Stimulation and inhibition of adenylyl cyclase by distinct 5-hydroxytryptamine receptors

5-Hydroxytryptamine (serotonin, 5-HT) stimulates basal adenylyl cyclase activity in membranes from guinea pig or rat hippocampi, but 5-HT inhibits forskolin-stimulated adenylyl cyclase activity in these same membranes. The opposing effects of 5-HT on adenylyl cyclase activity indicate that distinct 5-HT receptors, positively and negatively coupled to adenylyl cyclase, are present in these membranes. Stimulation of adenylyl cyclase activity is mediated by two distinct 5-HT receptors. The receptor with lower affinity for 5-HT, designated as RL, is apparently homologous with a 5-HT receptor present in rat collicular membranes, but it is not homologous with the stimulatory receptor characterized in neuroblastoma hybrid cell (NCB-20) membranes. The receptor with higher affinity for 5-HT is homologous with the 5-HT1A binding site. The magnitude of stimulation by 5-HT1A receptors is variable with respect to stimulation by RL and is sometimes completely absent. Inhibition of forskolin-stimulated adenylyl cyclase activity, in membranes from either rat or guinea pig hippocampus or rat cortex, is a functional correlate of the 5-HT1A binding site. This inhibitory response was used to determine the pharmacological characteristics of drugs that reportedly have high affinity for 5-HT1A binding sites, such as 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) and (-)pindolol. PAPP inhibited adenylyl cyclase activity in guinea pig hippocampal membranes with an EC50 value of 27 +/- 3 nM. (-)Pindolol was a partial agonist in inhibiting adenylyl cyclase activity in guinea pig and rat hippocampal membranes. Because of the low intrinsic activity of (-)pindolol, it was tested as an antagonist of the inhibition produced by 5-HT1A receptor agonists in rat hippocampal membranes. The Kb of (-)pindolol was 40 nM as measured by a Schild plot. (-)Propranolol was a simple competitive antagonist at the rat hippocampal receptor with a Kb value of 550 nM. In summary, guinea pig and rat hippocampal membranes possess two distinct populations of 5-HT receptors, a 5-HT receptor that mediates inhibition of adenylyl cyclase activity and is pharmacologically homologous with the 5-HT1A binding site, and a stimulatory receptor that appears to be homologous with the 5-HT receptor first characterized in infant rat collicular membranes.     

Lack of apparent receptor reserve at postsynaptic 5-hydroxytryptamine1A receptors negatively coupled to adenylyl cyclase activity in rat hippocampal membranes.

Previous studies have demonstrated the existence of a large receptor reserve for agonists at somatodendritic 5-hydroxytryptamine1A (5-HT1A) serotonin receptors in the raphe nuclei of the rat. 5-HT1A agonists with anxiolytic properties (e.g., buspirone, gepirone, and ipsapirone) display full intrinsic activity at these receptors but are partial agonists at postsynaptic 5-HT1A receptors, which suggests that the latter sites may be devoid of a receptor reserve. In the present studies, this was directly determined by examining the relationship between receptor occupancy and response at postsynaptic 5-HT1A receptors, in rat hippocampus, mediating the inhibition of forskolin-stimulated adenylyl cyclase activity, using the method of partial irreversible receptor inactivation. Rats were treated with vehicle or the irreversible antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), and 24 hr later hippocampi were removed for saturation analysis of [3H]8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) binding to 5-HT1A receptors or for adenylyl cyclase assays. EEDQ (1 and 6 mg/kg) dose-dependently reduced the maximal number of [3H]8-OH-DPAT binding sites by 68.5 and 80%, respectively, without altering the Kd. Concentration-response curves were generated for inhibition of forskolin-stimulated adenylyl cyclase activity by 5-HT and the selective 5-HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT). EEDQ treatment dose-dependently reduced the maximal inhibitory effect of 5-HT [percentage of inhibition: control, 23.6; EEDQ (1 mg/kg), 13.4; EEDQ (6 mg/kg), 8.9], without altering either the slope factor (1.01) or the EC50 (96.4 nM). Analogous results were obtained with DP-5-CT [percentage of maximal inhibition: control, 24.1; EEDQ (1 mg/kg), 15.2; EEDQ (6 mg/kg), 10.7), again without changes in slope factor (0.89) or EC50 (9.9 nM). Analysis of double-reciprocal plots of equieffective concentrations of agonist, followed by calculation of fractional receptor occupancy, revealed a linear relationship between receptor occupancy and response for both 5-HT and DP-5-CT (i.e., an absence of receptor reserve). The receptor specificity of the effect of EEDQ was demonstrated in two ways. First, it was shown that pretreatment of rats with the selective 5-HT1A partial agonist BMY 7378 (10 mg/kg) before EEDQ afforded substantial protection (about 75%) against loss of the inhibitory effect of DP-5-CT on forskolin-stimulated adenylyl cyclase activity. Second, EEDQ did not alter the inhibition of forskolin-stimulated adenylyl cyclase activity induced by the adenosine A1 receptor agonist phenylisopropyladenosine (PIA).(ABSTRACT TRUNCATED AT 400 WORDS)     

