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Biomedical subjects

H M Cox

Publications and source records attributed to H M Cox.

At least 37 records · Page 2Linked to original sources
PubMed1995

Receptors for calcitonin gene related peptide (CGRP) in gastrointestinal epithelia.

A pharmacological comparison of calcitonin gene related peptide (CGRP) receptors expressed in normal rat colon mucosa and two human adenocarcinoma cell lines has been undertaken. Using voltage-clamp techniques electrogenic ion transport was continuously monitored across either mucosal preparations or confluent epithelial monolayers grown on permeable supports. The data presented at this meeting show that CGRP receptors are preferentially located on the basolateral epithelial surface and that their stimulation by a variety of CGRP analogues results in enhanced CI- secretion mediated via a cyclic AMP dependent mechanism. Responses to rat alpha CGRP in rat descending colon mucosa and in the adenocarcinoma cell line Colony-29 are insensitive to the inhibitory effects of the C-terminal fragment human CGRP(8-37); however, significant inhibition of rat alpha CGRP responses was observed in the parent epithelial cell line HCA-7. This together with the subtle differences seen in agonist orders of potency in the three preparations indicates that different CGRP receptor subtypes exist in the basolateral domains of HCA-7 compared with rat colon and Colony-29 epithelia.

Animals↗
PubMed1994

Ultrastructural co-localization of neuropeptide Y and vasoactive intestinal polypeptide in neurosecretory vesicles of submucous neurons in the rat jejunum.

The localization of vasoactive intestinal polypeptide and neuropeptide Y in submucous nerves of rat jejunum was studied using both single-label pre-embedding immunocytochemistry and post-embedding double-label immunogold techniques. Vasoactive intestinal polypeptide-immunoreactive fibres and cell bodies were regularly observed in submucous plexus and a similar distribution was seen for neuropeptide Y. Varicose fibres were observed in single-label studies and when areas of specific interest were subjected to double-label immunogold protocols these immunoreactive profiles exhibited vesicles clearly stained for both vasoactive intestinal polypeptide and neuropeptide Y. Synaptic vesicles in immunopositive fibres observed close to the mucosa (and elsewhere in the submucosa) were dense-cored with an average diameter of 80 nm. Nerves associated with vascular elements only stained for neuropeptide Y, not for vasoactive intestinal polypeptide. These findings suggest that these two unrelated enteric peptides are co-released in the vicinity of the mucosal lining and the likely implications of such co-release are discussed.

Animals↗
PubMed1994

Calcitonin gene-related peptide receptors in human gastrointestinal epithelia.

1. The secretory responses to calcitonin gene-related peptide (CGRP) receptor agonists have been characterized in two human adenocarcinoma cell lines, namely HCA-7 and Colony-29 (Col-29) epithelia. These cells form polarized epithelial layers when grown on permeable supports and allow changes in electrogenic ion transport in response to agonists to be monitored continuously. 2. alpha-CGRP (rat and human sequences), rat beta-CGRP and human [Tyr0]CGRP applied to the basolateral surface were found to be full agonists, causing prolonged increases in short-circuit current. Concentration-response curves exhibited EC50 values of 0.6-1.5 nM in HCA-7 cells. The same agonists were less effective in Col-29 epithelia, the EC50 values ranging from 1 to 10 nM in these cells. [Cys(ACM)2,7]CGRP was effective in both cell lines and was more potent in HCA-7 cells. 3. CGRP receptors were preferentially located on the basolateral surface in both cell types. Addition of r alpha-CGRP to the apical domain produced significantly smaller secretory responses (8.1% in HCA-7 and 29.2% in Col-29) compared with those produced following basolateral application (100%). 4. In both cell lines r alpha-CGRP-elevated short-circuit current was inhibited by the loop diuretic piretanide (200 microM) and by somatostatin (100 nM). Pretreating epithelia with the cyclo-oxygenase inhibitor, piroxicam (5 microM) had no significant effect upon CGRP responses in either cell line. 5. Rat alpha-CGRP (0.2 nM) responses in HCA-7 epithelia were inhibited by the C-terminal fragment CGRP(8-37) (1 microM). Pretreatment of Col-29 cells with CGRP(8-37) did not, however, alter the size or profile of responses to r alpha-CGRP (1 nM).6. We conclude that high-affinity CGRP receptors exist on the basolateral surface of both cell lines,however they differ in their sensitivity to CGRP(8-37) and agonist orders of potency. Thus different CGRP receptor subtypes appear to predominate in these two epithelial cell types.

