Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations

ATP-sensitive K⁺ channels (K_ATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [³H]-P1075 ([³H]-N-cyano-N-(1,1-dimethylpropyl)-N-3-pyridylguanidine), a selective opener of K_ATP channels, in rat glomerular preparations.Equilibrium (saturation, competition) and kinetic experiments indicated that [³H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg^–1 glomerular protein. The association rate constant of the complex was 6,5×10⁷ M^–1 min^–1; dissociation occurred with a half-time of 6.2 min. Specific [³H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [³H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [³H]-P1075 binding was inhibited by representatives of the different classes of K_ATP channel openers and by sulphonylureatype blockers with inhibition constants similar to those obtained in rat aortic rings.It is concluded that rat glomerular preparations possess specific binding sites for K_ATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 m^–2) is much higher than that of K_ATP channels in smooth muscle.     

KATP channel openers: tissue selectivity of original 3-alkylaminopyrido- and 3-alkylaminobenzothiadiazine 1,1-dioxides

The present study was designed to further evaluate the biological effects and tissue selectivity of new 3-alkylaminobenzo- and 3-alkylaminopyridothiadiazine 1,1-dioxides bearing identical branched alkylamino chains at the 3-position. These original compounds were compared with their parent molecules; namely the K(ATP) channel openers diazoxide and pinacidil. All tested 3-alkylamino-substituted derivatives provoked a marked, concentration-dependent inhibition of the glucose-induced insulin secretion from rat pancreatic islets. The 3-alkylaminopyridothiadiazine 1,1-dioxides evoked a weak vasorelaxant activity whilst their 7-halo-substituted benzothiadiazine counterparts elicited pronounced, concentration-dependent, relaxations of rat aortic rings. The myorelaxant effect of the different drugs was tightly correlated with their capacity to increase (86)Rb outflow ((42)K substitute) from prelabelled and perifused rat aortic rings. EC(50)/IC(50) ratios (vascular/pancreatic) revealed a pronounced selectivity of the 3-alkylaminopyridothiadiazine 1,1-dioxides towards the pancreatic endocrine tissue. Structure-activity relationships suggest that, besides the requirement of an electronegative pole at the 7-position of the heterocycle, a minimal steric hindrance confers an optimal insulin-secreting cell selectivity. Lastly, radioisotopic, electrophysiological and pharmacological investigations indicate that the marked vasorelaxant properties of the 3-alkylaminobenzothiadiazine 1,1-dioxides are related to the activation of smooth muscle K(ATP) channels.     

SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil

1. The classical ATP sensitive K⁺ (K_ATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K⁺ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K⁺ channel openers, such as pinacidil and nicorandil.
2. In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the K_ATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method.
3. In the cell-attached recordings (145 mM K⁺ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K⁺ channel in both the transfected cells.
4. In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR_2B/Kir6.2 and SUR_2A/Kir6.2 channels (EC₅₀ of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR_2B/Kir6.2 channel >100 times more potently than the SUR_2A/Kir6.2 (EC₅₀ of ∼10 μM and >500 μM, respectively).
5. Thus, nicorandil, but not pinacidil, preferentially activates the K_ATP channels containing SUR_2B. Because SUR_2A and SUR_2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR_2B may play a critical role in the action of nicorandil on the vascular type classical K_ATP channel.

     

Diazoxide acts more as a PKC-epsilon activator, and indirectly activates the mitochondrial K(ATP) channel conferring cardioprotection against hypoxic injury

BACKGROUND AND PURPOSE: Diazoxide, a well-known opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has been demonstrated to exert cardioprotective effect against ischemic injury through the mitoK(ATP) channel and protein kinase C (PKC). We aimed to clarify the role of PKC isoforms and the relationship between the PKC isoforms and the mitoK(ATP) channel in diazoxide-induced cardioprotection. EXPERIMENTAL APPROACH: In H9c2 cells and neonatal rat cardiomyocytes, PKC-epsilon activation was examined by Western blotting and kinase assay. Flavoprotein fluorescence, mitochondrial Ca(2+) and mitochondrial membrane potential were measured by confocal microscopy. Cell death was determined by TUNEL assay. KEY RESULTS: Diazoxide (100 microM) induced translocation of PKC-epsilon from the cytosolic to the mitochondrial fraction. Specific blockade of PKC-epsilon by either epsilonV1-2 or dominant negative mutant PKC-epsilon (PKC-epsilon KR) abolished the anti-apoptotic effect of diazoxide. Diazoxide-induced flavoprotein oxidation was inhibited by either epsilonV1-2 or PKC-epsilon KR transfection. Treatment with 5-hydroxydecanoate (5-HD) did not affect translocation and activation of PKC-epsilon induced by diazoxide. Transfection with wild type PKC-epsilon mimicked the flavoprotein-oxidizing effect of diazoxide, and this effect was completely blocked by epsilonV1-2 or 5-HD. Diazoxide prevented the increase in mitochondrial Ca(2+), mitochondrial depolarization and cytochrome c release induced by hypoxia and all these effects of diazoxide were blocked by epsilonV1-2 or 5-HD. CONCLUSIONS AND IMPLICATIONS: Diazoxide induced isoform-specific translocation of PKC-epsilon as an upstream signaling molecule for the mitoK(ATP) channel, rendering cardiomyocytes resistant to hypoxic injury through inhibition of the mitochondrial death pathway.     