5-CT stimulation of adenylyl cyclase activity in guinea-pig hippocampus: evidence for involvement of 5-HT7 and 5-HT1A receptors.

1. A number of compounds, including the selective 5-HT7 receptor antagonist SB-258719, were investigated for their effect on [3H]-5-carboxamidotryptamine (5-CT) radioligand binding and 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes, in order to confirm the presence of functionally coupled 5-HT7 receptors in this tissue. 2. The [3H]-5-CT radioligand binding profile was consistent with binding predominantly to 5-HT7 receptors. The affinity of SB-258719 (pKi 7.2+/-0.1) was similar to its reported human 5-HT7 receptor affinity. 3. In the adenylyl cyclase functional assay, 5-CT was a potent and full agonist compared to 5-HT, whereas 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) was a partial agonist (intrinsic activity 0.4+/-0.1). The rank order of potency for agonists (5-CT>5-HT approximately 8-OH-DPAT) was consistent with activation of 5-HT7 receptors. SB-258719 (5 microM) and methiothepin (1 microM) surmountably antagonized the response to 5-CT, consistent with competitive antagonism. The pKB for SB-258719 (7.2+/-0.1) was in good agreement with its reported antagonist potency at the human cloned 5-HT7 receptor. 4. In the functional assay, WAY-100635 (100 nM) and cyanopindolol (1 microM) induced a biphasic 5-CT response curve, consistent with selective antagonism of a component of the response to 5-CT. The estimated pKB values for WAY-100635 and cyanopindolol (9.6 and 8.4 respectively) were in good agreement with their reported 5-HT1A receptor affinities. 5. The data are consistent with the presence of 5-HT7 receptors in guinea-pig hippocampus which are positively coupled to adenylyl cyclase. In addition, 5-HT7 receptor-mediated stimulation of adenylyl cyclase activity in this tissue appears to be augmented by a mechanism involving 5-HT1A receptor activation.     

Differential effects of centrally-active antihypertensives on 5-HT1A receptors in rat dorso-lateral septum, rat hippocampus and guinea-pig hippocampus.

1. The electrophysiological responses elicited by 5-hydroxytryptamine1A-(5-HT1A) receptor agonists in rat and guinea-pig CA1 pyramidal neurones and rat dorso-lateral septal neurones were compared in vitro by use of conventional intracellular recording techniques. 2. In the presence of 1 microM tetrodotoxin (TTX), to prevent indirect effects, 5-HT, N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT) and 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT) hyperpolarized the neurones from rat and guinea-pig brain. 3. The hypotensive drug flesinoxan, a selective 5-HT1A receptor agonist, hyperpolarized neurones in all three areas tested; however, another hypotensive agent with high affinity at 5-HT1A-receptors, 5-methyl-urapidil, hyperpolarized only the neurones in rat hippocampus and septum. 4. In guinea-pig hippocampal neurones, 5-methyl-urapidil behaved as a 5-HT1A-receptor antagonist. 5. The relative efficacies (5-HT = 1) of DP-5-CT, 8-OH-DPAT, flesinoxan and 5-methyl-urapidil at the three sites were: rat hippocampus, 1.09, 0.7, 0.5 and 0.24; rat septum, 0.88, 0.69, 0.82 and 0.7; guinea-pig hippocampus, 1.0, 0.69, 0.89 and 0, respectively. 6. It is concluded that the hypotensive agents flesinoxan and 5-methyl-urapidil appear to have different efficacies at 5-HT1A receptors located in different regions of the rodent brain. Whether these regional and species differences arise from receptor plurality or variability in intracellular transduction mechanisms remains to be elucidated.     

