Tissue fibronectin is an endogenous ligand for galectin-1

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.     

The effect of nociceptin, an endogenous ligand for the ORL1 receptor, on rat colonic contraction and transit

To assess the role of ORL1 (opioid receptor-like 1) receptor in the bowel movement, we investigated the effect of nociceptin on colonic contraction and transit in rats. Nociceptin (0.1-100 nM) concentration-dependently caused an immediate tonic contraction followed by rhythmic waves of contractions in the isolated colon. The response to nociceptin (10 nM) was not affected by the classical opioid receptor antagonists, naloxone, naltrindole and nor-binaltorphimine. Suppression of effect of inhibitory neurotransmitters using pituitary adenylate cyclase activating polypeptide(6-38) (PACAP-(6-38); 3 microM), vasoactive intestinal polypeptide(10-28) (VIP-(10-28); 3 microM) and N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) did not influence the nociceptin-induced contractions. In anesthetized rats, intravenous administration of nociceptin (1 microg/kg) or morphine (1 mg/kg) caused phasic contractions in the proximal colon. Pretreatment with naloxone (300 microg/kg, i.v.) abolished the contractions induced by morphine, but not by nociceptin. The rate of large intestinal transit was dose-dependently accelerated by nociceptin (0.03-3 microg/kg, s.c.), but was retarded by morphine (1.7-5 mg/kg, s.c.). These results indicate that stimulation of ORL1 receptor accelerates the colonic contraction and transit independently from opioid receptors.     

Evidence for an endogenous cAMP-adenosine pathway in the rat kidney

In the rat kidney, exogenous adenosine-3'-5'-monophosphate (cAMP) is converted to adenosine via the metabolism of cAMP to adenosine-5'-monophosphate by phosphodiesterase and adenosine-5'-monophosphate to adenosine by 5'-nucleotidase. Our purpose was to investigate whether in the rat kidney adenosine is synthesized from endogenous cAMP via the same pathway. Rat kidneys were perfused with Tyrode's solution, and stabilized for 3 hr to minimize basal renal purine secretion. In control experiments (n = 6), the renal venous secretion rate of adenosine, inosine, hypoxanthine and Sigmapurines (adenosine + inosine + hypoxanthine) did not change over the two 10-min experimental periods. In contrast, the beta adrenoceptor agonist (+/-)-isoproterenol (1 and 10 microM added to the perfusate) caused a significant (1-factor analysis of variance with repeated measures; n = 31) increase in the renal venous secretion of adenosine (P <.0001), inosine (P <.0007), hypoxanthine (P <.0007) and Sigmapurines (P <.0001) as measured by high-performance liquid chromatography with ultraviolet detection. The Sigmapurines was the most discriminating index of isoproterenol-induced changes in purine release, and the renal venous secretion of Sigmapurines was significantly (2-factor analysis of variance with repeated measures) attenuated by inhibition of beta adrenoceptors with propranolol (.1 microM, n = 6; P <.05), phosphodiesterase with 3-isobutyl-1-methylxanthine (1 mM, n = 5; P <.002) and 5'-nucleotidase with alpha, beta-methyleneadenosine-5'-diphosphate (0.1 mM, n = 5; P <.03). Our data indicate that activation of beta adrenoceptors increases purine biosynthesis in the rat kidney via a mechanism that involves phosphodiesterase and 5'-nucleotidase. These results support the existence of an endogenous cAMP-adenosine pathway in the rat kidney.     

Inhibition of endogenous nitric oxide synthesis activates particulate guanylyl cyclase in the rat renal glomeruli

