PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats

1. Microinjection of peptide YY (PYY, 7–46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY.
2. [Pro³⁴]rat(r)/porcine(p)PYY and [Pro³⁴]human(h)PYY (23–117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68±11 and 89±31 μmol 90 min⁻¹ respectively.
3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27±8 and 23±8 μmol 90 min⁻¹ respectively) and a higher dose (116 pmol) tended to reduce the response.
4. Pancreatic polypeptide (PP, 4–46 pmol), [Leu³¹,Pro³⁴]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY_3-36, pNPY_5-36 and pNPY_13-36 (25–168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion.
5. The rank order of potency of PYY⩾[Pro³⁴]r/pPYY=[Pro³⁴]hPYY>r/hNPY=pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu³¹,Pro³⁴]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.

     

Evidence for involvement of neuropeptide Y receptors in the regulation of food intake: studies with Y1-selective antagonist BIBP3226

1. Experiments were conducted to evaluate the effects of the novel non-peptide neuropeptide Y Y₁ receptor antagonist, BIBP3226 (N²-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide) on spontaneous, fasting-induced and NPY-induced food intake in rats. In addition to consumption of regular chow, the effects of BIBP3226 on consumption of highly palatable sweetened mash were monitored in a 1 h test on first exposure and after familiarization with novel food.
2. BIBP3226 (10.0 nmol, i.c.v.) had no effect on the consumption of regular chow, but reduced significantly the intake of highly palatable diet and the food intake stimulated by fasting (24 h). Neuropeptide Y (NPY, 1.0 nmol, i.c.v.) significantly increased the consumption of regular rat chow. This orexigenic effect of NPY was blocked by BIBP3226 (10.0 nmol, administered i.c.v. 5 min before NPY) at 30  min and 4  h, but not at 1 and 2  h. When animals were pretreated with diazepam (0.5 mg kg⁻¹, i.p., 20 min before NPY), BIBP3226 failed to suppress NPY-induced feeding.
3. An NPY Y₁ and Y₃ receptor agonist, [Leu³¹,Pro³⁴]NPY and a Y₅ receptor agonist human peptide YY_3–36 (hPYY_3–36, both 30 pmol), microinjected into the paraventricular nucleus of the hypothalamus (PVN) increased the consumption of regular rat chow. BIBP3226 (0.4 nmol, into the PVN) completely blocked the stimulatory effect of [Leu³¹,Pro³⁴]NPY but not that of hPYY_3–36. BIBP3226 (0.4 nmol) alone failed to modify the consumption of the regular chow. Higher doses of BIBP3226 (1.0 and 2.0 nmol) injected into the vicinity of the PVN reduced the consumption of the sweetened mash.
4. These results suggest that both the NPY Y₁ and Y₅ receptors in the PVN are involved in the regulation of food intake. The stimulatory effect of exogenous NPY is probably mediated through an NPY receptor subtype that is not identical with the Y₁ receptor (possibly Y₅ receptor). However, the NPY Y₁ receptors may mediate the effect of endogenous NPY in conditions of increased energy demand or on intake of highly palatable diets.

     

Receptor subtypes Y1 and Y5 are involved in the renal effects of neuropeptide Y

1. Systemic infusion of neuropeptide Y (NPY) reduces renal blood flow and can concomitantly increase diuresis, natriuresis and calciuresis in anaesthetized rats. The present study was designed to investigate whether the apparently contradictory NPY effects on renal blood flow and urine formation and composition are mediated by distinct NPY receptor subtypes.
2. NPY and its analogues, peptide YY (PYY), [Leu³¹, Pro³⁴]NPY and NPY₁₃₃₆, were infused in incremental doses of 0.3, 1 and 3 μg kg⁻¹ min⁻¹ for 45 min each and the results compared to those obtained in vehicle-infused rats. Renal blood flow was monitored in 15 min intervals, while urine excretion and composition were determined in 15 min collection periods. Plasma renin activity and aldosterone concentrations were measured at the end of the final infusion period.
3. Relative to vehicle NPY, PYY and [Leu³¹, Pro³⁴]NPY dose-dependently reduced renal blood flow and increased diuresis, natriuresis and calciuresis with roughly similar potency; NPY₁₃₃₆ slightly but significantly increased renal blood flow but had no effect on diuresis, natriuresis and calciuresis. None of the peptides significantly affected endogenous creatinine clearance or kaliuresis.
4. Plasma renin activity was significantly reduced by PYY. Quantitatively similar reductions were observed with NPY and [Leu³¹, Pro³⁴]NPY but failed to reach statistical significance with the given number of experiments. NPY₁₃₃₆ did not reduce plasma renin activity. None of the peptides significantly affected plasma aldosterone concentrations.
5. In another series of experiments infusion of PYY₃₃₆ (2 μg kg⁻¹ min⁻¹ for 120 min) did not reduce renal blood flow but significantly enhancd diuresis and natriuresis to a similar extent as the NPY 2 μg kg⁻¹ min⁻¹.
6. In a final series of experiments the Y₁-selective antagonist, BIBP 3226 (1 or 10 μg kg⁻¹ min⁻¹) dose-dependently antagonized reductions of renal blood flow elicited by bolus injections of NPY (0.130 μg kg⁻¹). BIBP 3226 (10 μg kg⁻¹ min⁻¹) also inhibited the effects of a 120 min infusion of NPY (2 μg kg⁻¹ min⁻¹) on renal blood flow but had only minor inhibitory effects on enhancements of diuresis and did not significantly affect enhancements of natriuresis.
7. We conclude that NPY reduces renal blood via a classical Y₁ subtype of NPY receptor. In contrast enhancements of diuresis, natriuresis and calciuresis occur via a distinct subtype which resembles the receptor that mediates NPY-induced enhancement of food intake.