Dihydroergotamine and its metabolite, 8'-hydroxy-dihydroergotamine,as 5-HT1A receptor agonists in the rat brain

1 In addition to stopping migraine attacks, dihydroergotamine (DHE) is an efficient drug for migraine prophylaxis. Whether 5-HT(1A) receptors could contribute to the latter action was assessed by investigating the effects of DHE and its metabolite, 8'-OH-DHE, on these receptors in the rat brain. 2 Membrane binding assays with [(3)H]8-OH-DPAT and [(3)H]WAY 100635 as radioligands showed that both DHE (IC(50)=28-30 nM) and 8'-OH-DHE (IC(50)=8-11 nM) are high-affinity 5-HT(1A) receptor ligands. 3 Both DHE and 8'-OH-DHE enhanced the specific binding of [(35)S]GTP-gamma-S to the dorsal raphe nucleus and the hippocampus in brain sections, but to a lower extent than 5-carboxamido-tryptamine (5-CT) in the latter area. 4 Both DHE (EC(50)=10.9+/-0.3 nM) and 8'-OH-DHE (EC(50)=30.4+/-0.8 nM) inhibited the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices. 5 Intracellular recording showed that 8'-OH-DHE was more potent than DHE to hyperpolarize CA1 pyramidal cells in rat hippocampal slices. 6 Both the stimulatory effects of DHE and 8'-OH-DHE on [(35)S]GTP-gamma-S binding and their electrophysiological effects were completely prevented by the selective 5-HT(1A) receptor antagonist WAY 100635. 7 As expected of 5-HT(1A) receptor partial agonists, DHE and 8'-OH-DHE prevented any subsequent hyperpolarization of CA1 pyramidal cells by 5-HT or 5-CT. 8 Through their actions at 5-HT(1A) auto- (in the dorsal raphe nucleus) and hetero-(notably in the hippocampus) receptors, DHE, and even more its metabolite 8'-OH-DHE, can exert both an inhibitory influence on neuronal excitability and anxiolytic effects which might contribute to their antimigraine prophylactic efficiency.     

Novel anxiolytics discriminate between postsynaptic serotonin receptors mediating different physiological responses on single neurons of the rat hippocampus

Summary The effects of buspirone on hippocampal pyramidal cells of the CA1 region were examined by means of intracellular recordings in in vitro hippocampal brain slices. Bath administration of buspirone elicited a long lasting hyperpolarization which was mediated by an increase in potassium conductance and resembled the hyperpolarizing component of the response to 5-HT (5-hydroxytryptamine). Buspirone, however, failed to mimic the depolarizing action of 5-HT or to reduce the calcium-activated afterhyperpolarization. Quantitative comparisons of the hyperpolarizing responses of 5-HT and buspirone revealed that the maximal hyperpolarization induced by buspirone was significantly smaller than that induced by 5-HT. Since the buspirone induced hyperpolarization was also accompanied by a surmountable antagonism of 5-HT responses, these results indicate that buspirone behaves as a partial agonist at a subpopulation of 5-HT receptors in the CA1 region of the hippocampus. Administration of the buspirone congeners gepirone and isapirone also elicited a hyperpolarization and reduced 5-HT responses, although they lack anti-dopaminergic activity, indicating that the effects observed with buspirone are unlikely to be mediated through dopamine receptors. These results indicated that novel anxiolytics can discriminate between functional 5-HT receptors. In conjunction with previous biochemical and electrophysiological studies, the present results suggest that their administration might alter the balance of serotonergic actions on postsynaptic neurons. Send offprint requests to R. Andrade at the above address     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).     