Nitric oxide (NO) plays a crucial role in the regulation of kidney function and metabolism. Our previous study showed that dexamethasone, one of several known selective inhibitors of inducible nitric oxide synthase (NOS), had a stimulatory effect on soluble guanylyl cyclase in the glomeruli of rat kidney. However, in the presence of dexamethasone, the atrial natriuretic factor (ANF)-dependent system remained suppressed. The aim of the present study was to investigate whether inhibition of synthesis of endogenous NO modulates the activity of the guanylyl cyclase system(s) in glomeruli. In these studies, rats were injected with a non-selective NOS inhibitor, N-omega-nitro-L-arginine methyl ester (NAME; NAME-group), or saline solution (controls; C-group). Creatinine clearance (C(Cr)), and plasma and urinary nitrate/nitrite (NOx-) levels decreased in the NAME-group, but plasma and urinary guanosine 3',5'-cyclic monophosphate (cGMP) contents were unchanged. In the presence of 0.1 microM ANF, synthesis of cGMP in the NAME-group exceeded threefold the cGMP production in the C-group. In addition, the pre-contracted glomeruli of the NAME-group were fully relaxed at 0.1 microM ANF, but glomeruli obtained from the C-group were relaxed in the presence of a 10 times higher dose of ANF. The increased sensitivity of glomeruli to ANF was possibly due to the more than doubled activity of particulate guanylyl cyclase (pGC) in the NAME-group in comparison with the C-group. In the presence of 100 microM sodium nitroprusside (SNP), soluble guanylyl cyclase (sGC) generated significantly lower cGMP production in the NAME-group than in the C-group (1.61 +/- 0.33 vs. 2.91 +/- 0.69 nmol/mg protein/10 min, respectively). These results demonstrate that inhibition of the synthesis of endogenous NO may also have an inhibitory effect on the activity of sGC. In addition, increased activity of the pGC and ANF-dependent system appears to be compensatory to the altered activity of soluble guanylyl cyclase.     

Evidence that guanylate cyclase is involved in modulating the resting tension and neurally-induced contraction of the guinea pig tracheal muscle

Electrical field stimulation of guinea-pig tracheal muscle strips produced a frequency-dependent biphasic response consisting of an initial cholinergic contraction followed by relaxation. Both phases of the response were of neural origin. In the presence of methylene blue, a guanylate cyclase inhibitor, the resting tension and the contraction were increased, but the accompanying relaxation was inhibited. However, in the presence of sodium nitroprusside, a guanylate cyclase activator, the resting tension was reduced and the contraction was inhibited, but the relaxation was prolonged and increased. Similarly, in the presence of either 3-isobutyl-1-methylxanthine, which promotes cyclic guanosine monophosphate (cGMP) accumulation, or 8-bromo-cGMP, an analogue of cGMP, the resting tension was reduced and the contraction was inhibited but the relaxation was prolonged and increased. From these results, it is concluded that guanylate cyclase is involved in modulating the resting tension and the neurally-induced contraction of guinea-pig tracheal muscle.     

Fas ligand is constitutively secreted by prostate cancer cells in vitro

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.     

Noxious stimulation decreases substance P binding in rat spinal dorsal horn: competition by endogenous ligand?

To determine the participation of NK-1 receptors in mediating inputs from noxious thermal stimulation, sustained noxious stimulation was applied to anaesthetized rats by immersing one hind paw in water for 1.5 min at different temperatures. After sacrificing the animal, lumbar spinal cords were removed and 20 microns sections were incubated with [125I]BH-substance P. Compared with unstimulated controls, binding was the least in rats given a 55 degrees C stimulus and sacrificed 1 min after the stimulus; the greatest reduction was found in the superficial dorsal horn. Rats sacrificed at 10 min showed intermediate binding levels. Groups given less intense stimuli showed smaller decreases in binding. It is suggested that the decrease in binding was due to occupation of receptors by endogenous ligand, which is consistent with the idea that noxious stimulation evokes the release of substance P at the spinal level.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Inter-regional trade in live fish as eggs, larvae or juveniles provides the potential for parallel movements of pathogens. Pathogens that exist in a carrier state and/or can be transmitted by vertical means pose the greatest threat since casual observation, and even periods of quarantine or pathogen inspections, may fail to indicate their presence. Additional complications arise with the movements of non-target species for which health examinations may not be required, or for which criteria for pathogen inspections have not been developed. Although international trade in salmonids has been responsible for most of the disease regulations currently in place, an equal or stronger effort should be expected with other species. At the same time, ensuring equal treatment of all trading partners with respect to the level and sophistication of the health examinations to which the product will be subjected is a major problem. There are several examples of past and potential pathogen movements with fish or fish products. Unfortunately, these are often confused by a poor understanding of the current situation in the region into which the animal or product has been imported. The technology, experience or extent of surveillance in the importing region may be insufficient to assess the situation. Distinguishing between exotic imported pathogens and unknown pathogens which are already present in indigenous fish stocks can therefore often be difficult. The author discusses examples of clearly-documented imports of pathogens, as well as the potential for the spread of agents which pose an equal or greater danger. In addition, the author discusses the confusion which often arises when the background into which these pathogens are to move is poorly understood in the importing region.     

