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Biomedical subjects

S Bevan

Publications and source records attributed to S Bevan.

At least 73 records · Page 4Linked to original sources
PubMed1995

Chromogranin A.

Chromogranin A (CgA) is the major member of the granin family of acidic secretory glycoproteins that are expressed in all endocrine and neuroendocrine cells. Granins have been proposed to play multiple roles in the secretory process. Intracellularly, granins play a role in targeting peptide hormones and neurotransmitters to granules of the regulated pathway by virtue of their ability to aggregate in the low-pH, high-calcium environment of the trans-Golgi network. Extra-cellularly, peptides formed as a result of proteolytic processing of granins regulate hormone secretion. Some conserved features of the mature CgA protein are polyglutamic acids, calcium-binding sites, and several pairs of basic amino acids. The first 2 features are important for its intracellular functions, and the latter characteristic suggested that peptides could be released from the molecule by precursor processing enzymes. Several biologically active peptides encoded within the CgA molecule, such as vasostatin, beta-granin, chromostatin, pancreastatin, and parastatin act predominantly to inhibit hormone and neurotransmitter release in an autocrine or paracrine fashion. The biosynthesis of CgA is regulated by many different factors, including steroid hormones and agents that act through a variety of signalling pathways. CgA biosynthesis and that of the resident hormone or neurotransmitter can be regulated differentially. The widespread distribution of CgA has made the measurement of circulating immunoreactive CgA a valuable tool in the diagnosis of neuroendocrine neoplasia, and CgA immunohistochemistry can help to identify the neuroendocrine nature of tumours. Recent molecular biology studies are identifying those elements in the CgA gene promoter responsible for its specific neuroendocrine cell expression.

Animals↗
PubMed1994

The discovery of capsazepine, the first competitive antagonist of the sensory neuron excitants capsaicin and resiniferatoxin.

Capsaicin and resiniferatoxin are natural products which act specifically on a subset of primary afferent sensory neurons to open a novel cation-selective ion channel in the plasma membrane. These sensory neurons are involved in nociception, and so, these agents are targets for the design of a novel class of analgesics. Although synthetic agonists at the capsaicin receptor have been described previously, competitive antagonists at this receptor would be interesting and novel pharmacological agents. Structure-activity relationships for capsaicin agonists have previously been rationalized, by ourselves and others, by dividing the capsaicin molecule into three regions--the A (aromatic ring)-, B (amide bond)-, and C (hydrophobic side chain)-regions. In this study, the effects on biological activity of conformational constraint of the A-region with respect to the B-region are discussed. Conformational constraint was achieved by the introduction of saturated ring systems of different sizes. The resulting compounds provided agonists of comparable potency to unconstrained analogues as well as a moderately potent antagonist, capsazepine. This compound is the first competitive antagonist of capsaicin and resiniferatoxin to be described and is active in various systems, in vitro and in vivo. It has recently attracted considerable interest as a tool for dissecting the mechanisms by which capsaicin analogues evoke their effects. NMR spectroscopy and X-ray crystallography experiments, as well as molecular modeling techniques, were used to study the conformational behavior of a representative constrained agonist and antagonist. The conformation of the saturated ring contraint in the two cases was found to differ markedly, dramatically affecting the relative disposition of the A-ring and B-region pharmacophores. In agonist structures, the A- and B-regions were virtually coplanar in contrast to those in the antagonist, in which they were approximately orthogonal. A rationale for agonist and antagonist activity at the capsaicin receptor is proposed, based on the consideration of these conformational differences.

Animals↗
PubMed1994

Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression.

Chromogranin A (CgA) is an acidic glycoprotein, which is widely expressed in endocrine and neuroendocrine cells. It plays multiple important roles in the process of regulated hormone secretion. The single copy human CgA gene was isolated from a human fetal liver gene library. The gene spans 15 kilobases and contains 8 exons. Exon I encodes the 5'-noncoding region and the majority of the signal peptide coding region. Exons II-V collectively encode the highly conserved amino-terminal domain (the beta-granin sequence). Exon VI encodes a variable domain within which is the chromostatin sequence, and exon VII encodes another variable domain, which contains the pancreastatin sequence. Exon VIII encodes the highly conserved carboxyl-terminal domain and the 3'-noncoding region. The human gene promoter has a consensus TATA box, cAMP response element, and Sp-I sequence. 2.3 kilobases of the upstream regulatory region of the human CgA gene directed efficient transcription of a reporter chloramphenicol acetyltransferase gene in several neuroendocrine cell lines, including human medullary thyroid C-cell tumor, mouse pituitary corticotroph, rat pituitary tumor, and rat pheochromocytoma. The promoter was virtually inactive in nonneuroendocrine cell lines. Transient transfection studies with deleted promoter constructs showed that sequences lying between -55 and +32 base pairs relative to the transcription initiation site, containing the consensus cyclic AMP response element and TATA box, were sufficient for neuroendocrine cell-specific expression.