5-Hydroxytryptamine-induced vasodilatation in the isolated perfused rat kidney: are endothelial 5-HT1A receptors involved?

Left kidneys obtained from male Wistar rats were perfused with Tyrode solution; the perfusion pressure was measured continuously and taken as an index of vascular resistance in the kidneys. 5-Hydroxytryptamine (5-HT; 3-50 nmol) caused dose-dependent dilator responses in kidneys preconstricted with noradrenaline (0.6 microM) and pretreated with ritanserin (10 nM) and ICS 205930 (10 nM). The 5-HT1 agonist 5-carboxamidotryptamine (5-CT; 16-64 nmol) also caused renal dilatations under similar conditions. The dilator responses to both 5-HT and 5-CT were antagonized by the non-selective 5-HT receptor antagonist metergoline (0.2 microM) and by the selective 5-HT1A receptor antagonist BMY 7378 (0.4 microM). The guanylate cyclase inhibitor methylene blue (30 microM) and the nitric oxide (NO) synthase inhibitor nitro-L-arginine (L-NNA; 100 microM) significantly attenuated the dilator responses to 5-HT and 5-CT. The 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.5-16 nmol) also caused dose-dependent dilator responses in preconstricted rat kidneys. These responses were antagonized by metergoline and BMY 7378 and significantly attenuated by the NO inhibitors hemoglobin (10 microM) and L-NNA. The renal dilator responses noted with the beta-adrenoceptor blocker tertatolol (1-32 nmol) were also antagonized by metergoline and BMY 7378 and significantly reduced by L-NNA and hemoglobin. Both 8-OH-DPAT and tertatolol (1-30 microM) significantly reduced the vasoconstrictor responses to angiotensin II (20 pmol). Our data indicate that 5-HT receptors located on the vascular endothelium of the renal circulation are involved in the dilator actions of 5-HT, 5-CT, 8-OH-DPAT and tertatolol, and suggest that these receptors resemble the 5-HT1A subtype.     

Centrally acting hypotensive agents with affinity for 5-HT1A binding sites inhibit forskolin-stimulated adenylate cyclase activity in calf hippocampus.

1. A number of centrally acting hypotensive agents and other ligands with high affinity for 5-hydroxytryptamine1A (5-HT1A) recognition sites have been tested on forskolin-stimulated adenylate cyclase activity in calf hippocampus, a functional model for 5-HT1A-receptors. 2. Concentration-dependent inhibition of forskolin-stimulated adenylate cyclase activity was elicited by the reference 5-HT1-receptor agonists (mean EC50 value, nM): 5-HT (22), 5-carboxamidotryptamine (5-CT, 3.2), 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 8.6), N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT, 2.3), 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine (PAPP or LY 165163, 20), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole (RU 24969, 20), buspirone (65) and ipsapirone (56). Emax amounted to 18-20% inhibition for all but the latter two agonists (14%). 3. The following hypotensive agents with high affinity for 5-HT1A sites were potent agonists in this system (mean EC50 value, nM): flesinoxan (24), indorenate (99), erythro-1-(1-[2-(1,4-benzodioxan-2-yl)-2-hydroxyethyl]-4-piperidyl )- 2-benzimidazolinone (R 28935, 2.5), urapidil (390) and 5-methyl-urapidil (3.5). The first two agents were full agonists, whereas the latter three acted as partial agonists with 60-80% efficacy. 4. Metergoline and methysergide behaved as full agonists and cyanopindolol as a partial agonist with low efficacy. Spiroxatrine and 2-(2,6-dimethoxyphenoxyethyl)aminomethyl- 1,4-benzodioxane (WB 4101) which bind to 5-HT1A sites with nanomolar affinity, were agonists and inhibited potently forskolin-stimulated adenylate cyclase in calf hippocampus, showing mean EC50 values of 23 and 15 nM, respectively. Spiroxatrine and WB 4101 yielded 90% and 50% efficacy, respectively. 5. Spiperone and methiothepin (each 1 microM) caused rightward shifts of the concentration-effect curve to 8-OH-DPAT, without loss of the maximal effect, as did the partial agonist cyanopindolol (0.1 microM) and the (-)- and (+)-enantiomers of pindolol (1 microM and 0.1 mM, respectively). 6. There was an excellent correlation (r = 0.90, P = 0.0001) between the pEC50 values (ranging from 6.4 to 8.7) of the 19 agonists tested at adenylate cyclase and their pKD for 5-HT1A recognition sites. Apparent pKB values of antagonists at adenylate cyclase and their pKD values for 5-HT1A binding sites were also significantly correlated. 7. This study further indicates that the 5-HT1A recognition site and the 5-HT receptor mediating inhibition of adenylate cyclase in hippocampus are the same.(ABSTRACT TRUNCATED AT 400 WORDS)     

