LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma.

BACKGROUND AND PURPOSE: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co-administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP). EXPERIMENTAL APPROACH: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper-reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05-1 mg·mL(-1) ) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure. KEY RESULTS: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper-reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co-administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL(-1) reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin. CONCLUSIONS AND IMPLICATIONS: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans.

Bronchoprotection in conscious guinea pigs by budesonide and the NO-donating analogue, TPI 1020, alone and combined with tiotropium or formoterol.

BACKGROUND AND PURPOSE: Inhaled corticosteroids, anticholinergics and ß2-adrenoceptor agonists are frequently combined for treating chronic respiratory diseases. We examine the corticosteroid, budesonide, and novel NO-donating derivative, TPI 1020, against histamine- and methacholine-induced bronchoconstriction and whether they enhance the ß2-adrenoceptor agonist formoterol or muscarinic antagonist tiotropium in conscious guinea pigs. EXPERIMENTAL APPROACH: Dunkin-Hartley guinea pigs received inhaled histamine (3 mM) or methacholine (1.5 mM) and specific airway conductance (sG(aw)) was measured before and 15 or 75 min after treatment with budesonide, TPI 1020, tiotropium or formoterol alone or in combinations. KEY RESULTS: Formoterol (0.7-10 µM) and budesonide (0.11-0.7 mM) inhibited histamine-induced bronchoconstriction and tiotropium (2-20 µM) inhibited methacholine-induced bronchoconstriction by up to 70.8 ± 16.6%, 34.9 ± 4.4% and 85.1 ± 14.3%, respectively. Formoterol (2.5 µM) or tiotropium (2 µM) alone exerted small non-significant bronchoprotection. However, when co-administered with TPI 1020 0.11 mM, which alone had no significant effect, there was significant inhibition of the bronchoconstriction (45.7 ± 12.2% and 79.7 ± 21.4%, respectively). Co-administering budesonide (0.11 mM) with tiotropium (2 µM), which alone had no effect, also significantly inhibited the methacholine bronchoconstriction (36.5 ± 13.0%), but there was no potentiation of formoterol against histamine. The NO scavenger, CPTIO, prevented the bronchoprotection by SNAPand TPI 1020. CONCLUSIONS AND IMPLICATIONS: TPI 1020 potentiated the bronchoprotection by formoterol and tiotropium. Budesonide also enhanced the effects of tiotropium but not formoterol. Combination of TPI 1020 with a long-acting ß2-adrenoceptor agonist or muscarinic receptor antagonist may therefore be a more potent therapeutic approach for treatment of chronic respiratory diseases.

Atypical P2X receptor pharmacology in two human osteoblast-like cell lines.

BACKGROUND AND PURPOSE: The expression and function of P2X(7) receptors in osteoclasts is well established, but less is known about their role in osteoblast-like cells. A study in P2X(7) receptor knockout mice suggested the involvement of these receptors in bone formation. We have investigated the expression and pharmacology of several P2X receptors in two human osteosarcoma cell lines to see if they could be involved in bone turnover in man. EXPERIMENTAL APPROACH: Reverse transcriptase-polymerase chain reaction and Western blotting were used to study P2X(2), P2X(4) and P2X(7) receptor expression at mRNA and protein levels, respectively, in human osteoblast-like cells. P2X(7) receptor pharmacology was studied by measuring pore formation in the presence of different agonists and antagonists using the YO-PRO 1 uptake method. KEY RESULTS: P2X(4) and P2X(7) receptor mRNA and protein were found to be expressed by these cell lines. No evidence was found for P2X(4)/P2X(7) receptor heteropolymerization. 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (DBzATP) was equipotent to ATP and the antagonists used were either ineffective or weakly blocked pore formation. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that P2X(4) and P2X(7) receptors are expressed by human osteoblast-like cells. The affinities of the different agonists suggest that the P2X(7) receptor is mainly responsible for pore formation although P2X(4) receptors may also be involved. The low affinity of DBzATP and the weak action of the antagonists support the previously described atypical pharmacology of the P2X(7) receptor in osteoblasts. Targeting the P2X(7) receptor in osteoblasts could represent a promising new treatment for bone diseases such as osteoporosis and rheumatoid arthritis.