Animals↗
PubMed1993

Pharmacological characterisation of neurokinin receptors mediating anion secretion in rat descending colon mucosa.

Substance P (SP), neurokinin A (NKA), neurokinin B (NKB), [Sar9, Met (O2)11]-SP (SMSP), senktide, [beta Ala8]-NKA(4-10) and neuropeptide gamma (NP gamma) all stimulate secretory responses in rat descending colon mucosa under voltage clamp conditions. Secretory responses (measured as short circuit current under voltage clamp conditions) were transient and those evoked by SP, SMSP, NKA and senktide were significantly reduced by pretreating tissues with the chloride channel blocker, diphenylamine carboxylate (DPC). Concentration-response curves showed varying degrees of sensitivity to tetrodotoxin (TTX). Senktide-induced secretion was virtually abolished by TTX, while NP gamma and [beta Ala8]-NKA(4-10) were not significantly altered. Rightward shifts of concentration-response curves were observed for SMSP, NKA and SP in TTX treated preparations compared with controls. NKA response curves in the presence of TTX were further inhibited by MEN10,207 and CP-96,345. GR71251, GR82334 and CP-96,345 all inhibited SMSP secretory responses with pA2 values of 5.8, 6.5 and 6.9 respectively. In conclusion three types of neurokinin receptor exist in preparations of rat colon mucosa and their relative location within neuronal and epithelial surfaces are discussed.

Animals↗
PubMed1992

Pituitary adenylate cyclase activating polypeptides, PACAP-27 and PACAP-38: stimulators of electrogenic ion secretion in the rat small intestine.

1. The effects of pituitary adenylate cyclase activating polypeptide (PACAP)-27 and PACAP-38 were investigated and compared with vasoactive intestinal polypeptide (VIP) responses in voltage clamped preparations of rat jejunum. Under these conditions electrogenic ion secretion was continuously recorded. 2. PACAP-27 is the most potent secretagogue described thus far, exhibiting a concentration-dependent dual secretory action. At low concentrations it stimulated rapid, transient secretory responses (not seen with either PACAP-38 or VIP) and these were inhibited by tetrodotoxin (TTX). At higher nM concentrations of PACAP-27 more prolonged secretory responses predominated which were insensitive to TTX. 3. In the presence of TTX, the concentration-response curve to PACAP-27 gave an EC50 value of 29.4 +/- 5.4 nM (n = 4) compared with 0.8 +/- 0.1 nM (n = 9) for PACAP-27 alone and 30.6 +/- 5.6 nM (n = 5) for PACAP-38. C-terminal fragments of PACAP-38 were not significantly effective. 4. Blockade of muscarinic and nicotinic receptors partially inhibited the low concentration effects of PACAP-27. Substance P desensitization and capsaicin pretreatment were effective at inhibiting the transient secretory PACAP-27 responses. Evidence is presented for selective, high affinity PACAP-27 receptors on submucous neurones innervating the mucosal region of the rat jejunum.

Adenylyl Cyclases↗
PubMed1991

The effects of capsaicin upon electrogenic ion transport in rat descending colon.

Preparations of rat descending colon mucosa have been used to record changes in short circuit current (SCC) under voltage clamp conditions. When added to the basolateral compartment capsaicin (8-methyl-N-vanillyl-6-nonenamide, 0.1-3 microM) caused an initial transient increase in SCC, followed by a more prolonged reduction in SCC, that lasted for 20-30 min. Repeated applications of 3 microM capsaicin caused desensitisation of the initial secretory response. The antisecretory effects (i.e. reduction in SCC from the original baseline) remained, although they were significantly reduced. In some preparations described as "non-responders", 3 microM capsaicin did not elicit a secretory response. No desensitization of the remaining antisecretory responses was observed in these tissues; in fact these reductions in SCC were consistently larger than those from tissues which responded with a secretory response. Tetrodotoxin (100 nM), hexamethonium (10 microM), and yohimbine (50 microM) had no significant effect upon either secretory or antisecretory responses. Ruthenium red (10 microM) abolished the secretory response to 3 microM capsaicin, but had no effect upon the antisecretory responses. Pretreatment of the tissues with 1 microM substance P (SP) resulted in significant desensitisation to the peptide and abolished the secretory response to 3 microM capsaicin. The antisecretory responses remained, and were significantly larger compared with responses from control tissues.

