfrog gallbladder. Histochemical staining for NADPH diaphorase (nitric oxide synthase) failed to demonstrate nerve fibers containing nitric oxide synthase in the bullfrog gallbladder. In vitro studies demonstrated that VIP had no effect on CCK-induced tension. However, in vitro studies using either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin demonstrated that both agents relaxed strips precontracted with CCK. The results of this study suggested that, while neither NO nor VIP had a role in modulating bullfrog gallbladder motility, cyclic AMP was capable of modulating bullfrog gallbladder motility.

The effect of body weight on the calcitonin gene-related peptide-induced relaxation of guinea pig gallbladder strips.

Calcitonin gene-related peptide (CGRP) has been reported to relax, in a concentration-dependent manner, cholecystokinin-induced tension in guinea pig gallbladder strips. It is now shown that this CGRP-induced relaxation is body weight dependent with the degree of relaxation inversely related to weight. Radioimmunoassay demonstrated that the amount of CGRP in the gallbladder increased with body weight. Receptor binding showed that the number of binding sites and the affinity of the receptor decreased with increasing body weight. CGRP may play a role in the normal pattern of gallbladder contraction and relaxation. Decreased motility, even between periods of complete emptying, has been demonstrated to have a role in gallbladder diseases. The decreased sensitivity of the gallbladder to CGRP may explain the increase in abnormalities with age.

Calcitonin gene-related peptide relaxes cholecystokinin-induced tension in Iguana iguana gallbladder strips.

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the iguana, Iguana iguana, gallbladder using immunocytochemistry. An intense reaction was observed in nerves located in the smooth muscle layers and associated blood vessels but no immunoreactive cell bodies were found. In vitro tension studies using gallbladder strips showed that chicken CGRP was more potent in relaxing cholecystokinin-induced tension than either human or rat CGRP. The use of glibenclamide and L-NG-nitro-arginine methyl ester suggested that this relaxation is mediated by either nitric oxide release from nerves stimulated by CGRP or by CGRP acting directly on the gallbladder smooth muscle.

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.

Calcitonin gene related peptide relaxes cholecystokinin-induced contraction in guinea pig gallbladder strips in vitro.

Calcitonin gene related peptide has been shown to relax vascular and intestinal smooth muscle. This study examines the effects of calcitonin gene related peptide on cholecystokinin-induced contraction of guinea pig gallbladder strips in vitro. Calcitonin gene related peptide was found to cause a dose-dependent relaxation of cholecystokinin-induced tension, which was blocked by the calcitonin gene related peptide receptor antagonist human calcitonin gene related peptide. Previous studies demonstrated that calcitonin gene related peptide acted directly on guinea pig gallbladder smooth muscle to inhibit acetylcholine- or KCl-induced contraction. The present results further confirm that calcitonin gene related peptide acts directly on the smooth muscle. In addition, the use of L-NG-nitroarginine methyl ester, glibenclamide, and other agents strongly suggests that calcitonin gene related peptide also acts by way of the nonadrenergic noncholinergic nervous system, to induce the relaxation of cholecystokinin-induced contraction observed in the guinea pig gallbladder strips.

Calcitonin gene-related peptide: an inhibitor of bullfrog (Rana catesbeiana) gallbladder contraction in vitro.

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the gallbladder of the bullfrog, Rana catesbeiana, using immunocytochemistry and confirmed by radioimmunoassay. An intense immunocytochemical reaction was observed in nerves located in the smooth muscle layers and associated with blood vessels. No immunoreactive nerve fibers were associated with ganglia, nor were immunoreactive cell bodies observed. Radioimmunoassay showed that 25.03 +/- 2.5 pmol/g tissue of CGRP-like material was present. In vitro tension studies using gallbladder strips showed that CGRP exerted an inhibitory effect on both acetylcholine- and cholecystokinin octapeptide-induced tension but had no effect on KCl-, norepinephrine-, or cerulein-induced tension. CGRP may act directly on the gallbladder smooth muscle to inhibit contraction.

Calcitonin gene-related peptide: an inhibitor of guinea pig gallbladder contraction.

Calcitonin gene-related peptide (CGRP) relaxes vascular and intestinal smooth muscle. This study localized CGRP in the guinea pig gallbladder, examined the effects of CGRP on KCl- and ACh-induced contraction, and determined CGRPs site of action in the gallbladder. The gallbladder of male Hartley guinea pigs was used in in vitro tension studies, radioimmunoassay, or immunocytochemical studies. Radioimmunoassay showed that 8.0 +/- 0.5 pmol/g of immunoreactive CGRP was present. Immunocytochemistry demonstrated that immunoreactive-CGRP nerve fibers occurred around blood vessels, in gallbladder smooth muscle layers, and were associated with ganglia. No immunoreactive cell bodies were observed, even after colchicine treatment. The in vitro tension studies showed that CGRP inhibits either KCl- or acetylcholine-stimulated contraction. CGRP may in part act directly on the gallbladder smooth muscle to inhibit contraction.

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.

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

Calcitonin 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.

Localization of calcium regulatory hormones in fish.

Immunocytochemical localization of hypocalcin, a hypocalcemic factor in the corpuscles of Stannius (CS), in American eels was examined at the light (ABC method) and electron microscopic (protein A-gold technique) levels with the specific antiserum raised against purified rainbow trout hypocalcin. Only type 1 cells in the CS were immunoreactive in the light microscopic immunocytochemistry. At the electron microscopic level, however, hypocalcin immunoreactivity was observed in secretory granules of both type 1 and type 2 cells. Our findings may indicate that type 1 cells are the main source of hypocalcin, but that type 2 cells also produce it, suggesting that the presence of two cell types reflects different physiological conditions of a single cell type, rather than functionally different cell types.In addition, we summarize our recent data on the localization of other calcium regulatory, or putative calcium regulatory, hormones in fish: parathyroid hormone, calcitonin and calcitonin gene-related peptide.

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.

Determination of calcitonin in reptilian serum by heterologous radioimmunoassay.

A heterologous radioimmunoassay for reptilian calcitonin using a commercially available kit for human calcitonin was used to determine circulating levels and brain levels of calcitonin in the green iguana, Iguana iguana. Cross-reactivity between anti-human calcitonin antibodies and iguana plasma was demonstrated. Removal of reptilian calcitonin from the plasma with affinity chromatography eliminated the cross-reactivity. Assays on blood samples indicated normal circulating levels of calcitonin to range between 600-975 pg/ml. Young female iguanas had higher blood levels than males. The use of this assay permits more detailed study of the secretion of calcitonin in reptiles in response to appropriate stimuli.

Pulp response after calcitonin treatment of direct exposures in the dog.

The hypocalcemic hormone, calcitonin, was used in directly pulp-capped teeth to determine whether or not calcitonin promoted healing. Two dogs were used. Twelve teeth received the calcitonin; nine control teeth were prepared, and five were treated with calcium hydroxide. Calcitonin did not increase either the rate of healing or the amount of secondary dentin formed. Calcitonin reduced the degree of inflammation in the treated tooth pulps.

Tooth replacement and growth in the young green iguana, Iguana iguana.

A study using eight rapidly growing young green iguanas (Iguana iguana; initial mean weight 68.0 ± 3.8 gm) examined the changes in the wave replacement of teeth, the increased size of the teeth, and the posterior migration of tooth positions over a period of 16 weeks. The teeth increase in width as the lizards grow. The tooth positions shifted posteriorly, providing adequate space for the larger replacement teeth. These observations suggest that the wave replacement of teeth allows for growth of the dentition in length and height adequate to maintain tooth size in proportion to the overall size of the individual.