Amino Acid Sequence↗
PubMed1994

Protons: small stimulants of capsaicin-sensitive sensory nerves.

The data reviewed in this article suggest that protons should no longer be considered simply as an unwanted by-product of anaerobic respiration that results from either an accumulation of inflammatory cells or a reduced oxygenated blood supply during ischaemia. A fall in extracellular pH can stimulate a subpopulation of sensory nerves by activation of ion channels. The available evidence indicates that most, if not all, of the activated neurones are also stimulated by capsaicin, and that protons and capsaicin share a common mechanism of neuronal activation. A proton should be viewed as a mediator that elicits a protective response with reflex cardiovascular and respiratory responses, which modulate systemic blood flow, and with the local release of sensory neuropeptides, which vasodilates the microvasculature and stimulates extravasation.

Animals↗
PubMed1993

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 1. The aromatic "A-region".

A series of analogues of capsaicin, the pungent principle of chilli peppers, was synthesized and tested in assays for capsaicin-like agonism in vitro. The results of these assays were compared with activities in an acute nociceptive model and a correlation was observed which established that the results of these in vitro assays were predictive of analgesia. Using a modular approach the structure-activity profile of specific regions of capsaicin congeners was established using an in vitro assay measuring 45Ca2+ uptake into neonatal rat dorsal root ganglia neurones. Substituted benzylnonanamides 2a-z and N-octyl-substituted phenylacetamides 4a-v were made to test the requirements for activity in the aromatic "A-region" of the molecule. Compounds with the natural substitution pattern (2b and 4c) and the corresponding catechols (2i and 4g) were the most potent, although the catechols were less potent in vivo. Other substitution patterns have reduced activity. These results have established stringent structural requirements for capsaicin-like activity in this part of the molecule.

Analgesics↗
PubMed1993

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 2. The amide bond "B-region".

A series of compounds incorporating replacements for the amide bond "B-region" moiety of capsaicin have been synthesized, including vanillylamides and esters, homovanillic acid amides and esters, ureas, and thioureas. These have been tested in an in vitro assay for agonism (45Ca2+ influx into dorsal root ganglia neurones), which is predictive of analgesic activity, to investigate the requirements in this region of capsaicin for activity. N-(4-Hydroxy-3-methoxybenzyl)-N'-octylthiourea (14a) emerged as the most potent analogue (EC50 = 0.06 microM). An operational model based on multiple hydrogen-bonding interactions is proposed to explain the structure-activity profile observed. In combination with studies on the other regions of the capsaicin molecule these results describe a picture of the molecular interactions of capsaicin with its putative receptor.

Analgesics↗
PubMed1993

Analogues of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 3. The hydrophobic side-chain "C-region".

Structural variants of the hydrophobic side chain ("C region") of the capsaicin molecule have been incorporated into a series of vanillylamides and vanillylthioureas. These compounds have been tested in an in vitro assay for agonism (45Ca2+ influx into dorsal root ganglia neurones), previously shown to be predictive of analgesic activity. The results of this study have established the requirement for a hydrophobic substituent of limited size (molar refractivity, MR, < 55) in order to obtain high potency. Combination of the information gained here about the "C-region" of the capsaicin molecule with the studies described in the preceding two papers provides a rational basis for the design of compounds of increased potency.

Analgesics↗
PubMed1993

Inhibition of the acute-phase response in a human hepatoma cell line.

The HuH-7 human hepatoma cell line was stimulated by IL-1 and IL-6 to increase the synthesis of acute-phase proteins, e.g. serum amyloid A (SAA), alpha 1 antichymotrypsin (ACT), alpha 1-protease inhibitor, alpha 1 acid-glycoprotein and haptoglobin, with the exception of the pentraxins (serum amyloid P and C-reactive protein). Haptoglobin and ACT were stimulated by IL-1 which has not been observed in some other hepatoma cell lines. The concentration of IL-1 required for stimulation of SAA was higher than that required for haptoglobin stimulation. IL-1 receptor antagonist was capable of inhibiting these responses and acted at a lower concentration to inhibit SAA than required to inhibit ACT or haptoglobin induction. Transforming growth factor beta (TGF beta) was also able to inhibit the response to IL-1 but had no effect on acute-phase protein responses to IL-6.

Acute-Phase Reaction↗
PubMed1993

Characterization of resiniferatoxin binding sites on sensory neurons: co-regulation of resiniferatoxin binding and capsaicin sensitivity in adult rat dorsal root ganglia.

