Mucosal glucagon-like peptide 1 (GLP-1) responses are mediated by calcitonin gene-related peptide (CGRP) in the mouse colon and both peptide responses are area-specific.

BACKGROUND: Glucagon-like peptide (GLP)-1 is an incretin hormone and its mimetics are proven antidiabetic and antiobesity drugs. GLP-1 exerts antimotility and mucosal proliferative activities but its epithelial ion transport effects are uncharacterized and these may contribute to the gastrointestinal (GI) disturbance, i.e., diarrhea experienced with some GLP-1 mimetics. Our aim was to establish GLP-1 agonist mechanisms and identify potential mucosal mediator(s) in the colonic tissue from C57BL/6J mice. METHODS: A tissue survey of GLP-1 responses (using exendin 4, Ex4) and α-calcitonin gene-related peptide (αCGRP) was undertaken, dividing the mouse colon into eight adjacent mucosal-submucosal preparations. Each preparation was voltage-clamped and changes in short-circuit current (Isc) measured. The involvement of submucosal neurons in GLP-1 agonism was tested using Ex(9-39) and tetrodotoxin (TTX), and CGRP receptors were blocked with BIBN4094. KEY RESULTS: Ex4 responses along the length of the colon were inhibited by the GLP-1 antagonist, Ex(9-39) or TTX, indicating neural mediation in all colonic regions. In the ascending colon, Ex4 increased Isc levels that were abolished by 10 nM BIBN4096, while in the descending colon it reduced Isc levels that were again BIBN4096-sensitive, but at 1 µM. The latter αCGRP response was dependent on epithelial Cl- conductance and Na+ /K+ -ATPase, and was partially (~25%) peptide YY-mediated, but was not nitrergic, somatostatin sst2 , or α2 -adrenoceptor-mediated. CONCLUSIONS AND INFERENCES: GLP-1 modulates epithelial ion transport indirectly by activating CGRP-containing submucosal enteric neurons in the mouse colon. This GLP-1-CGRP response was area-specific and could potentially contribute to the diarrheal side effect of certain GLP-1R therapeutics.

High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice.

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse molecular weight polyethylene glycols (PEGs) at two positions using promising lead structures while maintaining target activity. EXPERIMENTAL APPROACH: Modified peptides were synthesized by combined solid-phase and solution-phase peptide synthesis. Their potency was investigated in constitutively expressing human epithelial cells and isolated human colonic mucosa as well as receptor-transfected artificial cell lines. Human blood plasma and porcine liver homogenates were used to examine the in vitro stability of the analogues. The most promising variants were injected s.c. in C57BL/6JRj mice to monitor fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K22 (PEG22)]hPP2-36 and [K22 (PEG22),Q34 ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22 stabilizes hPP significantly while extending its biological activities and could be used in drug development prospectively.

L-arginine promotes gut hormone release and reduces food intake in rodents.

AIMS: To investigate the anorectic effect of L-arginine (L-Arg) in rodents. METHODS: We investigated the effects of L-Arg on food intake, and the role of the anorectic gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), the G-protein-coupled receptor family C group 6 member A (GPRC6A) and the vagus nerve in mediating these effects in rodents. RESULTS: Oral gavage of L-Arg reduced food intake in rodents, and chronically reduced cumulative food intake in diet-induced obese mice. Lack of the GPRC6A in mice and subdiaphragmatic vagal deafferentation in rats did not influence these anorectic effects. L-Arg stimulated GLP-1 and PYY release in vitro and in vivo. Pharmacological blockade of GLP-1 and PYY receptors did not influence the anorectic effect of L-Arg. L-Arg-mediated PYY release modulated net ion transport across the gut mucosa. Intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration of L-Arg suppressed food intake in rats. CONCLUSIONS: L-Arg reduced food intake and stimulated gut hormone release in rodents. The anorectic effect of L-Arg is unlikely to be mediated by GLP-1 and PYY, does not require GPRC6A signalling and is not mediated via the vagus. I.c.v. and i.p. administration of L-Arg suppressed food intake in rats, suggesting that L-Arg may act on the brain to influence food intake. Further work is required to determine the mechanisms by which L-Arg suppresses food intake and its utility in the treatment of obesity.

Gastrointestinal hormonal responses on GPR119 activation in lean and diseased rodent models of type 2 diabetes.

