Action of calcitonin gene-related peptide at the calcitonin receptor of the T47D cell line

Some effects of calcitonin (CT) can also be produced by calcitonin gene-related peptide (CGRP), an alternative product of the calcitonin gene. This might be mediated by interaction of CGRP at the CT-receptor site. The human breast cancer cell line T47D possesses well characterized CT-receptors (KD = 2.3 x 10(-10) M for 125I salmon CT). 50% inhibition of 125I-sCT binding was achieved with 10(-9) M sCT, 5 x 10(-6) M rat CGRP and 10(-5) M human CGRP. Half maximal cAMP production in T47D cells was seen with 6 x 10(-10) M sCT, 5 x 10(-6) M rCGRP and 10(-5) M hCGRP. Binding and displacement capacity as well as the biological activity of CT and CGRP seems to correlate well. These findings suggest that CGRP in pharmacological doses acts via the CT-receptor. This could be explained by the homology and conformational similarities between CT and CGRP.     

Effects of calcitonin gene-related peptide on neuromuscular transmission in the isolated rat diaphragm

The present study was undertaken to investigate a possible interaction between the cholinergic nerve neurotransmitter and CGRP on neuromuscular transmission in the isolated rat diaphragm. Electrical stimulation of the isolated phrenic nerve resulted in twitch contractions which were dose-dependently potentiated by CGRP in concentrations ranging from 1.2 x 10(-9) M to 3 x 10(-7) M. The potentiating action of CGRP (3 x 10(-7) M) disappeared in about 25 min. The same dose of CGRP 40 min later produced an augmentation of contraction amplitude similar to that observed prior to the administration of CGRP. The action of CGRP was dependent upon the stimulation pulse width ranging from 0.2 to 1.0 msec. Rat calcitonin (4.5 x 10(-7) M) caused a minimal change in the amplitude of twitch contractions. CGRP had no effect on the quiescent striated muscle. Twitch responses to direct electrical stimulation were also enhanced by CGRP (6 x 10(-8) M-6 x 10(-7) M) in the absence and presence of 10(-5) M d-tubocurarine. These results suggest that CGRP modulates the action of acetylcholine at the motor end plates of striated muscle.     

Modulation of cholinergic neurotransmitter release from myenteric plexus by somatostatin

Efflux of radiolabeled acetylcholine (Ach) was studied in vitro using myenteric plexus-longitudinal muscle strips from guinea pig small intestine. The data showed that somatostatin (6.0 x 10(-7) M) depressed resting output of Ach from enteric neurons and this inhibition was unaltered in the presence of naloxone (1.0 x 10(-6) M). The inhibition by somatostatin on field-stimulated Ach release was dose-dependent but this inhibition was never complete; there was a 40% fraction of total release remained resistant to somatostatin. Both caerulein (2.85 x 10(-9) M) and guanidine (3.0 x 10(-3) M) stimulated release of [H3]-Ach from plexus neurons. The release of Ach induced by guanidine or caerulein was also susceptible to inhibition by somatostatin (6.0 x 10(-7) M). This study provides functional evidence to further substantiate an inhibitory action on plexus cholinergic neurons by somatostatin.     

Suppression of insulin-stimulated glucose transport in L6 myocytes by calcitonin gene-related peptide

