Involvement of central beta2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats.

Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A(2) TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and beta(2)-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5-3 micromol/kg, i.v.) or its stable analogue methanandamide (0.75 micromol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 mumol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB(1) and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 mumol/kg each), and/or SQ 29548 (1 mumol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 mumol per animal i.c.v.) and by the thromboxane A(2) synthesis inhibitor furegrelate i.c.v. (1.8 micromol per animal). ICI 118551, MK-801 (1 micromol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central beta(2)-adrenergic, NMDA and thromboxane A(2) receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.

Urethane, but not pentobarbitone, attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic.

BACKGROUND AND PURPOSE: We examined whether cannabinoid CB(1) and histamine H(3) receptors resemble alpha(2)-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats. EXPERIMENTAL APPROACH: Effects of the cannabinoid agonist CP-55,940 and the H(3) receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with (3)H-noradrenaline. KEY RESULTS: The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB(1) or H(3) receptors and alpha(2) adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked (3)H-noradrenaline release. CONCLUSIONS AND IMPLICATIONS: Urethane, but not pentobarbitone, abolished the H(3) receptor-mediated vascular response in pithed rats and attenuated the CB(1) receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB(1), H(3) and alpha(2) receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.

Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin.

The aim of the present study was to challenge potential mechanisms of action underlying the inhibition of tumor cell proliferation by agmatine. Agmatine inhibited proliferation of the human hepatoma cells HepG2, the human adenocarcinoma cells HT29, the rat hepatoma cells McRH7777, and the rat pheochromocytoma cells PC-12. Inhibition of proliferation of HepG2 cells was associated with an abolition of expression of ornithine decarboxylase (ODC) protein and a doubling of mRNA content encoding ODC. In HepG2 cells, silencing of ODC-antizyme-1, but not of antizyme inhibitor, by RNA interference resulted in an increase of agmatine's antiproliferative effect. Thus, the distinct decrease in intracellular polyamine content by agmatine was due to a reduced translation of the synthesizing protein ODC but was not essentially mediated by induction of ODC-antizyme or blockade of antizyme inhibitor. In interaction experiments 1 mM L-arginine, 1 mM D-arginine, 1 mM citrulline, 100 microM N(omega)-nitro-L-arginine methyl ester, 1 and 10 microM sodium nitroprusside, and 1 microM N1-guanyl-1,7-diaminoheptane failed to alter agmatine's antiproliferative effect. Hence, the antiproliferative effect of agmatine in HT29 and HepG2 cells is due to an interaction with neither the NO synthases, the hypusination of eIF5A, nor an agmatine-induced reduction in availability of intracellular L-arginine. L-Arginine and citrulline, but not d-arginine, inhibited tumor cell proliferation by themselves. Their inhibitory effect was abolished after silencing of arginine decarboxylase (ADC) expression by RNA interference indicating the conversion to agmatine by ADC. Finally, in the four cell lines under study, agmatine-induced inhibition of cell proliferation was paralleled by an increase in intracellular caspase-3 activity, indicating a promotion of apoptosis.

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in guinea-pig vessels, but not in rat and mouse aorta.