BACKGROUND: G protein-coupled receptor 119 (GPR119) has emerged as a potential target for the treatment of type 2 diabetes (T2D) and tool compounds have been critical in the evaluation of GPR119 functions. METHODS: We synthesised a novel small-molecule GPR119 agonist, PSN-GPR119, to study GPR119 signalling activities in cells overexpressing GPR119. We measured GPR119-stimulated peptide hormone release from intestinal loops and oral glucose tolerance in vivo from lean (C57BL/6J mouse or Sprague-Dawley (SD) rat) and diabetic (ob/ob mouse or ZDF rat) models. To evaluate the direct effects of GPR119 agonism on gastrointestinal (GI) tissue, we measured vectorial ion transport (measured as ISC; short-circuit current) across rodent GI mucosae and from normal human colon specimens. RESULTS: GPR119 activation by PSN-GPR119 increased cAMP accumulation in hGPR119-overexpressing HEK293 cells (EC50, 5.5 nM), stimulated glucagon-like peptide 1 (GLP-1) release from GLUTag cells (EC50, 75 nM) and insulin release from HIT-15 cells (EC50, 90 nM). In vivo, PSN-GPR119 improved glucose tolerance by ~50% in lean mice or rats and ~60% in the diabetic ob/ob mouse or ZDF rat models. Luminal addition of PSN-GPR119 to isolated loops of lean rat small intestine stimulated GLP-1, glucose insulinotropic peptide (GIP) and peptide YY (PYY) release under basal (5 mM) and high glucose (25 mM) conditions. Activation of GPR119 also reduced intestinal ion transport. Apical or basolateral PSN-GPR119 addition (1 µM) to lean or T2D rodent colon mucosae reduced ISC levels via PYY-mediated Y1 receptor agonism. The GPR119 response was glucose sensitive and was abolished by Y1 receptor antagonism. Similarly, in human colon, mucosa PSN-GPR119 acted via a Y1-specific mechanism. CONCLUSIONS: Our results show that functional GPR119 responses are similar in lean and diabetic rodent, and human colon; that GPR119 stimulation can result in glucose lowering through release of intestinal peptide hormones and that PSN-GPR119 is a useful tool compound for future studies.

Endogenous PYY and GLP-1 mediate l-glutamine responses in intestinal mucosa.

BACKGROUND AND PURPOSE: l-glutamine (Gln) is an energy source for gastrointestinal (GI) epithelia and can stimulate glucagon-like peptide 1 (GLP-1) release from isolated enteroendocrine L-cells. GLP-1 and peptide YY (PYY) are co-secreted postprandially and both peptides have functional roles in glucose homeostasis and energy balance. The primary aim of this project was to establish the endogenous mechanisms underpinning Gln responses within intact GI mucosae using selective receptor antagonists. EXPERIMENTAL APPROACH: Mouse mucosae from different GI regions were voltage-clamped and short-circuit current (Isc) was recorded to Gln added to either surface in the absence or presence of antagonists, using wild-type (WT) or PYY-/- tissues. The glucose sensitivity of Gln responses was also investigated by replacement with mannitol. KEY RESULTS: Colonic apical and basolateral Gln responses (at 0.1 and 1 mM) were biphasic; initial increases in Isc were predominantly GLP-1 mediated. GLP-1 receptor antagonism significantly reduced the initial Gln response in the PYY-/- colon. The slower reductions in Isc to Gln were PYY-Y1 mediated as they were absent from the PYY-/- colon and were blocked selectively in WT tissue by a Y1 receptor antagonist. In jejunum mucosa, Gln stimulated monophasic Isc reductions that were PYY-Y1 receptor mediated. Gln effects were partially glucose sensitive, and Calhex 231 inhibition indicated that the calcium-sensing receptor (CaSR) was involved. CONCLUSION AND IMPLICATIONS: Gln stimulates the co-release of endogenous GLP-1 and PYY from mucosal L-cells resulting in paracrine GLP-1 and Y1 receptor-mediated electrogenic epithelial responses. This glucose-sensitive mechanism appears to be CaSR mediated and could provide a significant therapeutic strategy releasing two endogenous peptides better known for their glucose-lowering and satiating effects.

The Concise Guide to PHARMACOLOGY 2013/14: overview.

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

A role for neuropeptide Y in the gender-specific gastrointestinal, corticosterone and feeding responses to stress.