Functional coupling of a recombinant Human 5-HT5A receptor to G-proteins in HEK-293 cells

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT_5A receptor.
2. We have shown that ligand activation of the Human 5-HT_5A receptor results in functional coupling to G-proteins in HEK-293 cells.
3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [³⁵S]-GTPγS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT_5A receptor in any cell.
4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT_5A receptor cell line, suggesting a functional coupling to a Gαi, G-protein in the HEK-293 cell line.
5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT_5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

     

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
2. In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA₂ value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D₁ receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [³H]-N-methylscopolamine ([³H]-NMS) bound to striatal M₄ receptors with a monophasic inhibitory curve and a pK_i value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC₅₀ of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC₅₀=50 nM) and stimulating at higher concentrations (EC₅₀=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pK_i value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
5. As in rat striatum, in CHO cell membranes the displacement of [³H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
6. Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [³⁵S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [³⁵S]-GTPγS binding with a pA₂ value of 7.48.
7. These results show that at the striatal M₄ receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

     

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

1. Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y₂ receptor described here for the first time.
2. More than 70% of the hippocampal neurones were endowed with [¹²⁵I]-[Leu³¹,Pro³⁴]PYY Y₁-like receptor silver grain accumulations and Y₁ receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining.
3. Competition binding profiles revealed that specific [¹²⁵I]-[Leu³¹,Pro³⁴]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y₁ receptor sub-type with [Leu³¹,Pro³⁴]substituted NPY/PYY analogues>>C-terminal fragments=pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [¹²⁵I]-[Leu³¹,Pro³⁴]PYY of the newly cloned Y₄, Y₅ and Y₆ receptors.
4. The expression of the genuine Y₁ receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y₂-like/[¹²⁵I]-PYY_3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y₁ and the Y₂ receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.
5. Taken together, these results suggest that the Y₁ receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

     

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 the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes

1. SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [³H]-SB-204269 in rat forebrain membranes.
2. Specific [³H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (K_D) of 32±1 nM and a maximum binding capacity (B_max) of 253±18 fmol mg⁻¹ protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na⁺, K⁺, Ca²⁺ or Mg²⁺ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues.
3. Specific [³H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action.
4. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [³H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na⁺ or K⁺ channels or various other receptors did not demonstrate any affinity for the binding site.
5. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.

     

Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors

The cannabinoid receptor antagonist SR141716A has been suggested to be an inverse agonist at CB₁ receptors in some isolated intact tissues. We found that the basal incorporation of [³⁵S]-GTPγS in Chinese hamster ovary cells expressing human recombinant CB₁ and CB₂ receptors was inhibited by SR141716A (mean pEC₅₀s 8.26 and 6.00, respectively), whereas cannabinol (10 μM) had no significant effect at hCB₁ receptors but inhibited the binding at hCB₂ receptors. As cannabinol had no effect on basal [³⁵S]-GTPγS binding at hCB₁ at a concentration 100 fold higher than its binding affinity (K_i=0.1 μM), we conclude that endogenous cannabinoid receptor agonists are not a confounding factor and suggest the actions of SR141716A at the hCB₁ receptor, and the actions of SR141716A and cannabinol at the hCB₂ receptor, are due to inverse agonism.     

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.