Cannabinoids exert complex effects on blood pressure related to their interference with cardiovascular centres in the central nervous system and to their direct influence on vascular muscle, vascular endothelium and heart. In view of the relative lack of information on the occurrence of CB1 receptors on the vascular postganglionic sympathetic nerve fibres, the aim of the present study was to examine whether cannabinoid receptor ligands affect the electrically evoked tritium overflow in superfused vessels (tissue pieces) from the guinea-pig, the rat and the mouse preincubated with 3H-noradrenaline. The cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]1,4-benzoxazinyl](1-naphthalenyl) methanone) inhibited the evoked tritium overflow in the guinea-pig aorta, but not in that of the rat or mouse. The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB1 receptor antagonist rimonabant, yielding an apparent pA2 value of 7.9. The most pronounced (near-maximum) inhibition obtained at the highest WIN 55,212-2 concentration applied (3.2 microM) amounted to 40%. WIN 55,212-2 also inhibited the evoked overflow in guinea-pig pulmonary artery, basilar artery and portal vein, again in a manner sensitive to antagonism by rimonabant. The latter did not affect the evoked overflow by itself in the four vessels, but did increase the electrically evoked tritium overflow from superfused guinea-pig hippocampal slices preincubated with 3H-choline and from superfused guinea-pig retina discs preincubated with 3H-noradrenaline (labelling dopaminergic cells in this tissue). The inhibitory effect of 3.2 microM WIN 55,212-2 on the evoked overflow from the guinea-pig aorta was comparable in size to that obtained with agonists at the histamine H3, kappa opioid (KOP) and ORL1 (NOP) receptor (1 or 10 microM, producing the respective near-maximum effects) whereas prostaglandin E2 1 microM caused a higher near-maximum inhibition of 70%. Prostaglandin E2 also induced an inhibition by 65 and 80% in the rat and mouse aorta respectively, indicating that the present conditions are basically suitable for detecting presynaptic receptor-mediated inhibition of noradrenaline release. The results show that the postganglionic sympathetic nerve fibres in the guinea-pig aorta, but not in the rat or mouse aorta, are endowed with presynaptic inhibitory cannabinoid CB1 receptors; such receptors also occur in guinea-pig pulmonary artery, basilar artery and portal vein. These CB1 receptors are not subject to an endogenous tone and the extent of inhibition obtainable via these receptors is within the same range as that of several other presynaptic heteroreceptors, but markedly lower than that obtainable via receptors for prostaglandin E2.

Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors.

1 The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. 2 Under physiological conditions (135 mm sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 microm) similar to that in electrophysiological studies. 3 The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration-response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). 4 Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-d-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. 5 The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. 6 The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears to be an 'ideal' sodium substituent since it increases the signal-to-noise ratio without interfering with 5-HT binding.

Pharmacological characteristics of the specific transporter for the endogenous cell growth inhibitor agmatine in six tumor cell lines.

BACKGROUND AND AIMS: This study examined agmatine transport into six human intestinal tumor cell lines and compared the pharmacological properties of this transporter with those of the agmatine carrier previously characterized in human glioblastoma cells. METHODS: Carrier-mediated uptake was determined as specific accumulation of [(14)C]agmatine in the cells. The changes in intracellular agmatine concentration in the tumor cells after 24 h incubation with 1 mM agmatine was analyzed by high-performance liquid chromatography. RESULTS: Specific [(14)C]agmatine accumulation was found in the six human intestinal tumor cell lines Caco2, Cx1, Colo320, HT29, Colo205E, and SW480. Specific [(14)C]agmatine accumulation was inhibited by phentolamine, putrescine, spermine, clonidine, and decynium-22 but not by corticosterone, O-methylisoprenaline, or l-carnitine. Incubation with exogenous agmatine for 24 h increased intracellular agmatine content in all cell lines by a multiple of the basal endogenous content. Transfection of HEK293 cells with cDNA encoding either hOCT1, hOCT2, or hOCT3 did not enhance [(14)C]agmatine accumulation compared to nontransfected cells. CONCLUSION: All intestinal tumor cell lines investigated express a functional specific agmatine transporter which exhibit pharmacological characteristics similar to those of the agmatine transporter in glioblastoma cells. This agmatine carrier is not identical with any so far known organic cation transport system.

Molecular structure of the rabbit alpha2A-adrenoceptor: a contribution to the alpha2A-adrenoceptor versus I1 imidazoline receptor controversy.