BACKGROUND AND PURPOSE: Exposure to an acute stress inhibits gastric emptying and stimulates colonic transit via central neuropeptide Y (NPY) pathways; however, peripheral involvement is uncertain. The anxiogenic phenotype of NPY(-/-) mice is gender-dependent, raising the possibility that stress-induced gastrointestinal (GI) responses are female-dominant through NPY. The aim of this study was to determine GI transit rates, corticosterone levels and food intake after acute restraint (AR) or novel environment (NE) stress in male and female NPY(-/-) and WT mice. EXPERIMENTAL APPROACH: Upper gastrointestinal transit (UGIT) (established 30 min after oral gavage) and corticosterone levels were determined under basal or restrained conditions (30 min) and after treatment i.p. with Y(1) antagonist BIBO3304 or Y(2) antagonist BIIE0246. Faecal pellet output (FPO) was established after AR and treatment i.p. with NPY in the NE, as were colonic bead expulsion rates. KEY RESULTS: UGIT and FPO were similar in unrestrained male and female mice. NPY(-/-) females displayed significantly slower UGIT than NPY(-/-) males after AR, but both genders displayed significantly higher FPO and reduced food intake relative to WT counterparts. Peripheral NPY treatment increased bead expulsion time in WT mice. AR male NPY(-/-) mice had higher levels of corticosterone than male WT mice; whilst in AR WT mice, after peripheral Y(1) and Y(2) receptor antagonism in males, and Y(2) antagonism in females, corticosterone was significantly elevated. CONCLUSIONS AND IMPLICATIONS: NPY possesses a role in the gender-dependent susceptibility to stress-induced GI responses. Furthermore, NPY inhibits GI motility through Y(2) receptors and corticosterone release via peripheral Y(1) and Y(2) receptors.

Characterization of the EP receptor types that mediate longitudinal smooth muscle contraction of human colon, mouse colon and mouse ileum.

BACKGROUND: Prostaglandin E(2) (PGE(2) ) is an inflammatory mediator implicated in several gastrointestinal pathologies that affect normal intestinal transit. The aim was to establish the contribution of the four EP receptor types (EP(1-4) ), in human colon, that mediate PGE(2) -induced longitudinal smooth muscle contraction. METHODS: Changes in isometric muscle tension of human colon, mouse colon and mouse ileum were measured in organ baths in response to receptor-specific agonists and antagonists. In addition, lidocaine was used to block neurogenic activity to investigate whether EP receptors were pre- or post-junctional. KEY RESULTS: PGE(2) contracted longitudinal muscle from human and mouse colon and mouse ileum. These contractions were inhibited by the EP(1) receptor antagonist, EP(1) A in human colon, whereas a combination of EP(1) A and the EP(3) antagonist, L798106 inhibited agonist responses in both mouse preparations. The EP(3) agonist, sulprostone also increased muscle tension in both mouse tissues, and these responses were inhibited by lidocaine in the colon but not in the ileum. Although PGE(2) consistently contracted all three muscle preparations, butaprost decreased tension by activating smooth muscle EP(2) receptors in both colonic tissues. Alternatively, in mouse ileum, butaprost responses were lidocaine-sensitive, suggesting that it was activating prejunctional EP(2) receptors on inhibitory motor neurons. Conversely, EP(4) receptors were not functional in all the intestinal muscle preparations tested. CONCLUSIONS & INFERENCES: PGE(2) -induced contraction of longitudinal smooth muscle is mediated by EP(1) receptors in human colon and by a combination of EP(1) and EP(3) receptors in mouse intestine, whereas EP(2) receptors modulate relaxation in all three preparations.

Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y1 and Y2 receptors.

BACKGROUND AND PURPOSE: Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild-type, single and double peptide knockout mice, comparing mucosal responses with those from human colon. EXPERIMENTAL APPROACH: Mucosae were pretreated with a Y1 (BIBO3304) or Y2 (BIIE0246) receptor antagonist and changes in short-circuit current recorded. Colonic transit and colonic migrating motor complexes (CMMCs) were assessed in vitro and upper gastrointestinal and colonic transit measured in vivo. KEY RESULTS: Y receptor antagonists revealed tonic Y1 and Y2 receptor-mediated antisecretory effects in human and wild-type mouse colon mucosae. In both, Y1 tone was epithelial while Y2 tone was neuronal. Y1 tone was reduced 90% in PYY⁻/⁻ mucosa but unchanged in NPY⁻/⁻ tissue. Y2 tone was partially reduced in NPY⁻/⁻ or PYY⁻/⁻ mucosae and abolished in tetrodotoxin-pretreated PYY⁻/⁻ tissue. Y1 and Y2 tone were absent in NPYPYY⁻/⁻ tissue. Colonic transit was inhibited by Y1 blockade and increased by Y2 antagonism indicating tonic Y1 excitation and Y2 inhibition respectively. Upper GI transit was increased in PYY⁻/⁻ mice only. Y2 blockade reduced CMMC frequency in isolated mouse colon. CONCLUSIONS AND IMPLICATIONS: Endogenous PYY and NPY induced significant mucosal antisecretory tone mediated by Y1 and Y2 receptors, via similar mechanisms in human and mouse colon mucosa. Both peptides contributed to tonic Y2-receptor-mediated inhibition of colonic transit in vitro but only PYY attenuated upper GI transit.

