Differential agonist activity of somatostatin and L-362855 at human recombinant sst4 receptors

1. The operational characteristics of somatostatin (SRIF) sst₄ receptors are poorly understood. In this study, we have characterized human recombinant sst₄ receptors expressed in CHO cells (CHOsst₄) by radioligand binding and microphysiometry.
2. Increasing concentrations SRIF or other SRIF receptor ligands inhibited specific [¹²⁵I]-Tyr¹¹-SRIF binding in CHOsst₄ cell membranes with respective pIC₅₀ values of SRIF (8.82), L-362855 (7.40), BIM-23027 (<5.5) and MK-678 (<5.5).
3. These ligands displayed agonist activity, producing concentration-dependent increases in rates of extracellular acidification (EAR) with pEC₅₀ values of SRIF (9.6) and L-362855 (8.0), respectively. BIM-23027 and MK-678 were at least 1000 times weaker than SRIF. The SRIF maximum was about 40% of that observed with L-362855.
4. In the presence of SRIF (0.1–1 nM), concentration-effect curves to L-362855 were displaced to the right with a progressive reduction in the L-362855 maximum.
5. When cells were only exposed to a single maximally effective concentration of SRIF or L-362855, there was no difference in the magnitude of the agonist-induced increase in EAR. However, a second agonist challenge, 30 min later showed that responses to SRIF but not L-362855 were markedly desensitized.
6. When concentration-effect curves to SRIF and L-362855 were obtained by combining data from cells exposed to only a single agonist concentration, SRIF (pEC₅₀ 9.2) was approximately 20 times more potent than L-362855 (pEC₅₀ 8.0) but the maxima were the same. Responses to both SRIF and L-362855 were abolished by pertussis toxin.
7. SRIF and L-362855-induced increases in EAR were inhibited by N-ethyl isopropyl amiloride (10 μM) but were not modified by inhibitors of PKC (Go-6976), MAP kinase (PD-98059), tyrosine kinase (genistein) or tyrosine phosphatase (sodium orthovanadate).
8. The results suggest that SRIF-induced increases in EAR in CHOsst₄ cells involved activation of the Na⁺/H⁺ antiporter and were mediated via Gi/Go G proteins. Responses to SRIF, but not L-362855, were subject to marked desensitization which may be a consequence of differential activation of receptor-effector coupling pathways.

     

Outward current produced by somatostatin (SRIF) in rat anterior cingulate pyramidal cells in vitro

1. A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole-cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved.
2. SRIF (1–1000 nM) produced a concentration-dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba²⁺ (300 μM–1 mM), and was absent in the presence of a maximal concentration of the GABA_B receptor agonist, baclofen (100 μM). These observations suggest the outward current was carried by K⁺ ions.
3. SRIF analogues also elicited outward currents with a rank potency order of (EC₅₀, nM): octreotide (1.8)>BIM-23027 (3.7)>SRIF (20)=L-362,855 (20). BIM-23056 was without agonist or antagonist activity. Responses to L-362,855 were unlike those to the other agonists since they were sustained for the duration of the application.
4. The sst₂ receptor antagonist, L-Tyr⁸Cyanamid 154806 (1 μM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44±6% reversal) and L-362,855 (100 nM, 70±6% reversal) and fully reversed the response to BIM-23027 (10 nM). In contrast, L-Tyr⁸Cyanamid 154806 did not antagonize the response to baclofen (10 μM).
5. We conclude that SRIF activates a K⁺ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst₂ receptors.

     

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst_2(b) receptor (CHOsst_2(b)) and compared these to their effects in cells expressing the sst_2(a) receptor (CHOsst_2(a)). In contrast to the sst_2(a) receptor, the sst_2(b) receptor did not mediate inhibition of bFGF (10 ng ml⁻¹)-stimulated re-growth and cell proliferation. Rather, SRIF (0.1–1000 nM) and angiopeptin (0.1–1000 nM) stimulated basal re-growth and proliferation of CHOsst_2(b) cells in a concentration-dependent manner (estimated pEC₅₀ values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst₂ receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst_2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst_2(a) and sst_2(b) receptor splice variants mediate opposite effects on cell proliferation.     