     

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.

     

Endothelin receptor subtypes and their functional relevance in human small coronary arteries

1. The potent constrictor peptide endothelin (ET) has been implicated in various cardiovascular disorders including myocardial infarction and atherosclerosis. We have investigated the nature of ET receptor subtypes present on human small coronary arteries.
2. Small coronary arteries were mounted in a wire-myograph for in vitro pharmacology. To investigate the ET receptor subtypes present in different segments of the coronary vascular tree, arteries were grouped according to internal diameter. Responses in arteries with small internal diameters (mean 316.7±7.9 μm; Group B) were compared to those in larger arteries (mean 586.2±23.1 μm; Group A).
3. ET-1 consistently and potently contracted arteries from Group A and B, with EC₅₀ values of 1.7 (0.9–3.2) nM (n=15) and 2.3 (1.4–4.2) nM (n=14), respectively. No correlation was observed between ET-1 potency and internal diameter. The response to ET-1 was potently antagonized by the selective ET_A receptor antagonist PD156707 in both Group A and Group B, yielding pA₂ values of 8.60±0.12 (n=4–6) and 8.38±0.17 (n=4–6), respectively. Slopes from Schild regression were not significantly different from unity.
4. In contrast to ET-1, individual responses to ET-3 were variable. While all arteries from Group A responded to ET-3 (EC₅₀∼69 (23–210) nM) (n=12), no response was obtained in 5 of the 14 tested in Group B. Of those responding, many failed to reach a maximum at concentrations up to 1 μM. ET-1 was more potent than ET-3 in all arteries tested. A biphasic ET-3 response was observed in 8 arteries suggesting that a small ET_B population was also present in some patients. The selective ET_B receptor agonist sarafotoxin S6c had little or no effect up to 10 nM (n=4–6).
5. Responses to ET-1 and ET-3 were unaffected by removal of the endothelium in arteries from both groups suggesting a lack of functional, relaxant ET_B receptors on endothelial cells (n=5).
6. Using autoradiography, specific high density binding of the non-selective, ET_A/ET_B ligand [¹²⁵I]-ET-1 and selective ET_A ligand [¹²⁵I]-PD151242 was detected on the vascular smooth muscle layer of small intramyocardial coronary arteries (n=5). In contrast, little or no binding of the selective ET_B receptor ligand [¹²⁵I]-BQ3020 was observed (n=5). Similarly, [¹²⁵I]-ET-1 binding to vascular smooth muscle was absent in the presence of the selective ET_A receptor antagonist PD156707.
7. We conclude that human small epi- and intramyocardial coronary arteries express predominantly ET_A receptors and it is these receptors which mediate ET-induced contractions. A constrictor ET_B receptor population may exist in some patients. However, these receptors may have a limited role as contractions to ET-1 can be blocked fully by the selective ET_A receptor antagonist PD156707.

     

Neuropeptide Y potentiates via Y2-receptors the inhibitory effect of noradrenaline in rat locus coeruleus neurones

Summary Intracellular recordings were carried out in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). Pressure application of noradrenaline with various pulse durations inhibited the spontaneous frequency of action potentials and hyperpolarized the membrane. Neuropeptide Y (NPY), its C-terminal fragment NPY(16–36) and peptide YY (PYY), at a concentration of 0.1 µmol/l all, potentiated the effect of noradrenaline, while [Leu³¹, Pro³⁴]NPY (0.1 µmol/l) was inactive. These results are compatible with the presence of Y₂-type NPY-receptors at the cell somata of LC neurones. Correspondence to: P. Illes at the above address     

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.