Influence of a selective guanylate cyclase inhibitor, and of the contraction level, on nitrergic relaxations in the gastric fundus

1. The influence of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) on non-adrenergic non-cholinergic (NANC) relaxations and the possible role of a nerve-derived hyperpolarizing factor in NANC relaxation were investigated in the rat gastric fundus. 2. ODQ (10(-6) and 10(-5) M) concentration-dependently inhibited the short-lasting relaxations by NO (2 x 10(-6) M-10(-4) M) administered as a bolus without influencing the relaxation by 3 x 10(-8) M isoprenaline. The relaxation by an infusion of NO was reduced to the same extent by 10(-6) and 10(-5) M ODQ. 3. The electrically induced short-lasting and sustained relaxations (40 V, 1 ms, 0.5-16 Hz, 10 s trains at 2 min interval or cumulative increase in the frequency every 2 min) in NANC conditions were inhibited to a similar extent by 10(-6) and 10(-5) M ODQ, and by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-4) M). 4. ODQ (10(-6) M) and L-NAME (3 x 10(-4) M), administered after 5, 10 or 20 min of long-term stimulation, reversed the relaxation to a similar extent (approximately 50% at 2 Hz and 20% at 8 Hz). 5. When the tissues were contracted to 40% of maximum by adapting the concentration of prostaglandin F2alpha (PGF2alpha), the inhibitory effect of 3 x 10(-4) M L-NAME on relaxations induced by train and cumulative stimulation was the same as when tissues were contracted with 3 x 10(-7) M PGF2alpha. 6. The findings of this study illustrate that the relaxation by exogenous and endogenous NO in the rat gastric fundus is due to activation of soluble guanylate cyclase. During long-term electrical stimulation, the partial contribution of NO to NANC relaxation is maintained but it is small at higher frequencies of stimulation. Evidence for the contribution of a nerve-derived hyperpolarizing factor to NANC relaxation was not obtained.     

Inhibitors of NO-synthase and guanylate cyclase block the modulation of cholinoreceptor plasticity in snail neurons by 15-hydroxyeicosatetraenoic acid

Probable participation of two second messengers, NO and cGMP, in short- and long-latent effects of 15(S)-hydroxy-(5Z,8Z,11Z, 13E)-eicosatetraenoic acid (15-HETE, acyclic eicosanoid) on plasticity of somatic acetylcholine receptors on identified neurons of Helix lucorum was studied using two-electrode voltage clamp technique. It was shown that inhibitor of NO synthase N omega-Methyl-L-Arginine and inhibitors of soluble guanylate cyclase LY-83,583 and methylene blue disturbed short- and long-latent modulatory effects of 15-HETE on depression depth of the inward current evoked by rhythmic acetylcholine application on the neuron soma. We suppose that NO and cGMP participate in short- and long-latent effects of 15-HETE on plasticity of acetylcholine receptors.     

A capsaicin-receptor antagonist, capsazepine, reduces inflammation-induced hyperalgesic responses in the rat: evidence for an endogenous capsaicin-like substance

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the beta subunit-dependent differences in the receptor affinity for substance P.     

Anandamide, an endogenous cannabinomimetic substance, modulates rat brain protein kinase C in vitro

Anandamide (AnNH, N-arachidonoyl-ethanolamine) has been recently proposed as the endogenous ligand for mammalian brain cannabinoid receptor. Non-cannabinoid receptor-mediated, intracellular actions have been also found for this novel mediator. Here we present evidence for the modulation by anandamide of rat brain protein kinase C (PKC) activity in vitro. The ethanolamide of arachidonic acid (AA) was more active than the free acid in increasing phosphatidylserine (PS)-induced PKC activation (EC50 = 40 microM), but inhibited dioleylglycerol-induced potentiation of both Ca(2+)- and Ca2+/PS-induced PKC activation (IC50 = 8 microM and 30 microM, respectively). A dual modulatory action of anandamide on PKC, exerted by binding to the diacylglycerol regulatory site, is hypothesized in rat brain.     