Binding of [3H]resiniferatoxin was seen by autoradiography in sections of rat dorsal root ganglia and the superficial dorsal horn of the spinal cord. Membranes from rat dorsal root ganglia and spinal cord, but not other tissues, had saturable high-affinity binding sites for [3H]resiniferatoxin. A series of capsaicin analogues competed for these sites. The sites probably correspond to capsaicin receptors. Systemic pretreatment of rats with capsaicin caused loss of capsaicin sensitivity in sensory neurons and a reduction in binding of resiniferatoxin to rat dorsal root ganglia, measured by binding assays and autoradiography. Adult rat dorsal root ganglion neurons cultured without nerve growth factor also lost their capsaicin-sensitivity and showed reduced resiniferatoxin binding. Therefore, capsaicin responses in sensory neurons may be regulated by nerve growth factor through control of the number of capsaicin receptors.

Animals↗
PubMed1993

The role of calcium in capsaicin-induced desensitization in rat cultured dorsal root ganglion neurons.

The effects of capsaicin cytosolic Ca2+ concentration ([Ca2+]i) were measured in individual dorsal root ganglion neurons of the rat in culture. Capsaicin produced a rapid concentration-dependent (EC50 value of 72 nM) increase in [Ca2+]i which was entirely dependent on Ca2+ entry. Exposure of the neurons to a high concentration of capsaicin resulted in desensitization, but only in the presence of external Ca2+. Raising [Ca2+]i with a depolarizing concentration of potassium or the Ca2+ ionophore ionomycin did not reduce the response to a subsequent application of capsaicin. Capsaicin did not induce desensitization in Ca(2+)-free medium even if [Ca2+]i was simultaneously raised with a combination of ionomycin plus carbonyl cyanide m-chlorophenyl-hydrazone. Okadaic acid, a known inhibitor of protein phosphatases 1 and 2A, caused a transient dose-dependent (EC50 value, 100nM) rise in [Ca2+]i, but had no effect on either the responsiveness to capsaicin or capsaicin induced desensitization. The capsaicin antagonist capsazepine blocked the increase in [Ca2+]i evoked by capsaicin and prevented desensitization. These results suggest that desensitization requires the presence of extracellular Ca2+, cannot be mimicked by raising the concentration of [Ca2+]i and may involve Ca2+ entry through activated capsaicin-operated ion channels.

Animals↗
PubMed1993

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens.

1. The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically-evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2. In the rat bladder, capsazepine (3-30 microM) produced a concentration-dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10-30 microM) produced a non-competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 microM) produced a rightward shift of the curve to capsaicin while ruthenium red (3 microM) depressed the maximal response to the agonist. 3. At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat alpha-calcitonin gene-related peptide (alpha CGRP) in the vas deferens. 4. These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin-sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non-competitive antagonism.

Animals↗
PubMed1993

Effect of capsazepine on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) induced by low pH, capsaicin and potassium in rat soleus muscle.

1. We have determined the effect of the competitive antagonist capsazepine at the capsaicin receptor on the release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from rat isolated soleus muscle induced by capsaicin (1 microM), by superfusion with low pH medium (pH 5) or by KCl (80 mM). 2. Each one of the three stimuli tested produced a marked CGRP-LI release. Total evoked release (fmol g-1) was 482 +/- 69, 169 +/- 20 and 253 +/- 43 for capsiacin, low pH medium and KCL, respectively. 3. Prior application of capsiacin (10 microM for 30 min followed by 30 min of washout) to produce capasaicin desensitization in vitro abolished CGRP-LI release induced by the three stimuli. 4. Capsazepine (1-100 microM, 45 min preincubation) inhibited the evoked CGRP-LI release. Capsaicin-induced release was significantly inhibited by 77, 92 and 96% with 10, 30 and 100 microM capsazepine, respectively. Low pH-induced release was inhibited by 78, 84, 88 and 93% with 3, 10, 30 and 100 microM capsazepine, respectively. KCl-induced release was significantly inhibited by 55 and 93% with 30 and 100 microM (but not with 10 microM) capsazepine, respectively. 5. These findings demonstrate that capsazepine prevents low pH- and capsaicin-induced CGRP-LI release from rat soleus muscle at concentrations which do not affect the release evoked by KCl. These findings imply a relationship between the action of low pH and activation of the capsaicin receptor. At high concentrations, capsazepine produces a nonspecific inhibitory effect on CGRP-LI release from peripheral endings of the capsaicin-sensitive primary afferent neurone.

Animals↗
PubMed1992

Effects of insulin-like growth factor I on the rates of glucose transport and utilization in rat skeletal muscle in vitro.