     

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg⁹]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg⁹]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B₂ receptor antagonist, D-Arg⁰-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg⁹]-bradykinin was concentration-dependently inhibited by the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
7. These data demonstrate that bradykinin and [des-Arg⁹]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B₂-receptors and B₁-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

     

Characterization of [3H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxy- cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [³H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine ([³H]-DCG IV), was characterized in rat mGlu₂ receptor-transfected CHO cell membranes.
2. [³H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a K_d value of 160 nM and a B_max value of 10 pmol mg⁻¹ protein. Binding was not sensitive to Na⁺-dependent glutamate uptake blockers or Cl⁻-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [³H]-DCG IV binding.
3. Of the compounds observed to inhibit [³H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; K_i value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; K_i value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5′-O-(3-thiotriphosphate) (GTPγS) inhibited [³H]-DCG IV binding in a concentration-dependent manner, with an IC₅₀ value of 12 nM.
4. A highly significant correlation was observed between the potencies of compounds able to inhibit [³H]-DCG IV binding and potencies obtained for agonist activity in a GTPγ³⁵S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu₂ receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-α-methylserine-O-phosphate (MSOP).

     

Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization

1. We compared the binding properties of [³H]-desArg¹⁰-[Leu⁹]-kallidin, a radiolabelled kinin B₁ receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B₁ receptor.
2. The dissociation constant (K_D) of [³H]-desArg¹⁰-[Leu⁹]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B₁ receptor.
3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B₁ receptor agonist, desArg¹⁰-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B₁ receptor.
4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B₁ receptor.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

Tachykinin receptors in the guinea-pig isolated oesophagus: a complex system

1. The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea-pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar⁹,Met(O₂)¹¹]SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB, selective for NK₁, NK₂ and NK₃, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μM), thiorphan (1 μM) and amastatin (20 μM), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.
2. All agonists produced concentration-dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration-response curves of SP, [Nle¹⁰]NKA(4–10) and [MePhe⁷]NKB significantly leftwards and the concentration-response curve of NKB was shifted significantly rightwards. However, the EC₅₀ values were significantly different only for [Nle¹⁰]NKA(4–10) and NKB.
3. In the presence of the peptidase inhibitors, the EC₅₀ values of the selective agonists, [MePhe⁷]NKB (0.6 nM) and [Nle¹⁰]NKA(4–10) (66 nM) indicated the presence of both tachykinin NK₃ and NK₂ receptors. [MePhe⁷]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar⁹,Met(O₂)¹¹]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK₁ receptors were also present.
4. Assuming competitive antagonism, the NK₂-selective antagonist SR 48,968 (30 nM) gave apparent pK_B values of 8.13 and 8.65 for [Nle¹⁰]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK₂ receptors.
5. The NK₃-selective antagonist SR 142,801 (0.1 μM), suppressed responses to low (0.1–10 nM) concentrations of [MePhe⁷]NKB. These contractile responses to [MePhe⁷]NKB were also abolished by atropine (0.6 μM) suggesting that this response was mediated via cholinergic nerves.
6. It is concluded that the guinea-pig oesophagus is a complex system which has both NK₂ and NK₃ receptors and possibly some NK₁ receptors as well.

     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H₂ receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H₂ receptor were prepared and were used to infect insect Sf9 cells.
2. The His-tagged H₂ receptors expressed in insect Sf9 cells showed typical H₂ receptor characteristics as determined with [¹²⁵I]-aminopotentidine (APT) binding studies.
3. In Sf9 cells expressing the His-tagged H₂ receptor histamine was able to stimulate cyclic AMP production 9 fold (EC₅₀=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with K_i values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).
4. The expression of the His-tagged H₂ receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg⁻¹ protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×10⁶ copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [¹²⁵I]-APT binding up to 169±5% (mean±s.e.mean, n=3).
5. The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC₅₀ value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/10⁶ cells in the absence and 81±11 pmol/10⁶ cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3).
6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H₂ receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.
7. These experiments demonstrate the successful expression of His-tagged histamine H₂ receptors in insect Sf9 cells. The H₂ receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H₂ receptor protein is functional. These functional receptors are targeted to the plasma membrane.

