Notes: Abstract  The aim of this study was to examine the potencies of several recently identified selective somatostatin (SRIF)-receptor ligands as inhibitors of electrogenic ion transport in the rat distal colonic mucosa with the view to identifying the SRIF receptor type involved. Under basal conditions, cumulative administration of SRIF and SRIF28 decreased short circuit current (SCC), a measure of electrogenic ion transport, with EC50 values of 4 nM and 9 nM respectively. The peptidase inhibitors, phosphoramidon (1 μM) and amastatin (10 μM), had no effect on the potencies of either SRIF or SRIF28. The inhibitory action of SRIF on basal SCC was suppressed by piretanide and diphenylamine-2-carboxylate, compatible with the assumption that the Na+K+2Cl- co-transporter and Cl- channels, respectively, may be involved in this antisecretory action of SRIF. Tetrodotoxin (1 μM) had no effect on the antisecretory action of SRIF, suggesting that the process was not neuronally mediated. All of the SRIF analogues examined, with the exception of BIM-23056, maximally inhibited basal SCC to a similar extent as SRIF. Seglitide and octreotide were both more potent antisecretory agents than SRIF (respective EC50 values, 0.4 nM and 1.5 nM) suggesting that this effect was mediated by a receptor belonging to the SRIF1 receptor group. The most distinguishing feature of the rank order of agonist potencies was the high potency of the selective sst2 receptor ligand, BIM-23027 (EC50 value 0.32 nM), the weaker potency exhibited by the selective sst5 receptor ligand, L-362855 (EC50 value 21 nM), and the lack of agonist activity displayed by the selective sst3 receptor ligand, BIM-23056 (EC50 value 〉1000 nM). This profile is comparable with that observed in binding studies on the recombinant sst2 receptor. Forskolin-stimulated secretion was suppressed by SRIF analogues with the rank order of agonist potencies BIM-23027〉SRIF〉L-362855≫BIM-23056 which resembled that exibited under basal conditions. However, the absolute potencies of these agonists were lower (respective EC50 values 2 nM, 14 nM, 38 nM and 〉1000 nM) whilst the magnitude of inhibition was about three fold greater. BIM-23027 and SRIF (both 30 nM) also inhibited carbachol-stimulated increases in basal SCC by 60–70%, while a similar concentration of L-362855 inhibited these responses by 11%. BIM-23056 (1 μM) had no effect on carbachol-simulated secretion. Radioligand binding studies on rat colonic mucosal membranes using [125I]-Tyr11-SRIF suggested heterogeneity of SRIF binding sites. Thus, SRIF and SRIF28 competed for binding (IC50 values, 0.32 and 0.63 nM, respectively) with Hill slopes less than unity; while seglitide and BIM-23027 both maximally displaced only 30–40% of specific binding with apparent high affinity (respective pIC50 values, 10.1 nM and 10.0). In conclusion, SRIF decreases basal as well as both cAMP and Ca2+-dependent Cl- secretion in rat colonic mucosa. The rank order of agonist potencies suggests that receptors resembling the recombinant sst2 receptor mediate inhibition of basal and forskolin-stimulated secretion. Radioligand binding studies suggest that BIM-23027 interacts with a sub-population of [125I]Tyr11-SRIF binding sites in rat colonic mucosal membranes which probably correspond to the receptors mediating the antisecretory effects described here.