The binding of calcitonin gene-related peptide (CGRP) to L6 myocytes, the coupling of this receptor to adenylyl cyclase and the resultant effects on insulin-stimulated 2-deoxyglucose uptake were examined. L6 cells express specific binding sites for CGRP. Binding of human [125I]CGRP was inhibited by rat CGRP with an IC50 of approximately 10(-9) M. Synthetic human calcitonin at concentrations up to 10(-6) M had no effect on the binding of CGRP, suggesting that L6 cells express CGRP receptors, rather than calcitonin receptors which are also capable of binding CGRP. The CGRP receptor appeared to be coupled to adenylyl cyclase. Concentrations of CGRP greater than 3 x 10(-9) M increased the cellular content of cAMP. At 3 x 10(-8) M, CGRP increased cAMP 500-fold. CGRP at 10(-10) M and above suppressed the stimulation of 2-deoxyglucose uptake by insulin. Acute incubation of L6 cells with insulin stimulated 2-deoxyglucose uptake 1.6-fold, which was inhibited up to 70% by CGRP. Our results demonstrate that the specific binding of CGRP to L6 cells causes large increase in the cellular content of cAMP - and inhibition of insulin-stimulated 2-deoxyglucose uptake, but the differences in the dose-response curves suggest that the suppression of insulin action by CGRP cannot be solely explained by the increase in cAMP.     

Assessment of radiation exposure level for hydrobionts in some special industrial ponds at the "Mayak" PA

Evaluation of the radionuclide content in the ecosystem components (water, sediments, aquatic organisms) of industrial reservoirs-storages of liquid radioactive waste of the "Mayak" PA (reservoirs R-4, R-10, R-11, R-17, R-9) and the estimation of the absorbed dose rate in aquatic organisms of these reservoirs using the software package ERICA Assessment Tool 1.0 May 2009 have been performed. Gradient of the absorbed dose rate for the detected taxonomic groups of hydrobionts in the series of the studied reservoirs R-11 --> R-10 --> R-4 --> R-17 --> R-9 was almost equal to one order of magnitude. The estimated absorbed dose rate for phytoplankton ranged from 5.4 x 10(0) mGy/day (R-11) to 4.0 x 10(4) mGy/day (R-9), for zooplankton--from 6.4 x 10(-1) mGy/day (R-11) to 3.8 x 10(3) mGy/day (R-9), for zoobenthos (chironomids)--from 5.6 x 10(0) mGy/day (R-11) to 1.1 x 10(3) mGy/day (R-17), for fish (roach)--from 8.0 x 10(-1) mGy/day (R-11) to 1.9 x 10(1) mGy/day (R-4).     

Functional interaction between transforming growth factor alpha and capsaicin-sensitive sensory neurons in the rat stomach

BACKGROUND AND AIMS: Transforming growth alpha (TGFalpha) and sensory neurons have been shown to promote gastric mucosal protection and healing. Aims were to examine in vitro interactions between gastric sensory neurons, the sensory neuropeptide calcitonin gene-related peptide (CGRP), and TGFalpha. METHODS: Gastric mucosal/submucosal tissue fragments from Sprague-Dawley (SD) rats were incubated in short-term (30 min) culture. Peptide release into media and TGFalpha tissue content were measured by radioimmunoassay. RESULTS: TGFalpha (1 x 10(-8) to 1 x 10(-6) M) caused dose-dependent stimulation of CGRP release. Maximal CGRP release (+87%) was observed with 1 x 10(-6) M TGFalpha: 28.6+/-3.8 vs. control of 15.5+/-2.7 pg/g tissue; P<0.05. Both CGRP (1 x 10(-7) to 1 x 10(-5) M) and capsaicin (1 x 10-(8) to 1 x 10(-6)M) significantly inhibited basal TGFalpha release in a dose-dependent fashion that ranged from -20% to -39%. In contrast, capsaicin-induced sensory denervation caused significant increases in both basal TGFalpha release and TGFalpha tissue content. CONCLUSION: Function interactions between TGFalpha and gastric sensory neurons are suggested by the observations that (1) TGFalpha stimulated CGRP release from gastric sensory neurons; (2) CGRP and acute capsaicin treatment inhibited TGFalpha release and; (3) capsaicin-induced sensory denervation caused significant increases in both gastric TGFalpha basal release and tissue content.     

Characterization of functional receptors for somatostatin in rat pituitary cells in culture.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.     