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst₅ receptors (CHOsst₅), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers.
2. In VSMC, SRIF (0.1 nM–1 μM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC₅₀ 8.0–8.6) of the stimulated regeneration induced by sub-maximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml⁻¹), platelet-derived growth factor-BB (PDGF, 5 ng ml⁻¹) or endothelin-1 (ET-1, 100 nM). SRIF (pIC₅₀ 8.8) also inhibited bFGF-induced regeneration of CHOsst₅ cells.
3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml⁻¹) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM–1 μM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 μg ml⁻¹).
4. The sst₅ receptor-selective agonist, L-362,855 (pIC₅₀ 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst₅ cells whilst the sst₂ receptor-selective agonist, BIM-23027 (pIC₅₀ 6.8), was approximately 20 times weaker than SRIF.
5. The sst₅ receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst₅ cells (estimated pK_B values 8.8 and 8.3, respectively).
6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst₅ cells was abolished by pretreating cells with pertussis toxin (100 ng ml⁻¹) for 20 h.
7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst₅ receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive G_i/G_o proteins.

     

Identification and characterisation of heterogeneous somatostatin binding sites in rat distal colonic mucosa

We have previously shown that the somatostatin (SRIF) sst₂ receptor-selective peptide, BIM-23027, is a potent antisecretory agent in rat isolated distal colonic mucosa (RDCM) and in radioligand binding studies in RDCM membranes, it only maximally inhibited approximately 40% of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411). The aim of this study was to characterise the BIM-23027-sensitive and -insensitive SRIF binding sites in more detail and to compare their properties with those of the recombinant sst₂ receptor stably expressed in mouse fibroblast (Ltk^–) cells.SRIF-14, SRIF-28, CGP-23996 and D Trp⁸-SRIF-14 abolished [¹²⁵I]-Tyr¹¹-SRIF-14 binding (pIC₅₀ values, 8.7–9.7) but the competition curves had Hill slopes which were less than unity. Octreotide and L-362,855 inhibited binding over a wide concentration range (0.1 nM-1 M) and inhibition of binding was incomplete at the highest concentration studied. BIM-23056 (PIC₅₀ <6.5) was a weak inhibitor of [¹²⁵]-Tyr¹¹-SRIF-14 binding. GTPS decreased [¹²⁵I]-Tyr¹¹-SRIF-14 binding by 40%. Further binding experiments with [¹²⁵I]-Tyr¹¹-SRIF-14 were carried out in RDCM in the continuous presence of BIM-23027 (1 M). Under these conditions, seglitide had no effect on [¹²⁵I]-Tyr¹¹-SRIF-14 binding at concentrations up to 10 M, whilst SRIF-14 and SRIF-28 abolished specific [¹²⁵I]-Tyr¹¹-SRIF-14 binding in a manner which was consistent with the ligand binding to two sites. SRIF-14 and SRIF-28 displayed high affinity (pIC₅₀ values of 9.8 and 9.3 respectively) for approximately 70% of these binding sites and low affinity (pIC₅₀ values of 7.8 and 7.3) for the remaining sites. Octreotide, L-362,855 and BIM-23056 were weak inhibitors of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (PIC₅₀ <6.5). [¹²⁵I]-BIM-23027 labelled a single population of SRIF binding sites in RDCM membranes and mouse fibroblast (Ltk^–) cells stably expressing the human recombinant sst₂ receptor. There was a significant correlation between the affinitestimates of a range of SRIF analogues at inhibiting [¹²⁵I]-BIM-23027 binding in RDCM membranes and binding to the recombinant sst₂ receptor in Ltk^– cells, suggesting that the sites labelled by [¹²⁵I]-BIM-23027 in RDCM are similar to the sst₂ receptor. GTPS (100 M) decreased [¹²⁵I]-BIM-23027 binding in RDCM by 60%.The results from these studies demonstrate that [¹²⁵I]-Tyr¹¹-SRIF-14 labels a heterogeneous population of high affinity SRIF binding sites in RDCM membranes. The majority of these sites are insensitive to GTPS and display negligible affinity for the cyclic hexapeptides, BIM-23027 and seglitide. The remaining high affinity binding sites can be selectively labelled with [¹²⁵I]-BIM-23027, are sensitive to GTPS and show similar characteristics to the recombinant sst₂ receptor which appears to mediate the antisecretory effects of SRIF in the mucosa (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411).     

Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF₁-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [¹²⁵I]-Tyr¹¹-SRIF-14, with a pIC₅₀ of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF₁ receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst₂ subtype.
2. The stable radioligand, [¹²⁵I]-BIM-23027, bound with high affinity (K_d=13 pM, B_max=0.2 pmol mg⁻¹ protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4°C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15°C and 89% of the ligand dissociated at 37°C.
3. Equilibrium binding of [¹²⁵I]-BIM-23027 was partially (25%) inhibited by 10 μM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [¹²⁵I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 μM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4°C, 81% at 15°C and 93% at 37°C. Binding was totally inhibited by pretreatment of cells with pertussis toxin.
4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC₅₀ of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst₂ receptor, no rise in intracellular calcium concentration was observed with SRIF-14.
5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst₂ type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

     

Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization

1. Receptor-dependent internalization of somatostatin (SRIF) agonists has been a matter of controversy probably because [¹²⁵I]-Tyr¹¹-SRIF-14 is rapidly degraded. We have studied the internalization of a stable somatostatin analogue, [¹²⁵I]-BIM-23027, in a neuronal cell line, Neuro2A, which natively expresses somatostatin sst₂ receptors.
2. Incubation of Neuro2A cells with [¹²⁵I]-BIM-23027 at 37°C resulted in a time-dependent internalization of the ligand, which reached a maximum at 30 min. Acid-washing showed that cell-surface binding of the ligand accounted for only 34% of total binding at this time. internalization was dramatically reduced at 15°C.
3. internalization of [¹²⁵]-BIM-23027 was prevented by inclusion of unlabelled somatostatin receptor agonists in a concentration-dependent manner. The IC₅₀ values for inhibition of [¹²⁵I]-BIM-23027 internalization were approximately 100 fold lower than for inhibition of [¹²⁵I]-BIM-23027 binding to membrane homogenates but followed the same rank order of potencies.
4. Disruption of G-protein coupling by treatment with pertussis toxin caused a 60% reduction in internalization of ligand. A combination of antimycin (50 nM) and deoxyglucose (50 mM) pretreatment, which leads to a depletion of cellular ATP, decreased internalization of [¹²⁵I]-BIM-23027 by 66% of control and increased the proportion of surface-bound ligand. Hypertonic sucrose, which prevents clathrin-mediated endocytosis, reversibly abolished the internalization of ligand without increasing the proportion bound at the cell surface.
5. After internalization of [¹²⁵I]-BIM-23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37°C. This finding suggests that the intracellular content of [¹²⁵I]-BIM-23027 reaches a steady state which is determined by the rates of both internalization and recycling of the ligand. In contrast to studies in which the internalization of [¹²⁵I]-Tyr¹¹-SRIF-14 was examined, neither internalized nor recycled [¹²⁵I]-BIM-23027 was degraded to its component amino acids.
6. These findings indicate that the somatostatin agonist, [¹²⁵I]-BIM-23027, is internalized in a receptor-dependent manner which involves clathrin-coated pits in Neuro2A cells. Furthermore, much of the internalized ligand is rapidly recycled back to the extracellular medium without undergoing significant degradation.

     

Somatostatin receptors mediating inhibition of basal and stimulated electrogenic ion transport in rat isolated distal colonic mucosa

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 M) and amastatin (10 M), 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 M) 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 M) 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.     

[3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

1. The present study describes the direct labelling of A_2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 1.34 nM and 75 fmol mg⁻¹ protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [³H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A_2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.
3. Thermodynamic data indicated that [³H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A_2A adenosine receptors.
4. It was concluded that in human neutrophil membranes, [³H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A_2A adenosine receptors of other tissues. The receptors labelled by [³H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

Spermine modulation of specific [3H]-gabapentin binding to the detergent-solubilized porcine cerebral cortex α2δ calcium channel subunit