     

Gastroprotective Activities of Sennoside A and Sennoside B via the Up-Regulation of Prostaglandin E2 and the Inhibition of H+/K+-ATPase

Sennoside A (erythro) and sennoside B (threo) are dianthrone glycosides and diastereomers. We investigated their abilities to prevent the gastric lesions associated with diseases, such as, gastritis and gastric ulcer. To elucidate their gastroprotective effects, the inhibitions of HCl•EtOH-induced gastritis and indomethacin-induced gastric ulcers were assessed in rats. It was observed that both sennoside A and sennoside B increased prostaglandin E₂ (PGE₂) levels and inhibited H⁺/K⁺-ATPase (proton pump). In a rat model, both compounds reduced gastric juice, total acidity and increased pH, indicating that proton pump inhibition reduces gastric acid secretion. Furthermore, sennoside A and B increased PGE₂ in a concentration-dependent manner. In a gastric emptying and intestinal transporting rate experiment, both sennoside A and sennoside B accelerated motility. Our results thus suggest that sennoside A and sennoside B possess significant gastroprotective activities and they might be useful for the treatment of gastric disease.     

(+)-WAY 100135, a partial agonist,at native and recombinant 5-HT1B/1D receptors

1. We have studied the effects of the purportedly selective 5-HT_1A receptor antagonist (+)-WAY100135 on electrically stimulated 5-hydroxytryptamine (5-HT) efflux in the ventrolateral geniculate nucleus (vLGN), and its affinity at human 5-HT_1B and 5-HT_1D receptors stably expressed in Chinese hamster ovary (CHO) cells.
2. On short ‘pseudo single pulse'' stimulations (20 pulses at 100 Hz, 190 ms train duration), (+)-WAY100135 (1.0 μM) decreased 5-HT efflux in the vLGN to 68±8% of pre-drug values (P<0.01). This decrease could be blocked by the 5-HT_1D/1B receptor antagonist GR 127935 (50 nM). Conversely, when long stimulations (20 pulses at 20 Hz, 950 ms train) were used, (+)-WAY100135 had no effect on 5-HT efflux (84±8% of pre-drug values) although both methiothepin (200nM) and GR 127935 (50 nM) caused significant increases (to 175±18 and 130±10% of pre-drug values, respectively).
3. Paroxetine (100 nM), the selective 5-HT reuptake inhibitor, increased stimulated 5-HT efflux and re-uptake half-life (to 145±18% and 649±121%, respectively) on pseudo single pulse stimulations. When (+)-WAY 100135 was added in combination with the uptake blocker, the effect of paroxetine on stimulated 5-HT efflux was potentiated to 282±48% (P<0.01) without further effect on the 5-HT re-uptake half-life.
4. The affinity and intrinsic activity of (+)-WAY 100135 were determined at recombinant human 5-HT_1B and 5-HT_1D receptors expressed in CHO cells, by use of radioligand binding and [³⁵S]-GTPγS binding. (+)-WAY 100135 was a partial agonist at human 5-HT_1B and 5-HT_1D receptors with moderately high affinity for 5-HT_1D receptors (pEC₅₀=7.61).
5. In conclusion, (+)-WAY 100135 was found to be not a selective 5-HT_1A autoreceptor antagonist but may act as a partial agonist at the 5-HT_1B/1D receptor, displaying agonist or antagonist properties depending on the stimulation protocol used and the resultant 5-HT ‘tone'' at the receptor.

     

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.

     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

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.

     

Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg

1. The sea urchin egg homogenate is an ideal model to characterize Ca²⁺-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP₃- and ryanodine-sensitive Ca²⁺-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist.
2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca²⁺-releasing agent, NAADP, compared to inositol trisphosphate (InsP₃) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors.
3. NAADP-induced Ca²⁺-release was blocked by L-type Ca²⁺-channel blockers and by Bay K 8644, while InsP₃- and cyclic ADPR-induced Ca²⁺-release were insensitive to these agents. L-type Ca²⁺-channel blockers did not displace [³²P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion.
4. Similarly, a number of K⁺-channel antagonists blocked NAADP-induced Ca²⁺-release selectively over InsP₃- and cyclic ADPR-induced Ca²⁺-release. Radioligand studies showed that these agents were not competitive antagonists.
5. As has been shown for InsP₃ and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca²⁺-release mechanisms.
6. The presence of K⁺ was not essential for NAADP-induced Ca²⁺-release, since substitution of K⁺ with other monovalent cations in the experimental media did not significantly alter Ca²⁺ release by NAADP. On the contrary, cyclic ADPR and InsP₃-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K⁺. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca²⁺-release. While the alkaline condition permitted InsP₃ and cyclic ADPR-induced Ca²⁺-release, the acidic condition completely hampered both Ca²⁺-release mechanisms.
7. The present results characterize pharmacologically and biochemically the novel Ca²⁺-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.