Inhibition Of Forskolin-stimulated Adenylate Cyclase Research Articles

Receptor signaling and behavioral properties of EFF0311, a longer‐acting selective full D1 agonist as a potential treatment for Parkinson's disease

D1 dopamine full agonists like dihydrexidine (DHX) were previously used to show that D1, but not D2, agonists could match the efficacy of levodopa in the primate MPTP Parkinson's disease (PD) model and in humans with PD. Short duration of action, toxicity, and/or limited bioavailability have prevented a D1 agonist from being approved for clinical use, with only DHX (very short acting) available for limited investigator‐initiated clinical studies. We now report the properties of EFF0311, a DHX analog with greater D1 selectivity and nanomolar affinity for D1 and D5 receptors. EFF0311 had 100% intrinsic activity in stimulating adenylate cyclase via D1 receptors in expressed systems or brain. EFF0311 (relative to quinpirole) was far less potent at D2L‐mediated stimulation of arachidonic acid release than in inhibition of forskolin‐stimulated adenylate cyclase, consistent with functionally selective actions at D2L receptors. In the unilateral 6‐OHDA rat model of PD, EFF0311 effects lasted 3–5 times longer than DHX. These behavioral effects were completely blocked by a D1 antagonist, but not decreased by a D2 antagonist. The properties of EFF0311 (D1 full agonism, longer duration of action, atypical D2 actions) suggest it may be an ideal PD drug candidate.Grant support: MH082441, MH040537, PA Keystone Innovation Grant (RBM), NS042402 and PA Tobacco settlement fund (TS). RBM has a potential CoI.

Fish somatostatin sst3 receptor: comparison of radioligand and GTPγS binding, adenylate cyclase and phospholipase C activities reveals different agonist‐dependent pharmacological signatures

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.

Functional characterisation of the putative somatostatin sst2 receptor antagonist CYN 154806.

The two forms (DTyr8 and LTyr8) of the putative somatostatin sst2 receptor antagonist CYN 154806 (Ac-4NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-D/LTyr-NH2) were investigated on recombinant human somatostatin receptors and endogenous guinea-pig ileum receptors. In radioligand binding studies using the agonist radioligands [125I]LTT-SRIF-28, [125I][Tyr10]cortistatin-14, [125I]CGP 23996 and [125I][Tyr3]octreotide in Chinese hamster lung fibroblast (CCL39) and Chinese hamster ovary (CHO) cells expressing human somatostatin receptors (hsst1-5), CYN 154806 binds to sst2 receptors with nanomolar affinity (pKD=8.14-8.89), 40- to 4500-fold higher than for sst1, sst3 or sst4. High affinity was also demonstrated for sst5 receptors, particularly for LTyr8CYN 154806 where the sst5 affinity was higher than for sst2 receptors when using [125I]CGP 23996 and [125I][Tyr3]octreotide. Functional properties of the compounds were examined in Chinese hamster ovary (CHO) cells expressing human sst2 receptors, in (1) inhibition of forskolin-stimulated adenylate cyclase, (2) stimulation of serum response element-driven luciferase expression and (3) [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPS) binding. L- and DTyr8CYN 154806 showed full agonism at inhibition of forskolin-stimulated cAMP accumulation (pEC50=7.73 for both, Emax 104% and 78%, respectively), partial agonism at luciferase expression (pEC50=7.85 and 8.16, Emax=50% and 29%, respectively) and behaved as apparently silent antagonists at [35S]GTPS binding (no agonism observed, pKB=6.88 and 7.50, respectively). The agonist potential was confirmed in isolated guinea-pig ileum preparations via measurement of SRIF-induced inhibition of neurotransmission, where the L-isoform had marked agonism (pEC50=8.23, Emax=32%) whereas the D-isoform was apparently devoid of agonism. The present data suggest that CYN 154806 should be used with caution as an sst2 receptor antagonist tool, since it possesses intrinsic activity at sst2, and high affinity for both sst2 and sst5 receptors. The DTyr form, having lower intrinsic activity, especially in natural tissues, and greater selectivity for sst2 receptors, may be more reliable than LTyr CYN 154806.

Characterisation of human recombinant somatostatin receptors. 3. Modulation of adenylate cyclase activity.