1. The effects of insulin-like growth factor I (IGF-I) on the rates of glucose transport and utilization and its interaction with insulin were investigated in rat soleus muscle in vitro. IGF-I increased the rates of glucose transport, lactate formation, glycogen synthesis and the flux of glucose to hexose monophosphate, but it had no effect on the rate of glucose oxidation or glycogenolysis. 2. In the absence of insulin, low levels of IGF-I (0-30 ng/ml) increased the rate of glycolysis and the content of fructose 2,6-bisphosphate, but the content of glucose 6-phosphate remained unaltered; at higher levels of IGF-I (300-3000 ng/ml) the rate of glycolysis and the content of fructose 2,6-bisphosphate showed a further modest increase, but the content of glucose 6-phosphate doubled. Similar changes were seen when the level of insulin was increased from basal (0-0.4 ng/ml) to maximal (40 ng/ml). 3. Neither IGF-I nor insulin affected the contents of ATP, ADP, AMP, phosphocreatine or citrate. 4. Maximal concentrations of IGF-I increased the rate of lactate formation to a greater extent than did maximal concentrations of insulin. 5. In the presence of IGF-I, the rate of glucose utilization was less responsive to insulin. 6. The results suggest that, in rat skeletal muscle: (a) IGF-I increases the rates of glucose transport and utilization independently of insulin, and has a preferential effect on the rate of lactate formation; (b) the effects of IGF-I and insulin are not additive; (c) in addition to its effects on glucose transport, IGF-I increases the rate of glycogen synthesis and may stimulate glycolysis at the level of 6-phosphofructokinase; (d) changes in the content of fructose 2,6-bisphosphate may be part of the mechanism to regulate glycolytic flux in skeletal muscle in response to either IGF-I or insulin.

Animals↗
PubMed1992

Expression of functional bradykinin receptors in Xenopus oocytes.

mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108-15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 microM), with an initial inward current (10-20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 microM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 microM). mRNA from both NG108-15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately five-fold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108-15 cells.

Animals↗
PubMed1992

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1. Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2. Capsazepine (10 microM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 microM gamma-aminobutyric acid (GABA) and 5 microM adenosine 5'-triphosphate (ATP) were unaffected. 3. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 +/- 46 nM (mean +/- s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 microM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 +/- 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4. Capsazepine also inhibited the capsaicin- and RTX-evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM). No significant difference was noted in the apparent Kd estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits.6. Capsazepine, at concentrations up to 1O microM, had no significant effects on the efflux of 86Rb+ from cultured DRG neurones evoked either by depolarization with high (50 mM) K' solutions or by acidification of the external medium to pH 5.0-5.6. Similarly capsazepine had no significant effect on he depolarization (50 mM KCl)-induced efflux of [14C]-guanidinium from vagus nerve preparations.7. Ruthenium Red was also tested for antagonism against capsaicin evoked ['4C]-guanidinium release from vague nerves and capsaicin induced 45Ca2" uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10-500nM in DRG and 0.5-10microM in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex,non-competitive inhibition.

Adenosine Triphosphate↗
PubMed1992

Effects of in-vivo administration of insulin-like growth factor-I on the rate of glucose utilization in the soleus muscle of the rat.

This study investigated the effects of insulin-like growth factor-I (IGF-I) administered to rats in vivo on the soleus muscle isolated from these rats. In order to study the interactions between IGF-I and insulin, the soleus muscles were incubated in the presence of various concentrations of insulin. IGF-I (190-200 micrograms) was given twice daily; the rats were killed 1 h after one injection of IGF-I (acute administration) or after treatment with IGF-I for 10 days (prolonged administration). The level of IGF-I in plasma was increased by approximately 100% after acute administration and by around 30% after 10 days of treatment with IGF-I. Acute administration of IGF-I to the rats increased the flux of glucose to hexose monophosphate and the rates of lactate formation and glycogen synthesis in the soleus muscles; however, the responsiveness of these muscles to insulin was lost: the increase in the rate of glucose utilization by IGF-I at physiological concentrations of insulin (10 or 100 mU/l) was similar to that observed at maximal concentrations of insulin (1000 mU/l). Similar results were obtained after prolonged treatment of the rats with IGF-I; however, the increase in the rate of glucose utilization was less pronounced than when IGF-I was given acutely and the muscles were still capable of responding to insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

IL-1 receptor antagonist regulation of acute phase protein synthesis in human hepatoma cells.

The hepatoma cell line HuH-7 has recently been shown to synthesize serum amyloid A (SAA) in response to IL-1. IL-1 receptor antagonist (IL-1Ra) was able to completely inhibit the response of SAA to IL-1 but not the increase seen in response to IL-6. IL-1Ra was equally effective at inhibiting IL-1 alpha or IL-1 beta. At a 10-fold molar excess of IL-1Ra over IL-1 there was complete inhibition of the SAA response. Removal of IL-1 at 24 h rapidly reduced the SAA secreted over the next 24 h. Addition of IL-1Ra to the cells at this time was as effective as removal of IL-1 at inhibiting the subsequent secretion of SAA. IL-1Ra was less effective at inhibition of IL-1-induced haptoglobin secretion. We would conclude that IL-1Ra may play an important role in the regulation of acute phase protein synthesis.

Acute-Phase Proteins↗