Dietary trace amine-dependent vasoconstriction in porcine coronary artery.

BACKGROUND AND PURPOSE: The dietary trace amines tyramine and beta-phenylethylamine (beta-PEA) can increase blood pressure. However, the mechanisms involved in the vascular effect of trace amines have not been fully established. The purpose of this study was to evaluate whether trace amine-dependent vasoconstriction was brought about by tyramine and beta-PEA acting as indirect sympathomimetic agents, as previously assumed, or whether trace amine-dependent vasoconstriction could be mediated by recently discovered trace amine-associated (TAA) receptors. EXPERIMENTAL APPROACH: The responses to p-tyramine and beta-PEA were investigated in vitro in rings of the left anterior descending coronary arteries of pigs. KEY RESULTS: p-Tyramine induced a concentration-dependent (0.1-3 mM) vasoconstriction. The maximum response and pD(2) value for p-tyramine was unaffected by endothelium removal or pre-treatment with antagonists for adrenoceptors, histamine, dopamine or 5-HT receptors. beta-PEA also produced a concentration-dependent (0.3-10 mM) vasoconstriction which was unaffected by endothelium removal, beta-adrenoceptor or 5-HT receptor antagonists. A substantial, but reduced, response to beta-PEA was obtained in the presence of prazosin (alpha(1)-adrenoceptor antagonist), haloperidol (D(2)/D(3) dopamine receptor antagonist) or mepyramine (H(1) histamine receptor antagonist). The pD(2) value for beta-PEA was unaffected by any of the antagonists tested. CONCLUSIONS AND IMPLICATIONS: Vasoconstriction induced by p-tyramine does not involve an indirect sympathomimetic effect, although vasoconstriction caused by beta-PEA may occur, in part, by this mechanism. We therefore propose that trace amine-dependent vasoconstriction is mediated by phenylethylamine-specific receptors, which are closely related to or identical to TAA receptors. These receptors could provide a target for new antihypertensive therapies.

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors.

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.

Distribution of P2X(1) and P2X(3) receptors in the rat and human urinary bladder.

Adenosine 5'-triphosphate (ATP) is known to play a significant role as a neurotransmitter in smooth muscle. There is evidence to show that ATP can cause bladder contractions and may also be involved in the processing of sensory information in the urinary bladder. These effects are likely to be mediated by P2X receptors, namely P2X(1) and P2X(3), respectively. This study set out to investigate their distribution in rat and human urinary bladders. P2X(1) receptor immunoreactivity was found on detrusor muscle fibres and P2X(3) receptor immunoreactivity was found in the urothelium of both species. This is the first demonstration of a non-neuronal localisation for P2X(3) receptors. No clear evidence was found for the presence of P2X(3) receptors on calcitonin gene-related peptide-containing sensory nerves and therefore P2X(3) receptors may not have a direct role in the mediation of sensory responses to ATP in the urinary bladder.

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors.

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X(2/3) heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious 'warming' stimuli.

Apparent species differences in the kinetic properties of P2X(7) receptors.