The five human somatostatin receptor subtypes (hsst1-5) were stably expressed in CCL39 cells (Chinese hamster lung fibroblast cells) to study the inhibition of forskolin-stimulated adenylate cyclase (FSAC) activity induced by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and SRIF peptide analogues. Inhibition of FSAC was observed with all five receptors, although the maximal effects produced by SRIF14 varied from around 40% (sst1, sst2, sst4) to 67% (sst3, sst5) reflecting to some extent differences in receptor density (Bmax values published in accompanying paper, this journal). SRIF28 was slightly more potent than SRIF14 to inhibit FSAC at all five receptors, although the potency of the natural peptides SRIF14, SRIF28 and CST17 was generally similar with pEC50-values ranging from 7.5 to 8.7 depending on receptor and peptide. At SRIF1 receptors (sst2, sst3, sst5) most of the peptide analogues displayed full agonism (with some exceptions e.g. BIM 23056 at sst1-3 and sst5 receptors, and L362,855 and cycloantagonist SA at sst3 receptors), whereas at SRIF2 receptors these analogues tended to behave as partial agonists. BIM 23056 was an antagonist at sst3 receptors (antagonist binding constant pKB = 6.33), but not at other receptors. The AC inhibition profiles of sst1-5 receptors were compared with the different radioligand binding profiles as well as with [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding profile for sst2-5 receptors. High correlations were observed between FSAC inhibition, radioligand binding and [35S]GTPgammaS binding profiles at sst3, sst4 and sst5 receptors; by contrast, correlation coefficients at sst1 and sst2 receptors were low, and the binding profiles of [125I][Tyr10]CST14 correlated poorly. In line with these findings, the FSAC inhibition and [35S]GTPgammaS binding correlated poorly at sst2 receptors (sst1 receptors show no significant induction of [35S]GTPgammaS binding). The apparent lack of, or only weak, relationship between FSAC, radioligand or [35S]GTPgammaS binding observed for some SRIF receptors suggests that different active states may exist for these receptors, which may favour one transduction cascade over others.

PK 11195 blockade of benzodiazepine‐induced inhibition of forskolin‐stimulated adenylate cyclase activity in the striatum

1. The effects of benzodiazepine receptor antagonists on the inhibition of forskolin-stimulated adenylate cyclase (AC) activity by various benzodiazepine (BZ) and indoleamine agonists in the rat striatum were investigated. 2. A biphasic inhibition of forskolin-stimulated AC activity by the peripheral-type agonist, Ro5-4864, and a multiphasic inhibition by the non-selective BZ, diazepam, was observed. One phase of AC inhibition is consistent with a Gi-coupled receptor-mediated action, whereas the other phases appear to involve a direct effect on the enzyme itself. 3. While the central-type antagonist, flumazenil, had no effect on the ability of Ro5-4864 to inhibit AC activity, the peripheral-type receptor ligand, PK 11195, abolished the first phase of inhibition. 4. PK 11195 and pertussis toxin were found to block the inhibitory effect of various BZs and the indoleamines, melatonin and 2-iodomelatonin, on induced AC activity. 5. Saturation binding studies, conducted at 30 degrees C with [3H]-diazepam revealed a single binding site in the rat striatum (KD = 19.3 +/- 0.80 nM) which significantly decreased in affinity in the presence of GTP (KD = 30.5 +/- 2.6 nM; P < 0.05). No significant change in Bmax was observed. 6. These findings indicate the presence of Gi-coupled BZ receptors in the rat striatum. Thus, suppression of cyclic AMP production may contribute to the diverse neuropharmacological effects of BZs, melatonin and related drugs.

Characterization and distribution of somatostatin SS-1 and SRIF-1 binding sites in rat brain: identitity with SSTR-2 receptors

Somatostatin (SRIF) SS-1 binding sites were initially defined in radioligand binding studies performed in rat brain cerebral cortex membranes using [ 125I]204-090 (a radiolabelled Tyr 3 analogue of SMS 201-995, octreotide). SRIF-1 recognition sites were defined in binding studies performed with [ 125I]MK 678 (seglitide). Both SS-1 and SRIF-1 sites were characterized by their high affinity for SRIF-14, SRIF-28 and for cyclic peptides such as octreotide and seglitide, in marked contrast to SS-2 and SRIF-2 sites which have very low affinity for these synthetic SRIF analogues. In the present study, SS-1 and SRIF-1 radioligand binding studies were performed in rat cortex membranes and compared to results obtained in cloned Chinese hamster ovary cells expressing human SSTR-2 receptors using [ 125I]204-090 and/or [ 125I]MK-678. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-1/SRIF-1 binding sites and recombinant SSTR-2 receptors were very similar and correlated highly significantly ( r = 0.94–0.99); by contrast, correlation between SS-1 and SSTR-5 ( r = 0.44) or SSTR-3 binding ( r = 0.07) was not significant. Autoradiographic studies were performed in rat brain using both radioligands [ 125I]204-090 and [ 125I]MK-678 and compared with the distribution of SSTR-2 receptor mRNA determined using in situ hybridization. A clear overlap was observed between the distribution of SSTR-2 mRNA and binding sites labelled with both radioligands. SSTR-2 receptor-mediated inhibition of forskolin-stimulated adenylate cyclase in Chines hamster ovary cells by a variety of SRIF analogues and short synthetic peptides displayed a rank order of potency highly similar to their rank order of affinity at SS-1/SRIF-1 binding sites. It is concluded that SS-1 and SRIF-1 binding sites respectively labelled with [ 125I]204-090 and [ 125I]MK 678, both display the pharmacological profile of SSTR-2 receptors, that the distribution of [ 125I]204-090 and [ 125I]MK-678 binding sites in rat brain is superimposable and largely comparable to that of SSTR-2 mRNA expression. It is also shown that neither [ 125I]204-090 nor [ 125I]MK-678 label SSTR-3 or SSTR-5 receptors in rat brain. Finally, it is demonstrated that SSTR-2 receptors can very efficiently couple to adenylate cyclase activity in an inhibitory manner.