1. Recent studies have identified the [³H]-gabapentin-binding protein, purified from porcine cerebral cortical membranes, as the α₂δ subunit of voltage-sensitive calcium channels (Gee et al., 1996). The present study investigates the influence of the polyamine spermine on specific [³H]-gabapentin binding to detergent-solubilized porcine cerebral cortical membranes.
2. Spermine, spermidine, 1,10 diaminodecane, Mg²⁺ and Zn²⁺, all divalent cations, displaced [³H]-gabapentin binding to detergent-solubilized membranes in a concentration-dependent manner with a maximal inhibition of 65–75%. Radioligand binding studies showed that spermine did not directly interact with the [³H]-gabapentin-binding site. Spermine inhibited [³H]-gabapentin binding by interacting with a polyamine-sensitive allosteric site on the membrane protein. The steep concentration-dependence of spermine inhibition of [³H]-gabapentin binding may suggest multi-site co-operativity.
3. Prolonged dialysis of cerebral cortical membranes and Tween 20-solubilized membranes resulted in a >2.0 fold increase in [³H]-gabapentin binding. The increase in binding was due to the removal of a heat stable, low molecular weight (<12,000Da) endogenous molecule which influences [³H]-gabapentin binding competitively.
4. Dialysis of detergent-solubilized cerebral cortical membranes also resulted in a decrease in the maximum inhibition of [³H]-gabapentin binding by spermine. Since the rates of the increase in [³H]-gabapentin binding and the loss of the ability of spermine to inhibit [³H]-gabapentin binding on dialysis were different it was inferred that a second endogenous ligand was removed during dialysis.
5. During initial steps of purification of the [³H]-gabapentin-binding protein there was a decrease in the maximum inhibition of [³H]-gabapentin binding by spermine. The loss of the second endogenous molecule during initial purification would reasonably explain the reduction in inhibition of binding by spermine. However, spermine stimulation of [³H]-gabapentin binding to material that eluted from the gel-filtration column later in the purification scheme does not appear to be due to removal of a dialysable endogenous factor or to the dissociation of other calcium channel subunit(s).
6. Adding back dialysate, before or after boiling, to detergent solubilized membranes resulted in a dose-dependent restoration of the inhibition of [³H]-gabapentin binding and of the maximal inhibition [³H]-gabapentin binding by spermine. This result is consistent with the re-addition of two endogenous heat stable ligands.
7. The finding that [³H]-gabapentin binding to the pure α₂δ subunit was stimulated by spermine indicates that the α₂δ subunit of voltage-sensitive calcium channels bears a modulatory spermine site. Such a spermine site has not been identified before. Spermine stimulation of [³H]-gabapentin binding to the purified protein was reversed to inhibition after adding back dialysate. Thus the inhibitory spermine effect in membranes is also probably due to one or more modulatory sites on the α₂δ subunit.

     

D2 dopamine receptors and modulation of spontaneous acetylcholine (ACh) release from rat striatal synaptosomes

1. The effect of two D_3/2 dopamine receptor agonists, LY-171555 (quinpirole) and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) on spontaneous [³H]-acetylcholine ([³H]-ACh) release were investigated in rat striatal synaptosomes.
2. Quinpirole and 7-OH-DPAT inhibited in a concentration-dependent manner the basal efflux of [³H]-ACh with similar E_max (maximal inhibitory effect) values (29.95±2.91% and 33.19±1.21%, respectively). Significant differences were obtained between the pEC₅₀ (−log of molar concentration) of quinpirole (7.87±0.12) and 7-OH-DPAT (7.21±0.17; P<0.01).
3. Different concentrations (0.3–10 nM) of haloperidol (D_2/3 dopamine receptor antagonist) shifted to the right the concentration-response curves elicited by quinpirole and 7-OH-DPAT, without modifications in the E_max.
4. Slopes of a Schild plot obtained with haloperidol in the presence of quinpirole and 7-OH-DPAT were not signficantly different from unity (0.85±0.05 and 1.17±0.11, respectively) and consequently haloperidol interacted with a homogeneous receptor population. The pK_B values of haloperidol obtained from Schild regression were 9.96±0.15 (in presence of quinpirole) and 9.90±0.09 (in presence of 7-OH-DPAT).
5. Specific binding of [³H]-YM-09151-2 to membranes of striatal synaptosomes and cells expressing D₂ and D₃ dopamine receptors was inhibited by haloperidol. Analysis of competition curves revealed the existence of a single population of receptors. There were no differences between the estimated pK_i (−log of molar concentration) values for synaptosomes (8.96±0.02) and cells expressing D₂ receptors (8.81±0.05), but the pK_i value from cells expressing D₃ dopamine receptors differed significantly (8.48±0.06; P<0.01).
6. In conclusion, the data obtained in the present study indicate that quinpirole and 7-OH-DPAT, two D_3/2 dopamine receptor agonists, inhibit the spontaneous [³H]-ACh efflux and this effect is competitively antagonized by haloperidol and probably mediated through dopamine D₂ receptors.