Mechanisms of proton-induced stimulation of CGRP release from rat antrum

Mechanisms of acid-evoked CGRP release from gastric afferent nerves were investigated in rat antral mucosal/submucosal tissues. Low pH (pH 4.0, 5.0 and 6.0) stimulated antral CGRP release significantly and dose-dependently from rat antral fragments. Removal of extracellular calcium from the incubation medium resulted in significant inhibition (59%, P < 0.001) of acid (pH 4.0)-stimulated CGRP release. Conotoxin (1 x 10(-7) M), the selective blocker of N-type calcium channels, also significantly inhibited proton (pH 4.0)-induced CGRP release to values that were 74% below net stimulated levels. Neither nifedipine (1 x 10(-6) M), the L-type Ca(2+)-channel antagonist, nor indomethacin (1 x 10(-5) M), inhibitor of prostaglandin synthesis, altered acid-induced CGRP release. In contrast, ruthenium red (1 x 10(-5) M), capsaicin antagonist, almost completely prevented acid (pH 4.0)-stimulated CGRP release. Capsazepine (1 x 10(-4) M), a specific capsaicin receptor antagonist, also completely abolished acid-induced CGRP release. In conclusion, the results of these studies indicate that hydrogen ions are capable of evoking CGRP release from peripheral sensory neurons in rat antral mucosal/submucosal tissues. Proton-evoked CGRP release requires extracellular calcium and involves N-type calcium channels. Furthermore, acid appears to exert a capsaicin-like effect to evoke sensory neuropeptide release that is sensitive to capsazepine and ruthenium red. These data suggest that proton-induced antral CGRP release represents a direct action of hydrogen ions on mucosal/submucosal sensory dendritic nerve endings to effect local release of neuropeptide.     

N-oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRP8-37 (1 x 10(-6) M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRP8-37, RP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.     

Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity

The calcitonin peptides [calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin] share many biological actions, including activity on bone cells. In the present study, CT (10(-11) to 10(-9) M) stimulated [(3)H]thymidine incorporation in primary cultures of human osteoblasts (hOB), as already demonstrated for CGRP and amylin. RT-PCR analysis showed that the calcitonin receptor and the calcitonin receptor-like receptor are both expressed in hOB. In these cells, CT (10(-10) M) and amylin (10(-9) M), in contrast to CGRP (10(-8) M), did not increase cAMP production. All three peptides stimulated protein kinase C (PKC) activity. To evaluate PKC involvement in hOB proliferation, cells were incubated with phorbol 12,13-dibutyrate, a stimulator of PKC activity; cell proliferation was increased in a dose-dependent manner (EC(50) = 3.4 x 10(-8) M). Staurosporine (10(-9) M), a PKC inhibitor, blocked phorbol 12,13-dibutyrate-induced PKC activity and cell proliferation. Inhibition of PKC by staurosporine also counteracted the stimulatory effect of CT, CGRP, and amylin on hOB proliferation. From these data, it is deduced that the activation of PKC is important for hOB proliferation and that it is involved in the anabolic effect of CT peptides on bone.     

Localisation and neural control of the release of calcitonin gene-related peptide (CGRP) from the isolated perfused porcine ileum