Pharmacological identity between somatostatin SS-2 binding sites and SSTR-1 receptors

Somatostatin (SRIF) SS-2 binding sites were originally defined in rat brain cerebral cortex membranes using [ 125I]Tyr 11-SRIF-14 in the presence of 120 mM NaCl. These sites were characterized by their high affinity for SRIF-14 and SRIF-28, but very low affinity for cyclic peptides such as octreotide (SMS 201-995) and seglitide (MK 678). The characteristics of SS-2 sites are reminiscent of 125I]CGP 23996-labelled sites in rat brain which have been termed SRIF-2 sites. In the present study, the pharmacological profile of SS-2 sites was determined in radioligand binding studies performed in rat cortex membranes using [ 125I]SRIF-14 in the presence of 120 mM NaCl and compared to that of human SSTR-1 receptors expressed in human embryonic kidney (HEK 293) cells, using [ 125I]SRIF-14. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-2 binding sites and recombinant human SSTR-1 receptors were very similar and correlated highly significantly ( r = 0.99). However, SS-2 binding correlated also with binding to recombinant SSTR-4 receptors ( r = 0.91). Autoradiographic studies were performed using the radioligand [ 125I]CGP 23996 which has been claimed to label selectively SRIF-2 binding sites and compared with the distribution of SSTR-1 receptor mRNA determined using in situ hydridization in rat brain. Although some overlap was observed between the distribution of SSTR-1 mRNA and [ 125I]CGP 23996 binding sites, the latter were clearly more widespread, suggesting this ligand to label SSTR-1 and other sites. In addition, inhibition of forskolin-stimulated adenylate cyclase was investigated in HEK 293 cells transfected with human SSTR-1 receptors; a variety of SRIF analogues and short synthetic peptides behaved as agonists at adenylate cyclase and displayed a rank order of potency highly similar to that observed for these compounds at SS-2 binding sites. Seglitide acted as an antagonist at SSTR-1 receptor mediated inhibition of adenylate cyclase activity with a pK B of 4.42. It is concluded that the pharmacological profile of SS-2 binding sites resembles most closely that of SSTR-1 receptors (although similarities with SSTR-4 receptors were observed), that [ 125I]CGP 23996 labels presumably several SRIF receptors in rat brain, and that SSTR-1 receptors are negatively and efficiently coupled to adenylate cyclase activity.

Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine 1A receptor

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine 1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine 1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [ 3H]8-hydroxy-2-(di- n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine 1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine 1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine 1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine 1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine 1A receptors might differ from one neuronal cell type to another.

CDNA Cloning of an orphan opiate receptor gene family member and its splice variant

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned, μ, δ and κ receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67–72% homology with those encoded by μ1, δ1 and κ1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate ligands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.

Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5'-[beta-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp.

The effects of guanosine 5'-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5'-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.

Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin.

The effect of aluminium fluoride (AlF4-) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-125I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mM LiCl, AlF4- stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF4- increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF4(-)-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF4(-)-stimulated or the basal calcium levels. AlF4- blocked the increase in cyclic AMP stimulation by 1 microM forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF4- also attenuated the binding of [2-125I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF4- activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.