1. Apparent species differences in the responses of recombinant P2X(7) receptors to repeated application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. 2. Repeated application of 100 microM BzATP resulted in a progressive increase in current magnitude (current growth) at mouse and human, but not rat P2X(7) receptors. 3. Current growth was thought to reflect progressive dilation of the P2X(7) ion-channel to a pore permeable to large molecules (MW<900), suggesting that channel dilation was not occurring at the rat P2X(7) receptor. However, 100 microM BzATP produced a rapid influx of YO-PRO-1 (MW375) in cells expressing rat or human P2X(7) receptors. 4. There were, however, species differences in agonist potency such that 100 microM BzATP was a supra-maximal concentration at rat, but not human or mouse, P2X(7) receptors. Importantly, when sub-maximal concentrations of BzATP or ATP were examined, current growth occurred at rat P2X(7) receptors. 5. The rate of current growth and YO-PRO-1 accumulation increased with agonist concentration and appeared more rapid at rat and human, than at mouse P2X(7) receptors. 6. The potency of BzATP and ATP was 1.5 - 10 fold lower in naïve cells than in cells repeatedly exposed to ATP. 7. This study demonstrates that current growth occurs at mouse, rat and human P2X(7) receptors but only when using sub-maximal concentrations of agonist. Previously, current growth was thought to reflect the progressive increase in pore diameter of the P2X(7) receptor ion channel, however, the results of this study suggest a progressive increase in agonist potency may also contribute.

Evidence for P2X3 receptors in the developing rat brain.

P2X receptor-mediated responses to the ATP analogue, alpha,beta-methylene ATP, in rat brain cannot be accounted for by the receptor proteins known to be present. Such experiments are often performed on cells from neonates and, since differential developmental regulation of P2X1 and P2X2 receptor messenger RNAs has already been demonstrated, this is likely to be the case for other P2X receptors. This study was designed to address the possible existence of alpha,beta-methylene ATP-sensitive P2X3 receptors in rat brains of various ages using a P2X3 receptor-selective antibody. P2X3 receptor protein was found in discrete regions of the embryonic (E16) and neonatal rat brain (P7 and P14) but was not detectable in adult animals. This is the first demonstration of the presence of these receptors in brains from various ages of rat and the differential expression of these receptors in neonates may account for some reported electrophysiological responses to alpha,beta-methylene ATP.

Molecular cloning and functional characterization of a rat somatostatin sst2(b) receptor splice variant.

1. The mouse somatostatin (SRIF) sst2 receptor exists in two splice variants, sst2(a) and sst2(b), which differ in their intracellular carboxy-termini only. The murine sst2(b) receptor was reported to be less prone to agonist-induced desensitization as compared with the sst2(a) receptor. To determine whether a sst2(b) splice variant with similar functional characteristics exists in the rat, we have isolated a cDNA fragment from rat gastric mucosa encoding a sst2(b) receptor and expressed the full-length protein in CHO-K1 cells for functional characterization. 2. This study provides the first evidence for the occurrence in the rat of the sst2(b) receptor, which has a 15 amino acid carboxy-terminus differing in composition to the 38 amino acid C-terminus of the rat sst2(a) receptor. 3. In CHO-K1 cells expressing rat recombinant sst2(a) or sst2(b) receptors, SRIF caused concentration-dependent increases in extracellular acidification rates (EAR) with pEC50 values of 9.0 and 9.9, respectively. Pre-treatment with pertussis toxin (Ptx) caused a rightward displacement of the SRIF concentration-effect curves with pEC50 values of 8.3 (sst2(a) and 8.4 (sst2(b)). 4. SRIF (3 pM-3 nM) also caused concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation in CHO-sst2(a) cells (pIC50 10.5) and CHO-sst2(b) cells (pIC50 10.4). The degree of inhibition was less with higher concentrations of SRIF resulting in bell-shaped concentration-effect curves. Following pre-treatment with Ptx, the inhibitory effect of SRIF was abolished and SRIF caused only increases in cyclic AMP formation. 5. Both the SRIF-induced increases in EAR and inhibition of cyclic AMP formation were susceptible to agonist-induced desensitization, but this was less apparent following pre-treatment with Ptx. 6. This demonstrates that the operational characteristics of the recombinant rat sst2(a) and sst2(b) receptors are broadly similar. Both isoforms couple to Ptx-sensitive as well as -insensitive G proteins and are equally prone to agonist-induced desensitization.