     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A_2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([³H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
2. Saturation experiments revealed a single class of binding sites with K_d and B_max values of 0.85 nM and 35 fmol mg⁻¹ protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [³H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A_2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
3. Thermodynamic data indicate that [³H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A_2A-adenosine receptors.
4. It is concluded that in human lymphocyte membranes [³H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A_2A-receptor. The presence of A_2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.

     

Characterization of [3H]-prostaglandin E2 binding to prostaglandin EP4 receptors expressed with Semliki Forest virus

1. The human prostaglandin EP₄ receptor has been expressed by use of the Semliki Forest virus system.
2. In cell membranes [³H]-prostaglandin E₂ ([³H]-PGE₂) bound to a high affinity site with a K_d of 1.12±0.3 nM and a B_max of 3.1±0.3 pmol mg⁻¹ protein.
3. In competition studies the rank order of potency for prostaglandins was PGE₂  = PGE₁  ≈#62;  PGF_2α =PGI₂.
4. The binding of [³H]-PGE₂ to cell membranes was inhibited by approximately 60% by the addition of guanylnucleotides, suggesting that this proportion of the receptors was G-protein coupled.
5. [³H]-PGE₂ binding was increased by greater than 200% by the addition of divalent cations, with little change in the IC₅₀ of PGE₂.
6. In saturation studies removal of divalent cations and addition of GTPγS resulted in a 65% reduction in the B_max with no change in the K_d. These results are consistent with the ligand labelling two states of the receptor R*, a high affinity state and R*G, a high affinity G protein coupled state.

     

The agonist activities of the putative antipsychotic agents,L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor

1. Dopamine D₄ receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced.
2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D_4.4 receptor (HEK293/D₄ cells). [³H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response.
3. [³H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D₄ cell homogenates (B_max 505±90 fmol mg⁻¹ protein, pK_D 9.5±0.1, n=3). Inhibition of specific [³H]-spiperone binding was observed with spiperone (pK_i 9.6±0.1, n=3), clozapine (pK_i 7.4±0.1, n=4), L-745,870 (pK_i 8.5±0.1, n=3) and U-101958 (pK_i 8.9±0.1, n=3). By contrast, raclopride was very weak (pK_i<5, n=3).
4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent fashion (E_max 71±2% inhibition of forskolin-stimulated levels, pEC₅₀ 8.7±0.1, n=10). This effect was mimicked by the dopamine D₂-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).
5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D₄ cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC₅₀) values of L-745,870 and U-101958 were 9.0±0.2 (n=4) and 8.7±0.3 (n=3), consistent with pK_i values determined in radioligand binding studies.
6. Dopamine, L-745,870 and U-101958 (up to 1 μM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells.
7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D₄ cells could be antagonized by spiperone (pK_B 8.2–8.8) and clozapine (pK_B 7.1), but not by raclopride (pK_B<5). None of these antagonists had any significant agonist activity at concentrations up to 10 μM.
8. These results show that the putative dopamine D₄ receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D_4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D₄ receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

     

Modulation of spasmogen-stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle

1. The effects of isoenzyme-selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM).
2. Neither the PDE3-selective inhibitor ORG 9935, nor the PDE4-selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 μM) concentration-dependently (−log EC₅₀ (M), 6.55±0.15; n=3) increased cyclic AMP levels to about 70% of the maximal response to the β-adrenoceptor agonist isoprenaline.
3. Rolipram per se inhibited histamine-stimulated [³H]-inositol (poly)phosphate ([³H]-InsP_X) accumulation by >80% (−log EC₅₀ (M), 6.92±0.11; n=3). Although ORG 9935 (1 μM) had little effect on histamine-stimulated [³H]-InsP_X accumulation alone it greatly facilitated the inhibitory action of rolipram (−log EC₅₀ (M), 8.82±0.39; n=3). The effects of PDE3 and/or PDE4 inhibition on [³H]-InsP_X accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 μM).
4. The greater-than-additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol-mediated phasic contraction, additions of rolipram (10 μM) or ORG 9935 (1 μM) were without effect, whereas added together the inhibitors caused a significant (P<0.01) 40% reduction in the peak phasic contractile response.
5. The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP₃) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 μM) rolipram concentration-dependently inhibited carbachol-stimulated InsP₃ accumulation by ⩾50% (−log EC₅₀ (M), 6.77±0.21; n=4).
6. Carbachol (100 μM) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca²⁺ from the incubation medium prevented the carbachol-evoked decrease in cyclic AMP and this coincided with a greater inhibition (⩾80%) of the carbachol-stimulated InsP₃ response.
7. These data indicate that combined PDE3 and PDE4 inhibition has greater-than-additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor-mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP₃ generation.