Chloroamphetamine did not prevent the effects of chronic antidepressants on 5-hydroxytryptamine inhibition of forskolin-stimulated adenylate cyclase in rat hippocampus

Administration of p-chloroamphetamine (PCA, 10 mg/kg i.p. on two occasions) to rats resulted in a severe depletion of [ 3H]paroxetine binding sites, a measure of presynaptic serotonergic terminals, in both cortex and hippocampus, but did not affect [ 3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]8-OH-DPAT) binding or 5-hydroxytryptamine (5-HT)-induced inhibition of forskolin-stimulated adenylate cyclase in hippocampal membranes. Administration of either imipramine (15 mg/kg i.p. for 2 weeks) or lithium (0.2% for 2 weeks) to PCA-treated rats did not affect [ 3H]8-OH-DPAT binding but reduced the degree of inhibition of forskolin-stimulated adenylate cyclase by 5-HT in hippocampal membranes. It is concluded that the effects of imipramine and Li + on 5-HT 1A receptor-mediated responses in the hippocampus are exerted postsynaptically, possibly at a level distal to the receptor.

The 5‐HT1B Receptors

The 5-HT1B receptors have been identified by radioligand binding techniques predominantly in the basal ganglia of the rat and mouse brain. A number of 5-HT receptor agonists have been shown to display high affinity but limited selectivity for the 5-HT1B recognition site. These include 5-CT, 5-HT, RU 24969, TFMPP, MCPP, and CGS 12066B. Antagonists at the 5-HT1B site include the drugs metitepin, metergoline, cyanopindolol, isamoltane, and propranolol but none of these drugs are selective for this receptor. Functional correlates of 5-HT1B receptor activation have been most closely defined in vitro. These include inhibition of transmitter release, inhibition of forskolin-stimulated adenylate cyclase and actions on the mouse urinary bladder strip and the rat vena cava. Many functional correlates of 5-HT1B receptor activation in vivo have been proposed, but convincing evidence from antagonist studies is generally lacking. The development of selective 5-HT1B receptor agonists and antagonists will be a key step in defining the physiological role of this receptor site in the brain and periphery of the mouse and rat although it must be realized that these compounds, if they are developed, are unlikely to have functional effects in man since the 5-HT1B recognition site is absent in the human CNS. Nevertheless many of these studies on the 5-HT1B receptor may aid the development of drugs acting at the 5-HT1D site since this receptor has been identified as being the equivalent of the 5-HT1B site in species other than the rat and mouse.

The behavioural responses to 8-OH-DPAT, ipsapirone and the novel 5-HT1A receptor agonist Bay Vq 7813 in the pig

In pigs, behavioural responses were examined after administration of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), a full agonist at 5-hydroxytryptamine (5-HT) receptors of the 5-HT1A subtype, and the pyrimidinylpiperazine derivatives ipsapirone and Bay Vq 7813 (2-[4-(2-pyrimidinyl)-1-piperazinylpropyl]-1,2-benzisothiazol++ +-3(2H) one-1,1-dioxide), which act as partial agonists at 5-HT1A receptors. The most prominent behavioural response examined after 8-OH-DPAT, 0.5 mg/kg i.m., ipsapirone, 2-5 mg/kg i.m., and Bay Vq 7813, 0.5-2 mg/kg i.m. or i.v., were head shakes. The potency of the three drugs to induce this behaviour correlated with their activity at 5-HT1A receptors as determined by inhibition of forskolin-stimulated adenylate cyclase, substantiating that the head shake response has potential as a quantitative probe of in vivo receptor function. The 5-HT2/5-HT1C receptor antagonist ritanserin did not counteract the head shakes induced by ipsapirone, suggesting that neither 5-HT2 nor 5-HT1C receptors are involved in mediation of this response to this 5-HT1A receptor agonist in pigs. Once daily administration of Bay Vq 7813 or ipsapirone for 3-5 days led to a reduction in the head shake response. 1-Pyrimidinylpiperazine (1-PP), a pharmacologically active metabolite shared by ipsapirone, Bay Vq 7813, and related pyrimidinylpiperazine derivatives, did not induce behavioural alterations in pigs. The data provide further evidence that marked species differences exist in functional responses to 5-HT receptor ligands.

The local anaesthetic benzyl alcohol attenuates the α2-adrenoceptor-mediated inhibition of human platelet adenylate cyclase activity when stimulated by prostaglandin E1, but not that stimulated by forskolin

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.