Localization and functional expression of splice variants of the P2X2 receptor.

cDNAs encoding three splice variants of the P2X2 receptor were isolated from rat cerebellum. The first variant has a serine/proline-rich segment deleted from the intracellularly located carboxyl-terminal domain of the P2X2 subunit. The second and third variants have the splice site in the second half of the predicted first transmembrane domain. Either a 12-amino acid insertion or a six-amino acid deletion occurs at this position. cRNAs for these isoforms of the P2X2 subunit were injected into Xenopus laevis oocytes and tested for function. ATP evoked inward currents only with the splice variant [designated P2X2(b)] having the 69-amino acid deletion. The potencies of various agonists at the homomeric P2X2(b) receptor were not significantly different from those at the P2X2(a) homomeric channel. However, the P2X2(b) receptor showed significantly lower antagonist sensitivity. In contrast to the nondesensitizing P2X2(a) receptor, prolonged application of ATP produced a more rapid desensitization of the P2X2(b) receptor. When the P2X2(a) and P2X2(b) receptor responses were recorded in transfected mammalian cells, this difference was again found. The change in desensitization may be determined by proline/serine-rich segments and/or phosphorylation motifs that are removed from the tail region in formation of the P2X2(b) subunit. In situ hybridization of the three newly isolated isoforms of the P2X2 subunit was performed at the macroscopic and cellular levels; transcripts for two of them [P2X2(b) and p2x2(c)] but not the third [p2x2(d)], which carries the 12-amino acid addition, were present in many structures in the neonatal rat brain and on sensory and sympathetic ganglia. mRNA for the p2x2(d) splice variant was present only in the nodose ganglion, at a low level.

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

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

A quantitative autoradiographical study on the distribution of somatostatin sst2 receptors in the rat central nervous system using [125I]-BIM-23027.

The kinetic properties, steady state binding characteristics and autoradiographic distribution of the somatostatin (SRIF) sst2 receptor-selective ligand, [125I]-BIM-23027, have been investigated in the rat central nervous system. Analysis of kinetic, saturation and competition binding data in rat hippocampal membranes was consistent with [125I]-BIM-23207 binding to a single population of non-interacting binding sites. Competition studies, using different SRIF ligands suggested that [125I]-BIM-23027 was binding to sites similar to that of the recombinant sst2 receptor. The rank order of affinity for displacing specific binding was BIM-23027 = SRIF > L-362855 > > BIM-23056. There was a widespread distribution of [125I]-BIM-23027 binding sites in the rat central nervous system. The highest density of binding was observed in the dentate gyrus, medial habenular, amygdala, claustrum and lateral septum as well as in the piriform, cingulate and parietal cortex. The cervical and lumbar spinal cord also displayed moderate levels of binding localized to the substantia gelatinosa. The cellular localization of [125I]-BIM-23027 binding was found to be associated with dendritic terminal fields. In contrast, the cellular signal for sst2 receptor mRNA was restricted to cell somata. The widespread distribution of [125I]-BIM-23027 binding sites within the brain suggests that receptors similar to the recombinant sst2 receptor may mediate a variety of different physiological effects within the central nervous system.

Localization of P2X purinoceptor transcripts in the rat nervous system.

We used transcript-specific oligonucleotides to examine the localization in the rat nervous system of the corresponding mRNAs for the two P2X purinoceptor genes cloned recently from the rat vas deferens and PC12 cells. PC12 P2X purinoceptor mRNA was labeled in the olfactory tubercle, striatum, hypothalamus, hippocampus, dentate gyrus, amygdala, cortex, and cerebellum, whereas the vas deferens P2X purinoceptor-specific probes labeled the cerebellum and, at lower levels of expression, the striatum, hippocampus, and cortex. Both types of P2X purinoceptor transcript were found on cell bodies in the nodose and superior cervical ganglia. The presence of these two purinoceptor transcripts in the brain was confirmed by polymerase chain reaction. Two partial cDNAs, identical to sections of the PC12 or vas deferens P2X purinoceptor coding sequences, were amplified from neonatal brain and cerebellum poly(A)+ RNA, respectively. These findings are in broad agreement with earlier Northern blot studies on the PC12 P2X purinoceptor mRNA but differ from those for the vas deferens P2X purinoceptor mRNA, which had not previously been detected in adult brain. This difference is attributed to the low levels seen in the adult compared with the neonate and to the greater sensitivity of the methods used in the present study. The neonate medial habenula had low levels of transcripts for the PC12 but none for the vas deferens P2X purinoceptor. Because pharmacologically the recombinant PC12 P2X purinoceptor differs from the functional purinoceptor in the medial habenula, these results suggest the existence of other, unidentified, P2X purinoceptors in the rat nervous system.