In view of the high structural and pharmacological similarities between the alpha(2A)-adrenoceptors of humans and other mammalian species, it has been concluded, in particular, from experiments in rabbits that the (2A)-adrenoceptor is the exclusive site of action of central antihypertensive drugs, although the amino acid sequence of the alpha(2A)-adrenoceptor of just this species was unknown. Therefore, the aim of the present investigation was to determine the complete nucleotide sequence of the coding region of the rabbit alpha(2A)-adrenoceptor gene. Degenerate oligonucleotides corresponding to regions of the alpha(2A)-adrenoceptor conserved between rat and man were used in a polymerase chain reaction with genomic DNA prepared from rabbit. A 1,356-base pair product with an open reading frame of 1,353 base pairs was obtained that encodes a protein of 451 amino acids which is similar to the alpha(2A)-adrenoceptors of other mammals (man, pig, rat, mouse, guinea-pig and cattle) but not to their alpha(2B)- and alpha(2C)-adrenoceptor subtypes suggesting its classification as an alpha(2A)-adrenoceptor. However, the degree of amino acid sequence identity is, at best, only 80% and, thus, about 10% less than between the other mammalian species. Compared with the human sequence there are 81 substantial changes of amino acids. In conclusion, rabbit and human alpha(2A)-adrenoceptors substantially differ in their amino acid sequence which may explain the opposite pharmacodynamic properties of the central antihypertensive drug rilmenidine (alpha(2)-adrenoceptor agonism and antagonism, respectively) reported in the literature. Hence, the present study supports the view that experiments with central antihypertensive drugs in rabbits are not reliably predictive for the site of action of such drugs in man.

Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site.

Excised outside-out patches from HEK293 cells stably transfected with the human (h) 5-HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5-HT-induced current using the patch clamp technique. In addition, binding studies with radioligands for 5-HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5-HT-induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): 9-THC, WIN55,212-2, anandamide, JWH-015 and CP55940. The WIN55,212-2-induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212-3, an enantiomer of WIN55,212-2, did not affect the 5-HT-induced current. WIN55,212-2 did not change the EC50 value of 5-HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]-SR141716A and the CB1/CB2 receptor ligand [3H]-CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5-HT3A receptor cDNA, WIN55,212-2, CP55940, anandamide and SR141716A did not affect [3H]-GR65630 binding, but 5-HT caused a concentration dependent-inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5-HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5-HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Noradrenaline release-inhibiting receptors on PC12 cells devoid of alpha(2(-)) and CB(1) receptors: similarities to presynaptic imidazoline and edg receptors.

The aim of the present study was to classify release-inhibiting receptors on rat pheochromocytoma PC12 cells. Veratridine-evoked [3H]noradrenaline release from PC12 cells was inhibited by micromolar concentrations of the imidazoline and guanidine derivatives cirazoline, clonidine, aganodine, 1,3-di(2-tolyl)guanidine, BDF6143 and agmatine, and of the cannabinoid receptor agonist WIN55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-yl](1-naphthalenyl)methanone mesylate), but not by noradrenaline. The inhibitory effect of clonidine was antagonized by micromolar concentrations of rauwolscine and SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). The potencies of the agonists and antagonists were compatible with an action at previously characterized presynaptic imidazoline receptors. 1-Oleoyl-lysophosphatidic acid, but not sphingosine-1-phosphate, produced an inhibition of release that was antagonized by 30 microM rauwolscine, 1 microM SR141716A and 10 microM LY320135 as well as by pretreatment of the cells with 100 microM clonidine for 72 h. Polymerase chain reaction (PCR) experiments on cDNA from PC12 mRNA suggest mRNA expression of lysophospholipid receptors encoded by the genes edg2, edg3, edg5 and edg7, but not of receptors encoded by edg1, edg4, edg6 and edg8, and not of alpha(2A(-))nd CB(1) receptors. In conclusion, PC12 cells are not endowed with alpha(2)-adrenoceptors and CB(1) cannabinoid receptors, but with an inhibitory receptor recognizing imidazolines, guanidines and WIN55,212-2 similar to that on sympathetic nerves. The PCR results and the ability of 1-oleoyl-LPA to mimic these drugs (also with respect to their susceptibility to antagonists) suggest that the release-inhibiting receptor may be an edg-encoded lysophospholipid receptor.

Agmatine and putrescine uptake in the human glioma cell line SK-MG-1.