Animals↗
PubMed1990

The antisecretory effects of somatostatin and analogues in rat descending colon mucosa.

Somatostatin-14 (SS-14) and somatostatin-28 (SS-28) produce concentration dependent reductions in short-circuit current in rat colonic mucosa. EC50 values of 15.0 and 13.3 nM were obtained for SS-14 and SS-28 respectively while the N-terminal fragments of SS-28, namely somatostatin-(1-12) (SS1-12) and somatostatin-(1-14) (SS1-14) were inactive. Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) and cyclo(Pro-Tyr-D-Trp-Lys-Thr-Phe) were potent antisecretory peptides, like SS-14 and SS-28; while the putative somatostatin antagonist, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl]) exhibited neither agonist nor antagonist effects. Responses to SS-14 could be regulated by agents which affected the secretory state of the epithelium. Antisecretory effects of SS-14 were markedly attenuated by piroxicam and were restored following piroxicam plus either forskolin or vasoactive intestinal polypeptide (VIP). SS-14 also attenuated secretory responses produced by carbachol, substance P (SP), VIP and alpha- and beta-calcitonin gene related peptide (alpha-, beta-CGRP). Therefore, SS-14 exhibits broad spectrum antisecretory effects in rat descending colon mucosa.

Amino Acid Sequence↗
PubMed1990

The effects of neuropeptide Y and its fragments upon basal and electrically stimulated ion secretion in rat jejunum mucosa.

1. The effects of neuropeptide Y (NPY) and a range of C terminal fragments were investigated both on basal short circuit current (s.c.c.) and electrical field stimulated responses in voltage clamped preparations in rat jejunal mucosa. 2. Most of the NPY fragments tested had direct effects upon the mucosa, reducing baseline s.c.c. with EC50 values of 1 micron or more. NPY was 30 times more effective than any of the fragments tested and the order of potency was: NPY much greater than NPY (11-36) greater than or equal to (12-36) greater than or equal to (13-36) greater than or equal to (14-36). NPY (15-36), (16-36), (20-36) and (22-36) were still less effective and complete concentration-response curves could not be constructed. NPY (26-36), des amido NPY and the C-terminal flanking peptide of NPY (CPON) were all inactive and did not significantly alter responses to NPY. 3. Electrical field stimulation (EFS) of mucosal preparations elicited rapid transient secretory responses in the presence of hexamethonium and atropine. NPY and fragments attenuated these secretory responses and where concentration-response relationships could be compared at a given time point the following order of potency was obtained: NPY much greater than NPY (11-36) greater than NPY (13-36). Again NPY (26-36), des amido NPY and CPON were ineffective, while at single concentrations (300 nM) a graded attenuation of EFS responses was obtained with NPY (14-36) greater than or equal to NPY (15-36) greater than NPY (16-36) greater than or equal to NPY (20-36) greater than NPY (22-36). 4. The attenuation of EFS responses by these peptides was not dependent upon the basal secretory state. Pretreatment of tissues with piroxicam reduced s.c.c. and attenuated further reductions in s.c.c. by NPY, but had no effect upon NPY-mediated inhibition of electrically-stimulated secretory responses. 5. NPY fragments attenuated both basal and EFS generated secretion. Since fragments are effective these receptors must, by definition be Y2-like. NPY (11-36) and NPY (13-36) at 300nm and 1 microM did not significantly attenuate secretory responses to either carbachol (CCh) or substance P (SP). A 1 microM concentration of either fragment was equivalent in effect to 30nm NPY upon basal current, but NPY at this concentration significantly reduced both CCh- and SP-induced secretion. The reduced spectrum of fragment activity together with the different order and potency ratios obtained with these three peptides indicates a presynaptic action for NPY and the fragments.

Animals↗
PubMed1989

Antisecretory activity of the alpha 2-adrenoceptor agonist, xylazine in rat jejunal epithelium.