Autoradiographic distribution of [3H]L-NG-nitro-arginine binding in rat brain.

The distribution of nitric oxide synthase (NOS), the enzyme which produces nitric oxide, has previously been studied in the rat central nervous system (CNS) using the NADPH-diaphorase technique and anti-NOS antibodies. However, the former method may not always be selective for NOS while the latter is not quantitative. Therefore a selective, quantifiable method would be desirable. L-NG-Nitro-arginine, an inhibitor of NOS, is available in a tritiated form which we have shown to bind to NOS. We have now examined the regional distribution of [3H]L-NG-nitro-arginine binding in the rat CNS using autoradiography. [3H]L-NG-nitro-arginine specific binding was seen in a number of brain regions with the highest levels in the accessory olfactory bulb, the amygdaloid complex, the Islands of Calleja and the cerebellum. This regional distribution of [3H]L-NG-nitro-arginine binding sites in the rat CNS was, in general, similar to that seen with the NADPH-diaphorase method and anti-NOS antibodies, consistent with the view that all three methods identify NOS in the CNS. Thus, [3H]L-NG-nitro-arginine appears to be a useful radioligand for studying the distribution of NOS in the CNS as its binding is quantifiable and apparently selective for NOS.

Central pre- and postsynaptic 5-HT1A receptors in rats treated chronically with a novel antidepressant, cericlamine.

Biochemical and electrophysiological approaches were used to assess the possible changes in 5-hydroxytryptamine (serotonin) 5-HT1A receptors in the rat brain after a long-term treatment with cericlamine [2-(3,4-dichlorobenzyl)-2-dimethylamino-1-propanol], a novel serotonin reuptake inhibitor with antidepressant properties. Possible changes in other serotonin receptor binding sites (5-HT2A, 5-HT2C and 5-HT3) were also investigated after this treatment. Cericlamine was injected for 2 weeks at a dose (16 mg/kg i.p., twice daily) that ensured complete prevention of 4-methyl-alpha-ethyl-meta-tyramine-induced depletion of brain serotonin. In vitro binding and quantitative autoradiographic studies showed that neither 5-HT1A, 5-HT2A, 5-HT2C nor 5-HT3 receptor binding sites in various brain areas were affected by the 14-day treatment with cericlamine. Although forskolin-stimulated adenylate cyclase activity was significantly increased in hippocampal homogenates from cericlamine-treated rats, the reduction in this enzymatic activity due to 5-HT1A receptor stimulation by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was unchanged in these animals as compared with controls. In contrast, in vitro and in vivo electrophysiological recordings of serotoninergic neurons in the dorsal raphe nucleus revealed a clearcut functional desensitization of somatodendritic 5-HT1A autoreceptors. Thus the potency of 8-OH-DPAT and ipsapirone to depress the firing rate of these neurons in brain stem slices was significantly reduced after the 2-week treatment with cericlamine. In vivo, the potency of an injection of cericlamine to inhibit the discharge of serotoninergic neurons was also markedly less in rats that had been pretreated for 2 weeks with this drug as compared with controls. However, the inhibitory effects of systemically injected 8-OH-DPAT and ipsapirone on the electrical activity of serotoninergic neurons were as pronounced in cericlamine-treated rats as in controls. In addition, the reduction in serotonin synthesis due to an acute treatment with 8-OH-DPAT (0.1 or 0.3 mg/kg s.c.) was not significantly different in both groups of rats. These data support the idea that postsynaptic (in the hippocampus) and somatodendritic (in the dorsal raphe nucleus) 5-HT1A receptors are differently regulated in the rat brain, because only the latter receptors desensitized after a long-term blockade of serotonin reuptake by cericlamine. They also suggest that the inhibitory influence of systemically administered direct 5-HT1A agonists such as 8-OH-DPAT and ipsapirone on the electrical and metabolic activity of serotoninergic neurons does not result solely from the stimulation of somatodendritic 5-HT1A autoreceptors.