The pharmacological properties of a specific agmatine uptake mechanism were investigated in the human glioma cell line SK-MG-1 and compared with those of the putrescine transporter expressed by the same cells and with those of several other organic cation transport systems or ion channels reported in the literature. The specific accumulation of [14C]agmatine at 37 degrees C above nonspecific accumulation at 4 degrees C was energy-dependent and saturable with a Vmax of 64.3+/-3.5 nmol/min per mg protein and a Km of 8.6+/-1.4 microM. Specific accumulation was attenuated by replacement of extracellular Na+ by choline by 65%, not affected by lithium and enhanced by replacement by sucrose. Phentolamine, clonidine, 1,3-di(2-tolyl)guanidine, histamine, putrescine, spermine and spermidine were inhibitors of specific [14C]agmatine accumulation. In contrast, corticosterone, desipramine, O-methylisoprenaline, cirazoline, moxonidine, L-arginine, L-lysine, verapamil, nifedipine and CdCl2 at concentrations up to 10 mM failed to inhibit specific [14C]agmatine accumulation, thus excluding that the latter is mediated by amino acid or monoamine carriers, by Ca2+ channels or by the organic cation transporters OCT1, OCT2, OCT3, OCTN1 or OCTN2. The pattern of activity of inhibitory compounds was also different from that determined for specific putrescine accumulation found in the same cells (Km 1.3+/-0.1 microM, Vmax 26.1+/-0.4 nmol/min per mg protein) ruling out an identity of the specific [14C]agmatine and [14C]putrescine accumulation mechanisms. It is concluded that specific accumulation of agmatine in human glioma cells is mediated by a specific transporter whose pharmacological properties are not identical to those of the agmatine transporter previously identified in rat brain synaptosomes and to other so far known carrier mechanisms for organic cations and ion channels. The agmatine uptake system may be important for the regulation of the extracellular concentration of agmatine in man.

Novel histamine H(3)-receptor antagonists and partial agonists with a non-aminergic structure.

We determined the affinities of eight novel histamine H(3)-receptor ligands (ethers and carbamates) for H(3)-receptor binding sites and their agonistic/antagonistic effects in two functional H(3)-receptor models. The compounds differ from histamine in that the ethylamine chain is replaced by a propyloxy chain; in the three ethers mentioned below (FUB 335, 373 and 407), R is n-pentyl, 3-methylbutyl and 3,3-dimethylbutyl, respectively. The compounds monophasically inhibited [(3)H]-N(alpha)-methylhistamine binding to mouse cerebral cortex membranes (pK(i) 7.51 - 9.53). The concentration-response curve of histamine for its inhibitory effect on the electrically evoked [(3)H]-noradrenaline overflow from mouse cortex slices was shifted to the right by these compounds (apparent pA(2) 6.61 - 8.00). Only FUB 373 and 407 inhibited the evoked overflow by themselves (intrinsic activities 0.3 and 0.4); these effects were counteracted by the H(3)-receptor antagonist clobenpropit. [(35)S]-GTPgammaS binding to mouse cortex membranes was stimulated by the H(3)-receptor agonist (R)-alpha-methylhistamine in a manner sensitive to clobenpropit. Among the novel compounds only FUB 373 and 407 stimulated [(35)S]-GTPgammaS binding (intrinsic activities 0.6 and 0.4). In conclusion, the novel compounds are partial H(3)-receptor agonists (FUB 373 and 407) or H(3)-receptor antagonists; comparison with FUB 335 shows that the transition from antagonist to agonist is caused by a slight structural change. A protonated N atom in the side chain is not necessary for agonism at H(3) receptors, proposing a receptor-ligand interaction different from that of classical agonists.

Anandamide and methanandamide induce both vanilloid VR1- and cannabinoid CB1 receptor-mediated changes in heart rate and blood pressure in anaesthetized rats.