Notes: Abstract The aim of this study was to examine the potencies of several recently identified selective somatostatin (SRIF)-receptor ligands as inhibitors of electrogenic ion transport in the rat distal colonic mucosa with the view to identifying the SRIF receptor type involved. Under basal conditions, cumulative administration of SRIF and SRIF2g decreased short circuit current (SCC), a measure of electrogenic ion transport, with EC50 values of 4 nM and 9 nM respectively. The peptidase inhibitors, phosphoramidon (1 μM) and amastatin (10 μM), had no effect on the potencies of either SRIF or SRIF28. The inhibitory action of SRIF on basal SCC was suppressed by piretanide and diphenylamine-2-carboxylate, compatible with the assumption that the Na+K+2Cl− co-transporter and Cl− channels, respectively, may be involved in this antisecretory action of SRIF. Tetrodotoxin (1 μM) had no effect on the antisecretory action of SRIF, suggesting that the process was not neuronally mediated. All of the SRIF analogues examined, with the exception of BIM-23056, maximally inhibited basal SCC to a similar extent as SRIF. Seglitide and octreotide were both more potent antisecretory agents than SRIF (respective EC50 values, 0.4 nM and 1.5 nM) suggesting that this effect was mediated by a receptor belonging to the SRIF1 receptor group. The most distinguishing feature of the rank order of agonist potencies was the high potency of the selective sst2 receptor ligand, BIM-23027 (EC50, value 0.32 nM), the weaker potency exhibited by the selective sst5 receptor ligand, L-362855 (EC50 value 21 nM), and the lack of agonist activity displayed by the selective sst3 receptor ligand, BIM-23056 (EC50 value 〉 1000 nM). This profile is comparable with that observed in binding studies on the recombinant sst2 receptor. Forskolin-stimulated secretion was suppressed by SRIF analogues with the rank order of agonist potencies BIM-23027 〉 SRIF 〉 L-362855 〉 BIM-23056 which resembled that exibited under basal conditions. However, the absolute potencies of these agonists were lower (respective EC50 values 2 nM, 14 nM, 38 nM and 〉 1000 nM) whilst the magnitude of inhibition was about three fold greater. BIM-23027 and SRIF (both 30 nM) also inhibited carbachol-stimulated increases in basal SCC by 60–70%, while a similar concentration of L-362855 inhibited these responses by 11 %. BIM-23056 (1 μM) had no effect on carbachol-simulated secretion. Radioligand binding studies on rat colonic mucosal membranes using [125I]-Tyr11-SRIF suggested heterogeneity of SRIF binding sites. Thus, SRIF and SRIF28 competed for binding (IC50 values, 0.32 and 0.63 nM, respectively) with Hill slopes less than unity; while seglitide and BIM-23027 both maximally displaced only 30–40% of specific binding with apparent high affinity (respective pIC50 values, 10.1 nM and 10.0). In conclusion, SRIF decreases basal as well as both cAMP and Ca2+-dependent Cl− secretion in rat colonic mucosa. The rank order of agonist potencies suggests that receptors resembling the recombinant sst2 receptor mediate inhibition of basal and forskolin-stimulated secretion. Radioligand binding studies suggest that BIM-23027 interacts with a sub-population of [125I]Tyr11-SRIF binding sites in rat colonic mucosal membranes which probably correspond to the receptors mediating the antisecretory effects described here.

Notes: Abstract In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [3H]α,β-methylene ATP ([3H]αβmeATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [3H]αβmeATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl3 and AlCl3 (0.1 to 100 μM), produced the greatest increases in total binding of [3H]αβmeATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 μM and 1–10 mM, increased total binding of [3H]αβmeATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC50 values for potentiating binding of the radioligand were obtained: ZnCl2 (5.44), MnCl2 (4.52), CaCl2 (4.17), CoCl2 (4.06), MgCl2 (3.67) and BaCl2 (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand. The effects of ZnCl2, CaCl2 and MgCl2 were examined in saturation studies. In the absence of added divalent cations, [3H]αβmeATP labelled both high (pKd = 9.15) and low (pKd = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl2 (pKd = 9.56) and by 30 μM ZnCl2 (pKd = 9.46) but not by 3 mM MgCl2. The Bmax of the low affinity site for [3H]αβmeATP was increased (approximately 4 fold) by both 3 mM MgCl2 and 30 μM ZnCl2 but not by 3 mM CaCl2. The selective effect of CaCl2 on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl2 using a low concentration of [3H]αβmeATP (1 nM); the sites exhibited the binding characteristics expected of the P2x purinoceptor. The selective effect of MgCl2 on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl2 and using a high concentration of [3H]αβmeATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [3H]αβmeATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, αβmeATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [3H]αβmeATP binding sites than for the low affinity binding sites. Secondly, GTP-γ-S and pyrophosphate were selective ligands for the low affinity [3H]αβmeATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC50 values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [3H]αβmeATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity. In conclusion the present study has demonstrated that cations have a marked effect on the binding of [3H]αβmeATP in rat vas deferens. Of particular interest was the ability of CaCl2 to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl2 to increase the Bmax of the low affinity binding sites enabled these sites to be selectively labelled.