A functional comparison of recombinant and native somatostatin sst2 receptor variants in epithelia.

BACKGROUND AND PURPOSE: Somatostatin (SRIF-14) exerts broad spectrum antisecretory effects by activating the somatostatin 2 (sst(2)) receptor. The rat (r) sst(2) receptor exists in 'long' (sst(2a)) and 'short' (sst(2b)) forms that differ in their C termini, while a single human (h) sst(2a) exists. This study compares the characteristics of recombinant rsst(2a), rsst(2b) and hsst(2a) activation in human epithelia, and with native sst(2) responses in rat colon. EXPERIMENTAL APPROACH: Epithelial layers of each clone or rat colon were placed in Ussing chambers and short-circuit current (I (SC)) measured in response to SRIF-14 and chosen analogues. The relative potencies and ability to cause desensitization to SRIF-14 were assessed, and the affinities of the sst(2) antagonist, D-Tyr(8) CYN154806 for hsst(2a), rsst(2a) and native rat colon sst(2) receptors were established. KEY RESULTS: Basolateral SRIF-14 responses were transient in hsst(2a) and rsst(2a) epithelia, but prolonged in rsst(2b)-expressing cells. Activation of rsst(2a) resulted in significant desensitization to SRIF-14 and receptor phosphorylation, whereas the rsst(2b) receptor did neither. Sst(2)-preferred agonists (BIM23190C and BIM23027) reduced I (sc) with similar potency and both caused complete desensitization to SRIF-14. CYN154806 antagonized hsst(2a) and rsst(2a) receptors with pK (B) values of 7.9 and 7.8, respectively. In rat colon mucosa, CYN154806 blocked SRIF-14 responses with a pA (2) value of 8.2, and BIM23190C responses with a pK (B) of 8.4. CONCLUSIONS AND IMPLICATIONS: SRIF-14 caused rapid rsst(2a) receptor phosphorylation and desensitization of epithelial antisecretory responses, neither of which occurred with the rsst(2b) receptor. These mechanisms are most likely to be a prerequisite for sensitivity to sst(2)-analogues with radiotherapeutic potential.

Effect of ectopic expression of rat trefoil factor family 3 (intestinal trefoil factor) in the jejunum of transgenic mice.

To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([(3)H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitro studies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 +/- 60 versus 195 +/- 48 ohm.cm(2) in controls, p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration.

Multiple Y receptors mediate pancreatic polypeptide responses in mouse colon mucosa.

A functional study has been performed to characterise the Y receptors responsible for NPY, PYY and PP-stimulated responses in mouse colonic mucosal preparations. Electrogenic ion secretion was stimulated with VIP following which NPY, PYY and PP analogues were, to varying degrees, inhibitory. PYY(3-36), hPP, Gln(23)hPP and rPP were effective but less potent than full length PYY, NPY or their Pro(34)-substituted analogues, while the Y(5) agonist Ala(31), Aib(32)hNPY was the least active peptide tested. The Y(1) antagonists, BIBP3226 and BIBO3304 virtually abolished Pro(34)PYY and PYY responses while PYY(3-36) responses were selectively inhibited by the Y(2) antagonist, BIIE0246. A combination of BIBO3304 and BIIE0246 also partially attenuated hPP responses, leaving residual effects that were most probably Y(4)-mediated. Thus we conclude that Y(1), Y(2) and Y(4) receptors attenuate ion secretion in mouse colon.

Tachykinin NK(2) receptor mediates contraction and ion transport in rat colon by different mechanisms.