     

Characterization and localization of thromboxane A2 receptor in human and guinea-pig nasal mucosa using radiolabelled (+)-S-145

1. TxA₂ receptor (TP-receptor) antagonists such as S-1452 and Bay u 3405 have been shown to be effective in alleviating nasal blockage in patients with allergic rhinitis as well as guinea-pig allergic rhinitis models. The present study was conducted to examine the existence and localization of the TP-receptor in human and guinea-pig nasal mucosa by in vitro receptor binding autoradiography using radiolabelled (+)-S-145, which is a potent and specific TP-receptor antagonist with an extremely slow dissociation rate.
2. We ascertained the binding specificity of [³H]-(+)-S-145 in human and guinea-pig platelet membranes by comparing the ability of four TP-receptor ligands of U-46619, (+)-S-145, I-(+)-S-145 and Bay u 3405 to displace the specific binding of [³H]-(+)-S-145 and [³H]-U-46619. The rank order of potency (K_i) for the displacement was correlated highly with that determined from [³H]-U-46619 binding to the same preparations.
3. Quantitative autoradiography using a radioluminographic imaging plate system, in which the radioactivity of [³H]-(+)-S-145 is expressed as photostimulated luminescence (PSL) per area (mm²), revealed that specific binding of [³H]-(+)-S-145 to human and guinea-pig nasal mucosa was saturable. Scatchard analysis showed about three fold higher affinity and two fold greater maximal binding to the nasal mucosa of humans than that of guinea-pigs: the K_D and B_max values in human mucosa were 2.82±0.35 nM and 6.47±0.33 PSL mm⁻² and those in guinea-pig mucosa were 8.23±1.93 nM and 3.37±0.66 PSL mm⁻², respectively.
4. Specific [³H]-(+)-S-145 binding to cryostat sections of human and guinea-pig nasal mucosa was displaced by another TP-receptor antagonist, Bay u 3405, and a TP-receptor agonist, U-46619. The order of potency (K_i value: nM) was (+)-S-145 (2.5) > Bay u 3405 (15.4) >> U-46619 (359.6) in human nasal mucosa and (+)-S-145 (22.8) > U-46619 (49.8) ≈amp; Bay u 3405 (62.1) in guinea-pig nasal mucosa. These rank orders showed rather good correlation with those obtained for the respective platelet membranes.
5. Autoradiographs of human nasal mucosa demonstrated that specific [¹²⁵I]-(+)-S-145 binding sites mainly exist on the smooth muscle layers of venous sinusoids and arterioles in the lamina propria, with few or no binding sites in the epithelium and nasal gland.
6. We concluded that radiolabelled (+)-S-145 can be used as a TP-receptor ligand for autoradiographic study, and that the TP-receptor is exclusively located on smooth muscle layers of venous sinusoids and arterioles in the nasal mucosa. The potent vasoconstrictive activity of TxA₂ may cause reduction of local blood flow followed by mucosal oedema probably through mechanisms of vascular injury such as ischaemia-reperfusion.

     

Postnatal development of δ-opioid receptor subtypes in mice

1. The density and affinity of binding sites for the δ-selective opioid ligands [³H]-[D-Ala², Asp⁴]deltorphin (DELT-I), [³H]-[D-Ala²Glu⁴]-deltorphin (DELT-II), [³H]-[D-Pen²,D-Pen⁵]enkephalin (DPDPE), and [³H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice.
2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (B_max) labelled by [³H]-NTI was about one half that labelled by [³H]-DELT-I, [³H]-DELT-II and [³H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE B_max was 25% less than the deltorphin-II B_max.
3. In saturation experiments, specific binding of [³H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34% high affinity site, K_d=1.08 nM and 66% low affinity sites, K_d=39.9 nM).
4. DPDPE produced a biphasic inhibition of specific [³H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice.
5. In adult mouse brain labelled with [³H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites. DELT-I and DPDPE produced monophasic inhibition of specific [³H]-DELT-II binding to brain homogenates of adult mice.
6. These data suggest that a sub-population of δ-sites (probably the δ₂-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward.
7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three δ-agonists was: DELT-I> DELT-II> DPDPE in 10 day-old mice, and: DELT-I= DELT-II> DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the δ₂-subtype.