     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [³H]-L-2-amino-4-phosphonobutyrate ([³H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors.
2. Specific [³H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [³H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum.
3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [³H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [³H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [³H]-L-AP4 uptake into cerebellar membranes, [³H]-L-AP4 binding to brain sections was sensitive to guanosine-5′-O-(3-thio)trisphosphate-γ-S.
4. In the molecular layer of the cerebellar cortex, [³H]-L-AP4 binding showed a maximal binding density (B_max) of 0.52±0.06 pmol mg⁻¹ tissue and an affinity (K_d) of 346 nM. The rank order of affinity for displacement of [³H]-L-AP4 binding to rat brain sections was: L-AP4>L-serine-O-phosphate>glutamate>(L)-2-aminomethyl-4-phosphonobutanoate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.

     

Validity of (−)-[3H]-CGP 12177A as a radioligand for the ‘putative β4-adrenoceptor' in rat atrium

1. We have recently suggested the existence in the heart of a ‘putative β₄-adrenoceptor'' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β₁- and β₂-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)-[³H]-CGP 12177A ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (−)-[³H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand.
2. Increasing concentrations of (−)-[³H]-CGP 12177A, in the absence or presence of 20 μM (−)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β₁- and β₂-adrenoceptors (pK_D 9.4±0.1, B_max 26.9±3.1 fmol mg^-1 protein) and higher concentrations bound to the ‘putative β₄-adrenoceptor'' (pK_D 7.5±0.1, B_max 47.7±4.9 fmol mg⁻¹ protein). In other experiments designed to exclude β₁- and β₂-adrenoceptors, (−)-[³H]-CGP 12177A (1–200 nM) binding in the presence of 500 nM (−)-propranolol was also saturable (pK_D 7.6±0.1, B_max 50.8±7.4 fmol mg⁻¹ protein).
3. The non-conventional partial agonists (−)-CGP 12177A (pK_i 7.3±0.2), (±)-cyanopindolol (pK_i 7.6±0.2), (−)-pindolol (pK_i 6.6±0.1) and (±)-carazolol (pK_i 7.2±0.2) and the antagonist (−)-bupranolol (pK_i 6.6±0.2), all competed for (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol at the ‘putative β₄-adrenoceptor'', with affinities closely similar to potencies and affinities determined in organ bath studies.
4. The catecholamines competed with (−)-[³H]-CGP 12177A at the ‘putative β₄-adrenoceptor'' in a stereoselective manner, (−)-noradrenaline (pK_iH 6.3±0.3, pK_iL 3.5±0.1), (−)-adrenaline (pK_iH 6.5±0.2, pK_iL 2.9±0.1), (−)-isoprenaline (pK_iH 6.2±0.5, pK_iL 3.4±0.1), (+)-isoprenaline (pK_i<1.7), (−)-RO363 ((−)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK_i 5.5±0.1).
5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (−)-CGP 12177A or (−)-isoprenaline to the ‘putative β₄-adrenoceptor''. In competition binding studies, (−)-CGP 12177A competed with (−)-[³H]-CGP 12177A for one receptor state in the absence (pK_i 7.3±0.2) or presence of GTP (pK_i 7.3±0.2). (−)-Isoprenaline competed with (−)-[³H]-CGP 12177A for two states in the absence (pK_iH 6.6±0.3, pK_iL 3.5±0.1; % H 25±7) or presence of GTP (pK_iH 6.2±0.5, pK_iL 3.4±0.1; % H 37±6). In contrast, at β₁-adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)-isoprenaline.
6. The specificity of binding to the ‘putative β₄-adrenoceptor'' was tested with compounds active at other receptors. High concentrations of the β₃-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy - ethyl]amino]propyl]phenoxy]acetic acid, 6 μM), SR 58611A (ethyl{(7S)-7-[(2R)-2 - (3 - chlorophenyl) - 2 - hydroxyethylamino] - 5,6,7,8 - tetrahydronaphtyl2 - yloxy} acetate hydrochloride, 6 μM), ZD 2079 ((±)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 μM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 μM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 μM) caused less than 22% inhibition of (−)-[³H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol. Histamine (1 mM), atropine (1 μM), phentolamine (10 μM), 5-HT (100 μM) and the 5-HT₄ receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding.
7. Non-conventional partial agonists, the antagonist (−)-bupranolol and catecholamines all competed for (−)-[³H]-CGP 12177A binding in the absence of (−)-propranolol at β₁-adrenoceptors, with affinities (pK_i) ranging from 1.6–3.6 log orders greater than at the ‘putative β₄-adrenoceptor''.
8. We have established and validated a radioligand binding assay in rat atrium for the ‘putative β₄-adrenoceptor'' which is distinct from β₁-, β₂- and β₃-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β₄-adrenoceptor''.