(-)Tertatolol is a potent antagonist at pre- and postsynaptic serotonin 5-HT1A receptors in the rat brain.

The potential 5-HT1A antagonist properties of the beta-antagonist tertatolol were assessed using biochemical and electrophysiological assays in the rat. (+/-) Tertatolol bound with high affinity (Ki = 38 nM) to 5-HT1A sites labelled by [3H]8-OH-DPAT in hippocampal membranes. The (-)stereoisomer (Ki = 18 nM) was about 50-fold more potent than the (+)stereoisomer (Ki = 864 nM) to inhibit the specific binding of [3H]-8-OH-DPAT. As expected of a 5-HT1A antagonist, (-)tertatolol prevented in a concentration-dependent manner (Ki = 24 nM) the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal homogenates. Furthermore in vivo pretreatment with (-)tertatolol (5 mg/kg s.c.) significantly reduced the inhibitory influence of 8-OH-DPAT (0.3 mg/kg s.c.) on the accumulation of 5-hydroxytryptophan in various brain areas after the blockade of aromatic L-amino acid decarboxylase by NSD-1015 (100 mg/kg i.p.). In vitro (in brainstem slices; Ki approximately 50 nM) and in vivo (in chloral hydrate anaesthetized rats; ID50 approximately 0.40 mg/kg i.v.), (-)tertatolol prevented the inhibitory effects of the 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone and lesopitron on the firing rate of serotoninergic neurones within the dorsal raphe nucleus. In about 25% of these neurones, the basal firing rate was significantly increased by (-)tertatolol (up to +47% in vitro, and +30% in vivo). These data indicate that (-)tertatolol is a potent competitive antagonist at both pre (in the dorsal raphe nucleus)-and post (in the hippocampus)-synaptic 5-HT1A receptors in the rat brain.

5-HT3 receptors in the rat central nervous system are mainly located on nerve fibres and terminals.

Autoradiographic and membrane binding studies with [3H](R,S)- or [3H](S)-zacopride were performed in combination with lesions using various neurotoxins in an attempt to identify which neuronal cell types are endowed with 5-HT3 receptors in the rat central nervous system. Lesions of noradrenergic (by DSP-4), dopaminergic (by 6-hydroxydopamine) and serotonergic (by 5,7-dihydroxytryptamine) systems had little effect generally on the density of 5-HT3 receptors labelled with [3H](R,S)- or [3H](S)-zacopride in various regions of the brain and the spinal cord. The only exception was the amygdala where a significant loss (approximately -20%) of 5-HT3 receptors labelled by [3H](R,S)-zacopride was associated with the selective lesion of serotonergic fibres by 5,7-dihydroxytryptamine. Microinjection of kainic or ibotenic acid into the dorsal and ventral hippocampus reduced the density of 5-HT1A receptors labelled with [3H]8-OH-DPAT (approximately -45%) as expected from their known location on intrinsic neuronal cell bodies and/or dendrites. In contrast, the same lesion did not affect 5-HT3 receptors, suggesting their location on fibres 'en passage'. At the spinal level, 5-HT3 receptors were found to exist on primary afferent fibres terminating within the superficial layers of the dorsal horn, as shown by the marked reduction in the local autoradiographic labelling by [3H](S)-zacopride after either dorsal rhizotomy (-81%) or neonatal capsaicin treatment (-72%). These data suggest that 5-HT3 receptors in the central nervous system are generally located presynaptically on nerve terminals or fibres of non-monoaminergic neurones.