MDL 73005EF: partial agonist at the 5-HT1A receptor negatively linked to adenylate cyclase

MDL 73005EF has been recently described as a potent, highly selective 5-HT1A ligand. Although proposed to act predominantly as an antagonist (M. Hibert, A.K. Mir, G. Maghioros, P. Moser, D.N. Middlemiss, M.D. Trickleband and J.R. Fozard, 1988, The Pharmacological properties of MDL 73005EF: a potent and selective ligant at 5-HT1A receptors, Br. J. Pharmacol. 93, 2P), we have demonstrated that MDL 73005EF also acts as a highly efficacious partial agonist at the 5HT1A receptor, based on its ability to inhibit forskolin-stimulated adenylate cyclase in rat hippocampal membranes. Compared with two structurally related 5-HT1A partial agonists, the rank order of potency of MDL 73005EF in the FSC assay was comparable to affinity calculated by radioligand binding.     

Characterization of SB-269970-A, a selective 5-HT(7) receptor antagonist

The novel 5-HT(7) receptor antagonist, SB-269970-A, potently displaced [(3)H]-5-CT from human 5-HT(7(a)) (pK(i) 8.9+/-0.1) and 5-HT(7) receptors in guinea-pig cortex (pK(i) 8.3+/-0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT(7(a))/HEK293 membranes (pEC(50) 7.5+/-0.1) and SB-269970-A (0.03 - 1 microM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA(2) (8.5+/-0.2) for SB-269970-A agreed well with the pK(i) determined from [(3)H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC(50) of 8.4+/-0.2) was inhibited by SB-269970-A (0.3 microM) with a pK(B) (8.3+/-0.1) in good agreement with its antagonist potency at the human cloned 5-HT(7(a)) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT(7) receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min(-1) kg(-1)). Following a single dose (3 mg kg(-1)) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg(-1) i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED(50) 2.96 mg kg(-1) i.p.) and the non-selective 5-HT(7) receptor antagonist metergoline (0.3 - 3 mg kg(-1) s.c.), suggesting a role for 5-HT(7) receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg(-1)) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats.     

Antagonism of 5-hydroxytryptamine1A (5-HT1A) receptor-mediated modulation of adenylate cyclase activity by pindolol and propranolol isomers

The interactions of the stereoisomers of pindolol and propranolol with 5-hydroxytryptamine1A (5-HT1A) binding sites and adenylate cyclase activity were examined in rat hippocampus. (-)Pindolol and (-)propranolol displayed high affinity for 5-HT1A binding sites, and their affinities were not affected significantly by the addition of 10(-4) M GTP to the radioligand assay. The selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) decreased forskolin-stimulated adenylate cyclase activity. The (-)isomers of pindolol and propranolol did not affect basal or forskolin-stimulated activity but, at a concentration of 10(-5) M, they reversed the 8-OH-DPAT inhibition of the forskolin-stimulated cyclase activity. The (+)isomers were less potent in producing this effect. These data suggest that (-)pindolol and (-)propranolol are potent antagonists at 5-HT1A receptors in rat hippocampus.     

Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists

We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.     

Intrinsic activity of enantiomers of 8-hydroxy-2-(di-n-propylamino)tetralin and its analogs at 5-hydroxytryptamine1A receptors that are negatively coupled to adenylate cyclase

Although many different types of compounds have been tested for 5-hydroxytryptamine1A (5-HT1A) binding affinity, much remains to be learned about the structural requirements associated with 5-HT1A agonism, partial agonism, and antagonism. The present study uses the forskolin-stimulated adenylate cyclase (FSC) assay as a functional screen in rat hippocampal membranes to examine structure-activity relationships for a series of enantiomers of novel analogs of the prototypic 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The findings illustrate that there can be large enantiomeric differences in intrinsic activity at the 5-HT1A receptor, independent of enantiomeric effects on binding affinity. Generally, for each enantiomeric pair exhibiting stereoselective 5-HT1A binding, the enantiomer with the higher affinity also displayed the greater amount of 5-HT1A intrinsic activity in the FSC assay. Interestingly, the enantiomers of 8-OH-DPAT itself displayed stereoselective differences in intrinsic activity but not 5-HT1A affinity. Several of the compounds, namely (S)-UH-301, (2R,3R)-CM-12, and (1S,2R)-LEA-146, may have potential as prototypes for selective 5-HT1A antagonists, and (S)-UH-301 itself may be useful as a selective 5-HT1A antagonist. The FSC data presented here are in good agreement with reported measures of in vivo 5-HT1A activity, which were in part the basis of a recently proposed model for the 5-HT1A pharmacophore [J. Med. Chem. 34: 497-510 (1991)].     

Effects of DAU 6215, a novel 5-hydroxytryptamine3 (5-HT3) antagonist on electrophysiological properties of the rat hippocampus.

1. The aim of the present study was to test the effects of DAU 6215 (endo-N-(8-methyl-8-azabicyclo-[3.2.1]-octo-3-yl)-2,3-dihydro-2-ox o-1H- benzimidazole-1-carboxamide carboxamide hydrochloride), a newly synthesized, selective 5-hydroxytryptamine3 (5-HT3) antagonist, on the cell membrane properties and on characterized 5-HT-mediated responses of pyramidal neurones in the hippocampal CA1 region. 2. Administration of DAU 6215, even at concentrations several hundred fold its Ki, did not affect the cell membrane properties of pyramidal neurones, nor modify extracellularly recorded synaptic potentials, evoked by stimulating the Schaffer's collaterals. 3. Micromolar concentrations (15-30 microM) of 5-HT elicited several responses in pyramidal neurones that are mediated by distinct 5-HT receptor subtypes. DAU 6215 did not antagonize the 5-HT1A-induced membrane hyperpolarization and conductance increase, a response that was blocked by the selective 5-HT1A antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phtalamido)butyl- piperazine). Similarly, DAU 6215 did not affect the membrane depolarization and decrease in amplitude of the afterhyperpolarization, elicited by the activation of putative 5-HT4 receptors. 4. 5-HT increased the frequency of spontaneous postsynaptic potentials (s.p.s.ps) recorded in pyramidal neurones loaded with chloride. In agreement with previous observations, most of the s.p.s.ps were reversed GABAergic events, produced by the activation of 5-HT3 receptors on interneurones, because they persisted in the presence of the glutamate NMDA and non NMDA antagonists, D-aminophosphonovaleric acid (APV; 50 microM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX; 25 microM), and were elicited by the selective 5-HT3 agonist, 2-methyl-5-HT (2-Me-5-HT, 50 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential coupling of 5-HT1A receptors occupied by 5-HT or 8-OH-DPAT to adenylyl cyclase