By immunohistochemistry, CGRP-like immunoreactive (CGRP-LI) nerve fibres were found in the lamina propria along small vessels and in the lamina muscularis mucosae in the porcine ileum. Immunoreactive nerve cell bodies were found in the submucous and myenteric plexus. Upon HPLC-analysis of ileal extracts, CGRP-LI corresponded entirely to porcine CGRP plus smaller amounts of oxidised CGRP. Using isolated vascularly perfused segments of the ileum, we studied the release of CGRP-LI in response to electrical stimulation of the mixed extrinsic periarterial nerves and to infusion of different neuroblockers. In addition, the effect of infusion of capsaicin was studied. The basal output of CGRP-LI was 2.9+/-0.7 pmol/5 min (mean+/-S.D.). Electrical nerve stimulation (8 Hz) significantly increased the release of CGRP-LI to 167+/-16% (mean+/-S.E.M.) of the basal output (n=13). This response was unaffected by the addition of atropine (10(-6) M). Nerve stimulation during infusion of phentolamine (10(-5) M) with and without additional infusion of atropine resulted in a significant further increase in the release of CGRP-LI to 261+/-134% (n=5) and 240+/-80% (n=9), respectively. This response was abolished by infusion of hexamethonium (3x10(-5) M). Infusion of capsaicin (10(-5) M) caused a significant increase in the release of CGRP-LI to 485+/-82% of basal output (n=5). Our results suggest a dual origin of CGRP innervation of the porcine ileum (intrinsic and extrinsic). The intrinsic CGRP neurons receive excitatory input by parasympathetic, possibly vagal, preganglionic fibres, via release of acetylcholine acting on nicotinic receptors. The stimulatory effect of capsaicin suggests that CGRP is also released from extrinsic sensory neurons.     

The effect of adrenomedullin,amylin fragment 8-37 and calcitonin gene-related peptide on contractile force,heart rate and coronary perfusion pressure in isolated rat hearts

The effect of human adrenomedullin, human amylin fragment 8-37 (amylin 8-37) and rat calcitonin gene-related peptide (CGRP) on contractile force, heart rate and coronary perfusion pressure has been investigated in the isolated perfused rat hearts. Adrenomedullin (2x10(-10), 2x10(-9) and 2x10(-8) M) produced a significant decrease in contractile force and perfusion pressure, but only the peptide caused a decline in heart rate at the highest dose. Amylin (10(-9), 10(-8) and 10(-7) M) significantly increased and then decreased contractile force. Two doses of amylin (10(-8) and 10(-7) M) induced a significant increase in heart rate, however amylin did not change perfusion pressure in all the doses used. Rat alpha CGRP (10(-8), 10(-7) and 10(-6) M) evoked a slight decline in contractile force following a significant increase in contractile force induced by the peptide. CGRP in all the doses raised heart rate and lowered perfusion pressure. Our results suggest that adrenomedullin has negative inotropic, negative chronotropic and coronary vasodilator actions. Amylin produces a biphasic inotropic effect and evokes a positive chronotropy. CGRP causes positive inotropic, positive chronotropic and vasodilatory effects in isolated rat hearts.     

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.     

Protection of calcitonin gene-related peptide-mediated preconditioning against coronary endothelial dysfunction induced by reperfusion in the isolated rat heart

This study was designed to explore the protective effect of ischemic preconditioning on reperfusion-induced coronary endothelial dysfunction, with a focus on the role of calcitonin gene-related peptide (CGRP) in this effect, in the isolated perfused rat heart. Thirty minutes of global ischemia and 30 min of reperfusion significantly decreased heart rate, left ventricular pressure, and its first derivative and impaired vasodilator responses to acetylcholine. Ischemia-reperfusion did not affect vasodilator responses to sodium nitroprusside. Preconditioning induced by three cycles of 5 min of ischemia and 5 min of reperfusion produced a significant improvement in cardiac function concomitantly with an amelioration of vasodilator responses to acetylcholine. The protective effects of ischemic preconditioning were abolished by CGRP(8-37) (10(-7) M) , the selective CGRP receptor antagonist. After pretreatment with capsaicin (50 mg x kg(-1), s.c.) to deplete endogenous CGRP, the preconditioning effect was absent. Pretreatment with exogenous CGRP (5 x 10(-9) M) for 5 min induced a preconditioning-like protection. The present study suggests that the cardioprotection of ischemic preconditioning is related to the preservation of the coronary endothelial cell, and that the protective effect of preconditioning is mediated by endogenous CGRP in the isolated perfused rat heart.     