We have characterized the tachykinin NK(2) receptor-mediated contraction and vectorial ion transport responses in the muscularis mucosae and mucosa of the rat isolated distal colon, respectively. The tachykinin NK(2) receptor-selective antagonist nepadutant (c([(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta))) produced competitive antagonism of [betaAla(8)]neurokinin A-(4-10)-induced contraction (pK(B) = 9.3) in the muscularis mucosae, and insurmountable blockade of increases in short-circuit current (I(sc)) responses (pK(B) = 8.6) in the mucosa. However, this latter effect was completely reversed by washout of the antagonist. [betaAla(8)]Neurokinin A-(4-10)-induced contractions were unaffected by indomethacin (3 microM). In sharp contrast, I(sc) responses induced by [betaAla(8)]neurokinin A-(4-10) (100 nM) were inhibited (>70%) by indomethacin (3 microM), while I(sc) responses to substance P (3 microM) were unchanged. Our study provides the first evidence that in the same organ stimulation of tachykinin NK(2) receptors leads to two independent responses mediated by different effector mechanisms both of which are blocked (albeit with different kinetics) by the potent and selective tachykinin NK(2) receptor antagonist, nepadutant.

Constitutive neuropeptide Y Y(4) receptor expression in human colonic adenocarcinoma cell lines.

1. Three human adenocarcinoma cell lines, Colony-24 (Col-24), Col-6 and Col-1 have been studied as confluent epithelial layers able to transport ions vectorially in response to basolateral vasoactive intestinal polypeptide (VIP) and pancreatic polypeptides (PP). 2. Different species PP stimulated responses in Col-24 with Y(4)-like pharmacology. Bovine (b)PP, human (h)PP and porcine (p)PP were equipotent (EC(50) values 3.0--5.0 nM) while rat (r)PP, avian (a)PP and [Leu(31), Pro(34)]PYY (Pro(34)PYY) were significantly less potent. PYY was inactive. The PP pharmacology in Col-1 was comparable with Col-24. However, Col-6 cells were different; pPP had an EC(50) intermediate (22.0 nM) between that of bPP (3.0 nM) and hPP (173.2 nM), with aPP and rPP being at least a further fold less potent. 3. Deamidation of Tyr(36) in bPP (by O-methylation or hydroxylation) or removal of the residue resulted in significant loss of activity in Col-24. 4. GR231118 (1 microM) had no PP-like effects. In Col-24 and Col-1, GR231118 significantly attenuated bPP (30 nM) or hPP (100 nM) responses, but it did not alter bPP responses in Col-6. BIBP3226 and GR231118 both inhibited Y(1)-mediated responses which were only present in Col-6. 5. RT--PCR analysis confirmed the presence of hY(4) receptor mRNA in Col-24 and Col-1 epithelia but a barely visible hY(4) product was observed in Col-6 and we suggest that an atypical Y(4) receptor is expressed in this cell line.

PYY preference is a common characteristic of neuropeptide Y receptors expressed in human, rat, and mouse gastrointestinal epithelia.

This investigation describes the relative potencies of four peptide agonists, namely, peptide YY (PYY), [Leu3l,Pro34]PYY (Pro34pYY), neuropeptide Y (NPY), and [Leu31,Pro34]NPY (Pro34NPY), as antisecretory agents in human, rat, and mouse gastrointestinal preparations. The inhibition of agonist responses by the Y1-receptor antagonist BIBP 3226 was also tested in each preparation. An unexpectedly pronounced preference for PYY and Pro34PYY was observed in functional studies of two human epithelial lines stably transfected with the rat Y1 receptor (Y1-7 and C1Y1-6). NPY and Pro34NPY were at least an order of magnitude less effective than PYY in these functional studies but were only marginally less potent in displacement binding studies using membrane preparations of the same clonal lines. The orders of agonist potency obtained in Y1-7 and C1Y1-6 epithelia were compared with those obtained from a single human colonic adenocarcinoma cell line (Colony-6, which constitutively expresses Y1 receptors) and also from mucosal preparations of rat and mouse descending colon. Similar peptide orders of potency were obtained in rat and mouse colonic mucosae and Colony-6 epithelia, all of which exhibited PYY preference (although less pronounced than with Y1-7 and C1Y1-6 epithelia) and significant sensitivity to the Y1 receptor antagonist, BIBP 3226. We have compared the pharmacology of these five mammalian epithelial preparations and provide cautionary evidence against the reliance upon agonist concentration-response relationships alone, in the characterization of NPY receptor types.

Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line.