In anaesthetized rats activation of vanilloid receptors on sensory vagal nerves elicits rapid bradycardia and hypotension (Bezold-Jarisch reflex). Recent in vitro experiments revealed that the endogenous cannabinoid ligand anandamide acts as an agonist at the vanilloid VRI receptors. The present study was aimed at examining whether vanilloid VR1 receptors are involved in the cardiovascular effects of anandamide in the anaesthetized rat. Intravenous injection of anandamide, its stable analogue methanandamide and the vanilloid receptor agonist capsaicin produced a dose-dependent immediate and short-lasting decrease in heart rate and blood pressure with the following rank order of potencies: capsaicin > methanandamide > anandamide. This bradycardia was dose-dependently diminished by the selective vanilloid receptor antagonist capsazepine (0.3-3 micromol/kg) and the nonselective inhibitor of these receptors, ruthenium red (1-10 micromol/kg). Both antagonists reduced or tended to reduce the hypotension stimulated by the agonists. Following this bradycardia and hypotension (presumably evoked by the Bezold-Jarisch reflex; phase I), capsaicin, anandamide and methanandamide led to a brief vasopressor effect (phase II). Subsequently both anandamides, but not capsaicin, induced a more prolonged decrease in blood pressure (phase III). Capsazepine and ruthenium red (at doses up to 3 tmol/kg and 10 micromol/kg, respectively) failed to affect these changes in blood pressure. The cannabinoid CB1 receptor antagonist SR 141716 at 3 micromol/kg abolished the prolonged decrease in blood pressure (phase III) induced by anandamide and methanandamide, but had no effect on the reflex bradycardia and hypotension (phase I) and on the subsequent vasopressor effect (phase II) evoked by capsaicin, anandamide and methanandamide. In conclusion, the endogenous cannabinoid receptor agonist anandamide and its stable analogue methanandamide induce reflex bradycardia and hypotension (phase I) by activating the vanilloid VRI receptor. Whereas the mechanism underlying the brief vasopressor effect (phase II) is unknown, the prolonged hypotension (phase III) results from stimulation of the cannabinoid CB1 receptor.

Modified 5-HT3A receptor function by co-expression of alternatively spliced human 5-HT3A receptor isoforms.

Serotonin (5-HT) exerts fast excitatory responses by activation of 5-HT3 receptors, irrespective of whether they are homomerically composed of 5-HT3A subunits or heteromerically assembled of 5-HT3A and 5-HT3B subunits. Here we describe a short, truncated (h5-HT3AT) and a long (h5-HT3AL) splice variant of the human 5-HT3A (hS-HT3A) receptor subunit. The deduced protein of the short isoform consists of 238 amino acids (aa) with a single transmembrane domain (M1). Compared to the known 5-HT3A receptor, the long isoform contains 32 additional aa in the extracellular loop between M2 and M3. Both splice variants are co-expressed together with the 5-HT3A subunit in the amygdala and hippocampus, whereas in the placenta only the short variant is co-expressed. Both splice variants, when expressed in transfected human embryonic kidney (HEK) 293 cells, are not able to form functional homomeric receptors, but modify 5-HT response at heteromeric h5-HT3A receptors. Co-expression of the short variant considerably decelerates the desensitization of the 5-HT3 receptor; thus, heteromeric assemblies of h5-HT3A and the h5-HT3AT subunit exhibit 5-HT-induced cation fluxes which are much larger than those of homomeric hS-HT3A receptors. In contrast, heteromeric complexes containing the h5-HT3AL subunit display reduced cation fluxes. In conclusion, the splice variants increase the functional diversity of 5-HT3 receptors.

Nuclear factor-kappaB mediates simultaneous induction of inducible nitric-oxide synthase and Up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages.

The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.

Pharmacological properties of the naturally occurring Phe-124-Cys variant of the human 5-HT1B receptor: changes in ligand binding, G-protein coupling and second messenger formation.