Inhibition of adenylate cyclase in rat brain synaptosomal membranes by GTP and phenylisopropyladenosine is enhanced in hypothyroidism

1. Synaptosomal membranes were isolated from rats made hypothyroid by treatment with propylthiouracil and a low iodine diet. 2. When assayed in the presence of 100 mM-Na+, inhibition of forskolin-stimulated adenylate cyclase by GTP was enhanced in membranes from hypothyroid animals. 3. Hypothyroidism also enhanced inhibition of adenylate cyclase by phenylisopropyladenosine (with 100 mM-Na+ and 10 microM-GTP present). 4. Hypothyroidism did not increase binding of the A1 adenosine receptor agonist phenylisopropyladenosine to synaptosomal membranes; rather, the maximum binding was slightly decreased without any change in the KD. 5. The effect of GTP in modifying the displacement of the antagonist [3H]diethylphenylxanthine from synaptosomal membranes by unlabelled phenylisopropyladenosine was more pronounced in the hypothyroid state. 6. These findings are consistent with hypothyroidism causing modification of the brain adenylate cyclase system at the level of the coupling protein Gi.

5-HT1D receptors in guinea-pig and pigeon brain

The characteristics of high affinity [3H]5-HT (5-hydroxytryptamine) binding to non 5-HT1A non 5-HT1C sites were examined in crude membranes prepared from different regions of guinea-pig and pigeon brains. The coupling of these sites to adenylate cyclase was examined, and its pharmacological profile investigated. In the presence of 100 nmol/l 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) and 100 nmol/l mesulergine, [3H]5-HT labelled with nanomolar affinity an apparently homogeneous population of recognition sites in guinea-pig and pigeon brain membranes. The rank order of affinities of agonists and antagonists (5-CT (5-carboxamidotryptamine) greater than 5-HT greater than RU 24969 (5-methoxy-3-(1,2,3,6-tetrahydro-4- pyridinyl)-1H indole succinate) greater than yohimbine greater than or equal to rauwolscine greater than DP-5-CT (N,N dipropyl-5-carboxamidotryptamine) greater than or equal to mianserin greater than 8-OH-DPAT greater than mesulergine greater than SDZ 21-009 ((+/-)-4(3-tert-butyl-amino-2-hydroxypropoxy)-indol-2 carbonic acid isopropyl ester) greater than (-)propranolol), as well as their individual pKD values, were very similar to those at porcine caudate 5-HT1D sites and clearly different from those at rat cortex 5-HT1B sites. In the substantia nigra of the guinea-pig the 5-HT receptor-mediated inhibition of forskolin-stimulated adenylate cyclase had a pharmacological profile fully comparable to that of 5-HT1D binding sites (5-CT greater than 5-HT greater than yohimbine greater than RU 24969 greater than 8-OH-DPAT greater than SDZ 21-009 = isamoltane greater than (-)pindolol greater than (-)propranolol).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic electroconvulsive shock on D 1 and D 2 dopamine receptor-mediated activity of adenylate cyclase in homogenates of striatum and limbic forebrain of rat

Stimulation of adenylate cyclase by dopamine in homogenates of the striatum was unaltered in rats which had received either a single or a series of 10 electroconvulsive shocks. However, stimulation by the selective D 1 dopamine receptor agonist SKF 38393 was significantly increased in homogenates of striatum from rats which had received chronic electroconvulsive shock, compared to those which received sham treatment. In homogenates of the limbic forebrain, stimulation by both 100 μM dopamine and by 4 μM SKF 38393 was significantly increased after chronic electroconvulsive shock. The activity of D 2 receptors, as measured by inhibition of forskolin-stimulated adenylate cyclase, in the presence of the d 1 receptor antagonist SCH 23390, was unaltered by chronic electroconvulsive shock in either area of the brain. The selective effect of chronic electroconvulsive shock in increasing the activity of D 1 receptors may account both for the increase, in dopamine-mediated behaviour, seen after chronic electroconvulsive shock and for the antiparkinsonian effects of this treatment.

Chronic MAO A and MAO B inhibition decreases the 5-HT 1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase

The effect of chronic administration of various monoamine oxidase (MAO) inhibitors on the ability of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to inhibit forskolin-stimulated adenylate cyclase activity was studied. Groups of 12 rats were given either saline, (E)-β-fluoromethylene-m-tyrosine (MDL 72394 0.25 mg/kg p.o.), clorgyline (1 mg/kg p.o.), selegiline (1 mg/kg p.o.) or tranylcypromine (5 mg/kg p.o.) once a day for 21 days. Biochemical determinations were made 72 h after the final dose. MDL 72394 and tranylcypromine produced a nonselective inhibition of MAO but clorgyline and selegiline selectively inhibited MAO A and MAO B respectively. All treatments that inhibited MAO A also increased tissue levels of 5-HT. Chronic treatment with MDL 72394, clorgyline or tranylcypromine reduced the ability of 8-OH-DPAT to inhibit forskolin-stimulated adenylate cyclase activity. These data suggest that chronic nonselective and chronic MAO A inhibition causes a down-regulation of the 5-HT 1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity.

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.