Xylazine, an alpha 2 adrenoceptor agonist, reduces short circuit current (SCC) in epithelial preparations of rat jejunum. The alpha 2 antagonist yohimbine, abolished this response while prazosin was without effect. The cyclooxygenase inhibitor, piroxicam, also attenuated xylazine responses indicating that the antisecretory effects were dependent upon endogenous eicosanoid formation. If the secretory state of piroxicam treated tissues was restored by addition of either forskolin, vasoactive intestinal polypeptide (VIP), prostaglandin E2 (PGE2) or isobutyl-1-methyl-xanthine (IBMX) then subsequent additions of xylazine were effective in reducing SCC. All these agents are known to increase SCC and cause Cl secretion by elevating intracellular cAMP. In addition, xylazine was also able to inhibit the Ca2+-mediated secretory responses of carbachol (CCh) and substance P (SP) in rat jejunum. This ability of xylazine to inhibit cAMP- and Ca2+-mediated secretion may indicate that alpha 2 adrenoceptors interact with more than one type of G protein or alternatively suggests a more general interaction between second messenger systems within epithelia of the small intestine.

Adrenergic alpha-Agonists↗
PubMed1989

Effects of alpha- and beta-calcitonin gene-related peptides upon ion transport in rat descending colon.

Both alpha- and beta-calcitonin gene-related peptide (CGRP) exhibit concentration-dependent dual actions upon rat colonic epithelia. At low concentrations (i.e. 0.03-10 nM) both CGRPs produce antisecretory effects that are abolished by pretreating tissues with tetrodotoxin (TTX). At higher concentrations (30-200 nM) secretory responses predominate and these effects are not abolished by TTX. It is suggested that the mediator of the antisecretory actions of CGRPs is somatostatin.

Animals↗
PubMed1988

Neuropeptide Y antagonises secretagogue evoked chloride transport in rat jejunal epithelium.

Neuropeptide Y (NPY) inhibits electrogenic Cl secretion in rat jejunal epithelium under voltage clamp conditions. This effect is dependent upon endogenous eicosanoid formation since it is blocked by the cyclooxygenase inhibitor, piroxicam, which itself has an inhibitory action upon chloride secretion. A number of chloride secretagogues have been examined for their ability to restore the antisecretory effects of NPY. Data presented here shows that NPY responsiveness is restored, in piroxicam pretreated tissues, by vasoactive intestinal polypeptide (VIP), forskolin, prostaglandin E2 (PGE2) isobutyl-1-methyl-xanthine (IBMX) and dibutyryl cAMP added prior to the neuropeptide. While all these agents cause chloride secretion by elevating intracellular cAMP, NPY is also effective in inhibiting the secretory effects of carbachol (CCh) and substance P (SP), agents believed to act by raising intracellular calcium (Cai). Although there is evidence that NPY can inhibit adenylate cyclase, its ability to attenuate chloride secretion brought about by secretagogues acting through both adenylate cyclase and calcium mechanisms, implies that NPY has either a more general fundamental mechanism or has multiple interactions with different second messenger systems.

Animals↗
PubMed1988

The effect of neuropeptide Y and peptide YY on electrogenic ion transport in rat intestinal epithelia.

1. Neuropeptide Y (NPY), peptide YY (PYY) and, to a lesser extent, human pancreatic polypeptide (HPP) reduced short-circuit current (SCC) in a concentration-dependent manner in epithelial preparations of rat jejunum and descending colon. 2. From concentration-response curves in the jejunum EC50 values of 3 nM for PYY and 10 nM for NPY were obtained. HPP was much less potent, the threshold concentration being around 100 nM, and NPY 13-36 was inactive. 3. Repeated exposure of jejunal preparations to either NPY or PYY led to a rapid desensitization. Cross-desensitization to the actions of these two peptides was also observed. Neither tetrodotoxin (TTX) nor phentolamine affected responses to either NPY or PYY on the jejunum. 4. Responses to both peptides were inhibited by the presence of transport inhibitors, particularly diphenylamine-2-carboxylate (DPC, a chloride channel blocker) and piretanide (Na+-K+-2Cl- co-transport inhibitor). These results may indicate that the reduction in SCC caused by the neuropeptides is due to a net increase in chloride movement in the apical to basolateral direction. 5. 36Cl-flux studies identified an inhibition of chloride secretion as the predominant mechanism of action of NPY and PYY, together with a smaller stimulation of chloride absorption. No significant changes in the movement of 22Na were seen in either direction. 6. The cyclo-oxygenase inhibitors piroxicam (5 microM) and indomethacin (5 microM) significantly reduced the responses to both NPY and PYY in rat jejunum. From this and other evidence it was concluded that the peptides depended for their effect on the endogenous formation of eicosanoids, the prevention of which attenuated the SCC reduction due to the peptides.