Circulatory, respiratory and acid-base balance changes produced by anesthetics in the rat

14C-D,L-verapamil was administered intravenously (10 mg) and orally (80 mg) to five volunteer patients. Plasma concentrations of verapamil, which were determined by mass fragmentography, declined bi-exponentially with half-lives of the chi-phase ranging from 18 to 35 min and of the beta-phase from 170 to 440 min. The apparent volume of distribution ranged from 270 to 460 litre and plasma clearance from 730 to 1980 ml/min. Following oral administration absorption was almost complete as judged from the area under the curve (AUC) of 14C-activity and cumulative urinary excretion of 14C. After intravenous infusion of verapamil about 80% of the radioactivity administered could be recovered in urine and faeces within 5 d. Despite its almost complete absorption after oral administration verapamil was shown to undergo extensive first pass metabolism as the bioavailability was only 10 to 22%. Rapid biotransformation had occurred as only a small percentage of AUC of 14C was seen to correspond to unchanged verapamil after both intravenous and oral administration.     

Transfer effects produced by overtraining in the rat.

In each of 2 experiments, 2 groups of 12 male hooded rats were trained on an orientation discrimination in the jumping stand, one group (CT) being trained to criterion and the other being given 150 trials of overtraining (OT). In Exp I the 2 groups did not differ when shifted to a black-white discrimination, but the OT group was superior in learning the reversal of the black-white problem. This result suggests that the beneficial transfer resulting from overtraining does not derive from an increased tendency for the OT Ss to attend to the overtrained dimension. Exp II tested the hypothesis that overtraining produces positive transfer by reducing the Ss' tendency to take up position habits. The OT and CT groups were presented with a choice between oblique stimuli with neither alternative being rewarded. Both groups took up position habits, the OT slightly more rapidly than the CT, and thus the hypothesis was not confirmed. (15 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Leptin is an endogenous protective protein against the toxicity exerted by tumor necrosis factor

Decrements in muscle strength as a result of prolonged bed rest are well defined, but little is known about potential countermeasures for preventing loss of strength under this condition. The purpose of this study was to determine whether testosterone administration would preserve protein balance and muscle strength during prolonged bed rest. Ten healthy men (age, 36 +/- 2 yr; height, 177.2 +/- 3.4 cm; weight, 80.5 +/- 3.9 kg; mean +/- SE) were admitted to our in-patient metabolic unit. After a 1-week ambulatory run-in period, each subject was confined to bed for 28 days at 6 degree head-down tilt while receiving a daily oral dose of T3 (50 microg/day). During the bed rest/T3 period, six of the men were randomized to receive testosterone enanthate by i.m. injection (T; 200 mg/week) while four received placebo in a double blind fashion. Nitrogen balance was determined throughout, and whole body [13C]leucine kinetics were assessed at baseline and on day 26 of bed rest. Before bed rest and on the third day of reambulation, the muscle strength of the knee extensors and flexors and shoulder extensors and flexors was determined at 60 degrees/s on a Cybex isokinetic dynamometer. Despite improved [13C]leucine kinetics and maintenance of nitrogen balance and lean body mass in T-treated subjects, little preservation of muscle strength, particularly in the knee extensors, was noted. Muscle strength [reported as the best work repetition in foot-pounds (FtLb)] for right knee extensors declined (P = 0.011) similarly in both groups; from 165 +/- 15 to 126 +/- 18 FtLb in T-treated men and from 179 +/- 22 to 149 +/- 13 FtLb in placebo-treated men. Overall, there was less of a decline in extension and flexion strength of the shoulder compared to the knee, with no benefit from T. These results suggest that in the absence of daily ambulatory activity, T administration will not increase or, in the case of this bed rest model, preserve muscle strength.