New methoxy-chroman derivatives, 4[N-(5-methoxy-chroman-3-yl)N- propylamino]butyl-8-azaspiro-(4,5)-decane-7,9-dione [(+/-)-S 20244] and its enantiomers, (+)-S 20499 and (-)-S 20500, with potent agonist properties at central 5-hydroxytryptamine1A receptors.

The potential interaction of the new methoxy-chroman derivatives: (+/-)-S 20244 (4-[N-(5-methoxy-chroman-3-yl)N-propylamino]butyl-8-azaspiro- (4,5)-decane-7,9-dione) and its enantiomers (+)-S 20499 and (-)-S 20500 with central 5-hydroxytryptamine1A (5-HT1A) receptors was assessed using biochemical and electrophysiological tests in the rat. In vitro binding assays revealed that these drugs bound with high affinity to 5-HT1A sites in hippocampal membranes (Ki: 0.19 nM for (+)-S 20499, 0.95 nM for (-)-S 20500 and 0.35 nM for the racemate (+/-) S 20244). As seen with the prototypical 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin, (+/-)-S 20244, (+)-S 20499 and (-)-S 20500 inhibited forskolin-activated adenylate cyclase in hippocampal homogenates with potencies corresponding to their respective affinities for 5-HT1A sites. The maximal inhibitory effect of the chroman derivatives was not additive with that of 8-hydroxy-2-(di-n- propylamino)tetralin and could be competitively reduced by 5-HT1A antagonists such as (-)-propranolol and (+/-)-tertatolol. Electrophysiological recordings within the dorsal raphe nucleus both in vitro (in brain-stem slices) and in vivo (in chloral hydrate anesthetized rats) showed that (+)-S 20499, (+/-)-S 20244 and (-)-S 20500 induced, in that order of (decreasing) potency, a dose-dependent reduction in the spontaneous firing of serotoninergic neurons. In vitro, as well as in vivo, the inhibitory influence of the chroman derivatives on the discharge frequency of serotoninergic neurons could be competitively antagonized by (+/-)-tertatolol. Finally, oral administration of increasing doses of the most potent enantiomer, (+)-S 20499, induced a marked reduction in the rate of 5-HT turnover, without affecting that of dopamine, in various brain areas. All these biochemical and electrophysiological data indicate that (+)-S 20499 is a highly potent agonist at both presynaptic (i.e., somatodendritic) and postsynaptic 5-HT1A receptors in the rat brain.

Inhibition of 5-hydroxytryptamine neuronal activity by the 5-HT agonist, DOI.

Systemic, intra-raphe and microiontophoretic administration of the 5-hydroxytryptamine (5-HT)1C/5-HT2 agonist (1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI) inhibited the firing of 5-HT neurones in the dorsal raphe. DOI administered systemically and directly into the raphe also decreased the extracellular concentration of 5-hydroxytryptamine (5-HT) in the frontal cortex. In contrast, the administration of DOI directly into the frontal cortex did not significantly alter the concentration of frontal cortical extracellular 5-HT. The reduction of the firing rate of 5-HT neurons in the dorsal raphe and extracellular 5-HT concentration in the frontal cortex induced by systemic administration of DOI could not be blocked by the 5-HT2 antagonist ketanserin, ritanserin (5-HT2/5-HT1C antagonist) or the putative 5-HT1A antagonist, pindolol. These results suggest that the inhibition of 5-HT neuronal firing seen with administration of DOI is mediated via an action within the dorsal raphe and at least in close proximity to the 5-HT neurone cell bodies. The decrease in frontal cortical extracellular concentration of 5-HT release was not due to a direct action in the frontal cortex itself and may possibly be as a result of the decrease in the firing rate of the 5-HT neurones in the dorsal raphe. The mechanism of action of DOI to produce these effects is, however, unclear and warrants further investigation.