Summary Human serotonin (5-hydroxytryptamine, 5-HT)-1A receptors have been transfected in NIH-3T3 cells, and their coupling to adenylyl cyclase was analysed depending on (1) the number of receptor expressed, (2) the experimental conditions used, (3) the nature of the agonists. Two monoclonal cell lines were used, expressing low (45 fmol/mg) and high (500 fmol/mg) levels of 5-HT_1A receptor. Two methods were tested to study the negative coupling of the transfected 5-HT_1A receptors to adenylyl cyclase: (1) measurement of CAMP production in intact cells, (2) measurement of adenylyl cyclase activity in vitro on membrane preparations. Studies on intact cells revealed that an increase in the receptor concentration was followed by (1) an increase in the efficacies of 5-HT, 5-CT (5-carboxamidotryptamine) and 8-hydroxy2-(di-n-propylamino)tetralin (8-OH-DPAT), (2) a 2 to 3-fold increase in the potency of 5-CT and 8-OH-DPAT, but no change in the potency of 5-HT. In membrane preparations, 8-OH-DPAT dose-response curve was shifted leftwards when the receptor concentration became higher whereas the corresponding shift was smaller for 5-HT and absent for 5-CT. Surprisingly, on membrane preparations, 8-OH-DPAT was a partial agonist relative to 5-HT. The relative efficacy of 8-OH-DPAT was lower in the clone expressing the lowest level of receptor. This partial agonist behavior of 8-OH-DPAT could be modulated by the ionic conditions under which the adenylyl cyclase activity was measured. When physiological intracellular concentrations of Na⁺, Mg²⁺ and K⁺ were used, 8-OH-DPAT became an almost full agonist relative to 5-HT.These data indicate that (1) the classical pharmacological models do not exactly fit with characteristics of the negative coupling between transfected 5-HT_1A receptors and adenylyl cyclase; (2) on membranes, the experimental procedures (ionic conditions) can modify this coupling differently depending on the nature of the agonist.Abbreviations 5-HT 5-hydroxytryptamine (serotonin) - 8-OHDPAT 8-hydroxy-2-(di-n-propylamino)-tetraline - 5-CT 5-carboxamidotryptamine - IBMX isobutyl methyl xanthine - BSA bovine serum albumine - EC₅₀ half maximal efficacy - DXM dexamethasone - PTX Bordetella pertussis toxin - G protein GTP-binding protein Correspondence to A. Varrault at the above address     

Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.     

Pharmacology of 5-hydroxytryptamine-1A receptors which inhibit cAMP production in hippocampal and cortical neurons in primary culture

Serotonin (5-hydroxytryptamine, 5-HT) inhibited the formation of cAMP promoted by vasoactive intestinal polypeptide, plus forskolin, in mouse hippocampal and cortical neurons in primary culture. The rank order of potencies of classical 5-HT1 agonists in inhibiting cAMP formation in hippocampal neurons was 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) greater than 5-carboxamidotryptamine (5-CT) greater than d-lysergic acid diethylamide greater than 5-HT greater than 5-methoxy-N,N-dimethyltryptamine (5-MeO-N,N-DMT) greater than RU 24969 greater than ipsapirone greater than bufotenine greater than buspirone [half-maximal efficacy (EC50) = 7, 18, 30, 52, 90, 102, 100, 110, and 128 nM, respectively]. All the tryptamine derivatives substituted in position 5 of the indol were potent agonists [5-HT, 5-CT, 5-MeO-N,N-DMT, 5-methoxytryptamine, and bufotenine], whereas tryptamine, N-methyltryptamine, and N,N-dimethyltryptamine were poor agonists. The most potent antagonists tested were spiperone, (+/-)-pindolol, (+/-)-cyanopindolol, WB4101, and methiothepin, the affinity of spiperone for this receptor being 22 nM. In contrast, ketanserin, a specific 5-HT2 antagonist, and 5-HT3-selective drugs (ICS 205 930 and MDL 72222) were very weak in antagonizing the 5-HT-inhibited cAMP formation. The pharmacological profiles of 5-HT receptors mediating the inhibition of cAMP formation indicate that these receptors correspond to the 5-HT1A-binding site subtypes. Experiments with the Bordetella pertussis toxin indicate that the 5-HT1A receptor mediating inhibition of cAMP production involves a pertussis toxin-sensitive GTP-binding protein. In the absence of VIP, cAMP formation could be stimulated through a 5-HT receptor, but the specific 5-HT1A agonists, 8-OH-DPAT and RU 24969 did not stimulate cAMP production. These results suggest that in mouse embryonic hippocampal neurons, the 5-HT1A receptors, which are negatively coupled to adenylate cyclase, are distinct from the receptor positively coupled to this enzyme. The pharmacological characterization of the 5-HT receptor negatively coupled to adenylate cyclase in mouse embryonic cortical neurons indicates that it differs from the 5-HT1A receptor found in hippocampal neurons. Its main differences with the 5-HT1A receptor in hippocampal neurons are as follows: 1) 8-OH-DPAT was only a poor partial agonist in cortical neurons, whereas it was the best full agonist in hippocampal neurons; and 2) metergoline and methysergide as well as the anxiolytic drugs, ipsapirone and buspirone, which were potent agonists in hippocampal neurons, were competitive antagonists in cortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological characterization of 5-hydroxytryptamine-induced hyperpolarization of the rat superior cervical ganglion.