1. Short-circuit current (I(SC)) responses to carbachol (CCh) were investigated in Colony 1 epithelia, a subpopulation of the HCA-7 adenocarcinoma cell line. In Krebs-Henseleit (KH) buffer, CCh responses consisted of three I(SC) components: an unusual rapid decrease (the 10 s spike) followed by an upward spike at 30 s and a slower transient increase (the 2 min peak). This response was not potentiated by forskolin; rather, CCh inhibited cyclic AMP-stimulated I(SC). 2. In HCO3- free buffer, the decrease in forskolin-elevated I(SC) after CCh was reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl- anions were replaced by gluconate, both Ca2+- and cyclic AMP-mediated electrogenic responses were significantly inhibited. 3. Basolateral Ba2+ (1-10 mM) and 293B (10 microM) selectively inhibited forskolin stimulation of I(SC), without altering the effects of CCh. Under Ba2+- or 293B-treated conditions, CCh responses were potentiated by pretreatment with forskolin. 4. Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3- free medium, without affecting the 2 min peak. The enhanced 10 s spike was inhibited by prior addition of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses. 5. In epithelial layers prestimulated with forskolin, the muscarinic antagonists atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, both at 100 nM) abolished subsequent 10 microM CCh responses. Following addition of p-fluoro hexahydro-sila-difenidol (pF-HHSiD, 10 microM) or pirenzepine (1 microM), qualitative changes in the CCh response time-profile also indicated a rightward shift of the agonist concentration-response curve; however, 1 microM gallamine had no effect. These results suggest that a single M3-like receptor subtype mediates the secretory response to CCh. 6. It is concluded that CCh and forskolin activate discrete populations of basolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl- permeability of the apical membrane may limit their combined effects on electrogenic Cl- secretion. In addition, CCh activates a Ba2+-sensitive apical K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3- secretion through a mechanism at least partially dependent on carbonic anhydrase.

Structure-activity relationships with neuropeptide Y analogues: a comparison of human Y1-, Y2- and rat Y2-like systems.

A structure-activity study utilising 36 synthetic Ala-analogues of the 36-residue oligopeptide neuropeptide Y (NPY) has been performed with mucosal preparations from the rat jejunum (Y2-like receptor) and compared with receptor displacement binding in the human neuroblastoma cell lines, SMS-KAN, (Y2-receptors) and SK-N-MC cells (Y1-receptors). Each amino acid of the natural sequence was replaced by L-alanine, and the four intrinsic alanine residues at position 12, 14, 18 and 23 were replaced by glycine. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated using membranes prepared from either SMS-KAN or SK-N-MC cells. The activity of each Ala-NPY analogue was assessed in mucosal preparations of rat jejunum, where NPY and PYY exert antisecretory responses which are Y2-like in pharmacology. Fourteen analogues with L-alanine replacements at position 3, 5, 8, 13, 20, 21, 22, 26, 27, 28, 29, 30, 34 and 36 were selected, none of which exhibited any antagonism of NPY responses. An order of agonist potency showed [Ala3] NPY and [Ala30] NPY equipotent with NPY, a 4-20-fold loss of activity with [Ala5] NPY, [Ala13] NPY, [Ala20] NPY, [Ala21] NPY and [Ala22] NPY; a 50-100-fold loss of activity, [Ala8] NPY, [Ala27] NPY, [Ala28] NPY and [Ala36] NPY, while [Ala34] NPY was inactive. This structure-activity relationship is similar to, but not the same as that observed in Y2-expressing SMS-KAN cells.

Inhibition of cyclic AMP-dependent chloride secretion by PP receptors and alpha 2-adrenoceptors in a human colonic epithelial cell line.

The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14-28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The alpha 2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50342 nM) through the alpha 2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers. Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylaxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 microM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit, chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 microM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca(2+)- and cAMP-dependent mechanisms involved in epithelial ion transport.

Disaster relief work: nurses and others in bushfire territory.

This paper describes one aspect of a study into the experiences in long-term healing of a community following the 1983 Ash Wednesday bushfire. Forty participants were interviewed, of whom 26 were residents and 14 disaster relief workers. The paper concentrates on the experiences of the latter, describing how they came to understand the bushfire affected the community and how they managed disaster work. For novices it was a profoundly difficult experience, for which they received little help and had to manage with whatever skill they drew on in their 'normal' working lives, mixed with a good deal of intuition. The paper suggests that health workers in vulnerable areas require preparation for a likely disaster; that 'outsiders' need to deal through existing community groups and individuals to gain access to those in need of their skills, and that they also require preparation for helping 'insiders' who are themselves victims of the catastrophe.