Adrenergic receptors are not mediators in the lipolytic effect of somatostatin in rat adipocytes

Beta-adrenergic receptors have been purported to act as possible mediators in the lipolytic effect of somatostatin in vivo. Investigations with isolated rat adipocytes studying the lipolytic activity of somatostatin (1.7 x 10(-7) M), glucagon (8.1 x 10(-8 M) and norepinephrine (10(-6) M), have shown that the lipolytic effect stimulated by somatostatin is not altered by 10(-5) M propranolol (beta-antagonist); is significantly enhanced by 10(-5) M isoproterenol (beta-agonist) and is not altered by the addition of 10(-6) M phenoxybenzamine (alpha-antagonist) or 10(-6) M phenylephrine (alpha-agonist). Similar results were found when lipolysis was stimulated by glucagon, whereas the lipolytic effect stimulated by norepinephrine was blocked by propranolol. These results indicate that the direct lipolytic effect of somatostatin on isolated rat adipocytes does not seem to be mediated through mechanisms involved with adrenergic receptors.     

Effect of calcitonin gene-related peptide on glucose and gastric inhibitory polypeptide-stimulated insulin release from cultured newborn and adult rat islet cells

Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that is present in peripheral cells of islets and in nerves around and within islets. CGRP can inhibit insulin secretion in vitro and in vivo. Whether the inhibitory action of CGRP is mediated by somatostatin or by nerve terminals is, however, not known. The objective of this study was to examine the effect of CGRP on insulin secretion, using cultured newborn and adult rat islet cells which did not contain nerve terminals. In adult rat islet cells, CGRP (10(-10) to 10(-8) M) significantly inhibited glucose-stimulated and gastric inhibitory polypeptide (GIP)-potentiated insulin secretion, but in newborn rat islet cells, CGRP did not inhibit glucose-stimulated insulin secretion. Inhibition of glucose-stimulated and GIP-potentiated insulin release was dependent on the glucose concentration during the prestimulation period. CGRP did not stimulate release of somatostatin. These findings suggest that rat CGRP can act directly on beta-cells through a specific receptor that is absent in newborn rat beta-cells.     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.     

Characterization of binding sites for calcitonin gene related peptide in abalone gill.

Target organs for calcitonin gene related peptide were investigated in the abalone. To elucidate the function of this neuropeptide in the biomineralization process, we have localized, in different tissues from abalone, specific binding sites for human calcitonin gene related peptide (hCGRP). Highest binding was observed in gill membranes where two classes of affinity components were identified. The affinity constants and the number of binding sites per mg of proteins for the site I were 5 X 10(9)M(-1)and 8.8 x 10(10). For the site II, the affinity constant was 1.34 X 10(7) M(-1)and the number of binding sites per mg of proteins was 1.1 x 10(12). In contrast, no specific calcitonin binding could be detected in every tested tissue, but the similar displacement of the 125I-labeled CGRP binding with unlabeled hCGRP and sCT suggested that, in the abalone, the identified receptor could belong to a third class receptor subtype, that recognize both hCGRPalpha and sCT. These data suggest that, during evolution, the role of CGRP in gill function is particularly well conserved and that this neuropeptide is likely to participate in the control of hydromineral metabolism in aquatic species.     

The effects of somatostatin on the arachidonate cascade of platelets

Somatostatin (10(-9) M) significantly elevated the synthesis of thromboxane B2 in rat platelets. The transformation of arachidonic acid to active lipoxygenase metabolites was suppressed by somatostatin (10(-9) and 10(-8) M). The ratio of the lipoxygenase/cyclooxygenase products was significantly reduced by the polypeptide (10(-9) and 10(-8) M) in rat platelets. Higher concentrations (10(-7), 10(-6) and 10(-5) M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor - proaggregatory cyclooxygenase products was stimulated by the polypeptide (10(-9) and 10(-8) M), while the formation of vasodilatator - antiaggregatory cyclooxygenase metabolites was induced by higher concentrations of somatostatin (10(-7) and 10(-6) M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose.     

Light- and electron-microscopical localization of calcitonin,calcitonin gene-related peptide,somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Summary Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light-and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.