Animals↗
PubMed1987

The effect of angiotensin II upon electrogenic ion transport in rat intestinal epithelia.

Epithelial sheets from rat jejunum and descending colon have been shown to respond to angiotensin II (AII) when studied under short-circuit conditions and bathed on both sides with Krebs-Henseleit solution. The octapeptide AII elicited increases in short-circuit current (SCC) in preparations of jejunum and decreases in SCC in the descending colon; both responses occurred when the peptide was applied to the basolateral surface, but not when applied to the apical solution. Responses in both tissues were highly specific, being inhibited by a range of AII antagonists with the following order of potency: [Sar1. Thr8]-AII greater than [Sar1. Leu8]-AII greater than [Sar1. Ile8]-AII greater than [Sar1. Ala8]-AII greater than [Des,Asp1. Ile8]-AII in rat jejunum. AII responses were not affected by alpha- or beta- adrenoceptor antagonists, atropine or tetrodotoxin. AII responses were totally inhibited by the chloride channel blocker, diphenylamine-2-carboxylate (DPC) while cotransport inhibitors e.g. piretanide and frusemide significantly reduced the size of AII responses in colon and jejunum. These patterns of activity suggest that in the jejunum the responses result from electrogenic chloride secretion. Although AII responses in colon were sensitive to DPC the transporting ions have not yet been identified. Both piroxicam and indomethacin inhibited the increase in SCC elicited by AII in the jejunum, and the reduction in SCC caused by AII in the colon. Taken together these results indicate that eicosanoids are involved in AII responses in both tissues. This is the first study to demonstrate a direct, electrogenic effect for AII on transporting epithelia from the gastrointestinal tract. The responses are most probably initiated by All interacting with previously identified specific All receptors within the epithelial membranes.

Angiotensin I↗
PubMed1986

Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding.

Angiotensin II↗
PubMed1984

Inactivation of [125I]-angiotensin II binding sites in rat renal cortex epithelial membranes by dithiothreitol.

Preincubation of renal epithelial membranes with DTT produced a dose-dependent inhibition of specific [125I]-angiotensin binding, with an IC50 of 1 mM and total loss of binding at 5-10 mM DTT. Inactivation of specific [125I]-angiotensin II binding by DTT was temperature sensitive; the t1/2 at 22 degrees was 6 min compared with 30 min at 4 degrees. A rapid inactivation rate was dependent on the presence of NaCl. In the presence of 120 mM NaCl the t1/2 for inactivation by DTT was 6 min and 33 min in the absence of NaCl. Protection against DTT inactivation was obtained by preincubating membranes with unlabelled angiotensin II greater than angiotensin I greater than renin substrate while the dipeptide, Ileu-His was only effective in protecting the binding site at high concentrations (10 mM). Preincubations with DTT (1 mM) caused a 43% decrease in Bmax from 217.0 +/- 39.5 to 123.7 +/- 30.9 fmol bound/mg protein while the KD was not significantly affected.

Angiotensin II↗
PubMed1984

Location of [125I]-angiotensin II receptors on rat kidney cortex epithelial cells.

Angiotensin II (AII) stimulates active Na+ extrusion from Na+ loaded renal cortex slices. Specific high affinity [125I]-AII binding sites in partially purified basolateral and brush-border epithelial membranes exhibit a KD of 0.88 nM and Bmax of 321.13 fmol mg-1 protein. Separation and purification of brush-border membranes yielded high affinity [125I]-AII binding sites with KD of 1.02 nM and Bmax of 56.6 fmol mg-1 protein. Angiotensin II receptors of the same affinity are present on renal cortex brush-border and basolateral membranes but a greater proportion are located on the latter. These receptors may be involved in the direct control of Na+ and water transport by AII.

Alkaline Phosphatase↗