1 A study has been made of the pharmacology of 5-hydroxytryptamine (5-HT)-induced hyperpolarization responses recorded extracellularly from the rat isolated superior cervical ganglion (SCG). 2 Hyperpolarization responses induced by 5-HT (1 X 10(-8)-1 X 10(-4) M) in the presence of MDL 72222 (1 X 10(-5) M) were not antagonized by phentolamine (1 X 10(-6) M), prazosin (1 X 10(-7)-3 X 10(-7) M), haloperidol (1 X 10(-6) M) or ketanserin (1 X 10(-7)-1 X 10(-6) M). However, the latter two compounds both potentiated and increased the persistence of the hyperpolarization induced by moderate to high concentrations of 5-HT. Spiperone (1 X 10(-7) M) caused similar effects. All further experiments were performed in the presence of ketanserin (1 X 10(-6) M) as well as MDL 72222. 3 8-Hydroxy-2(di-n-propylamino)-tetralin (8-OH-DPAT; 1 X 10(-7)-1 X 10(-4) M) and ipsapirone (3 X 10(-5)-3 X 10(-4) M) behaved as weak hyperpolarizing agonists on the SCG. However, at concentrations below those required to produce hyperpolarization, both compounds acted as unsurmountable antagonists of 5-HT-induced hyperpolarization. 4 5-Carboxamidotryptamine (5-CT; 1 X 10(-9)-1 X 10(-5) M) mimicked the hyperpolarizing activity of 5-HT on the SCG. The EC50 for 5-CT was approximately 9 fold lower than that for 5-HT. 5 Spiperone (1 X 10(-7) - 1 X 10(-5) M) behaved as a reversible competitive antagonist of hyperpolarization responses induced by 5-HT with a pKB value of 7.40 +/- 0.09. Spiperone (1 X 10(-7)-1 X 10(-6) M) also caused concentration-dependent rightward displacement of the 5-CT concentration-hyperpolarization response curve. In this case, the pKB was 7.80 +/- 0.05. 6 (+/-)-Cyanopindolol (3 X 10(-7)-3 X 10(-6) M) caused non-parallel rightward displacements of the 5-HT concentration-response curve. Against 5-CT, (+/-)-cyanopindolol (3 X 10(-7)-3 X 10(-6) M) caused a concentration-independent rightward displacement of the concentration-response curve, accompanied by a large increase in the maximum response. 5-CT-induced hyperpolarization recorded in the presence of (+/-)-cyanopindolol (3 X 10(-7) M) was not significantly antagonized by methiothepin (1 X 10(-6) M) or methysergide (1 X 10(-6) M). 7 It is concluded that 5-HT-induced hyperpolarization of the rat SCG is mediated via a 5-HT1-like receptor which resembles the 5-HT1A binding site.(ABSTRACT TRUNCATED AT 400 WORDS)