The aim of this study was to analyse whether substitution of phenylalanine in position 124 of the human (h) 5-HT1B receptor by cysteine, a naturally occurring variant of this receptor, modifies not only ligand binding, but also G-protein coupling and second messenger formation. Stably transfected rat C6 glioma cells, which express either the h5-HT1B variant receptor (VR) or the wild-type receptor (WTR) were used. In saturation experiments with [3H]5-carboxamidotryptamine ([3H]5-CT), the maximum binding (Bmax) of the VR amounted to only 60% of that to WTR. In competition experiments with 1 nM [3H]5-CT, the following 5-HT receptor ligands exhibited a higher affinity for the mutant receptor than for the WTR: L-694,247, 5-CT, 5-HT, sumatriptan (agonists listed at decreasing order of potency) and SB-224289 (a selective h5-HT1B receptor inverse agonist with competitive antagonistic properties). In contrast, the mixed 5-HT1B/1D receptor antagonist GR-127935 exhibited equal affinity for both isoforms. The efficacy of L-694,247, 5-CT, 5-HT and sumatriptan in stimulating [35S]GTPgammaS binding (a measure of G protein coupling) to membranes of cells expressing the VR was approximately 50-65% lower compared to membranes of cells expressing the WTR, but their potency was 2.8-3.6-fold higher. SB-224289, which decreased [35S]GTPgammaS binding when given alone, but not GR-127935, was more potent in antagonizing the stimulatory effect of 5-CT on [35S]GTPgammaS binding to membranes expressing the VR compared to membranes expressing the WTR. In whole cells expressing the VR, 5-CT and sumatriptan inhibited the forskolin-stimulated cAMP accumulation 3.2-fold more potently than in cells expressing the WTR. In conclusion, our data suggest that the Phe-124-Cys mutation modifies the pharmacological properties of the h5-HT1B receptor and may account for pharmacogenetic differences in the action of h5-HT1B receptor ligands. Thus, the sumatriptan-induced vasospasm which occurs at low incidence as a side-effect in migraine therapy may be related to the expression of the (124-Cys)h5-HT1B receptor in patients with additional pathogenetic factors such as coronary heart disease.

Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects.

The patch-clamp technique was used on excised (outside-out) patches to characterize h5-HT3A receptors stably transfected in HEK 293 cells and to compare the effects of the barbiturate anaesthetics methohexital and pentobarbital on this ligand-gated cation channel. At negative membrane potentials 5-HT induced inward currents in a concentration-dependent manner (EC50=8.6 microM, Hill coefficient =1.5). The mean peak current induced by 30 microM 5-HT was -110 pA at -100 mV. The 5-HT3A receptor antagonist ondansetron (0.3 nM) reversibly inhibited the 5-HT (30 microM) signal by 70% and at 3 nM it abolished the response. Methohexital and pentobarbital inhibited 5-HT-induced (30 microM) currents in a concentration-dependent manner. The maximal inhibition with a given methohexital or pentobarbital concentration was reached when the respective drug was applied 45 s prior to and during the 2-s 5-HT pulse (IC50 values=95 microM and 127 microM, Hill coefficient = -1.0 and -1.6, respectively). Although the barbiturates were, thus, equipotent, their effects differed substantially with respect to the dependence on the time schedule of application to the patches: the potency of methohexital was virtually maximal when the drug was applied exclusively 45 s before the agonist pulse, but its inhibitory potency decreased considerably when it was exclusively applied during the 2-s 5-HT pulse (IC50=380 microM). Conversely, pentobarbital was almost maximally potent in inhibiting the 5-HT signal when it was exclusively coapplied with this agonist, but its inhibitory potency was considerably lower (IC50 approximately 500 microM) when applied exclusively 45 s before 5-HT. Another difference between both barbiturates involves the rate of inactivation of 5-HT3 receptor-mediated currents: whereas high concentrations of methohexital (> or = 300 microM) were necessary to induce moderate (< or = twofold) acceleration of this parameter, pentobarbital produced such an effect at all concentrations and the extent of acceleration increased with increasing concentration (1.5- to fivefold). In conclusion, two barbiturates, chemically closely related but of different lipophilicity, clearly differ with respect to the kinetics of their effect on 5-HT3 receptor channels; one possible explanation involves drug access to an amphipathic site of action via both an aqueous and a hydrophobic pathway. Pentobarbital, in contrast to methohexital, inhibits hS-HT3A receptor-mediated currents at anaesthetic concentrations (approximately 90 microM).

Species-specific pharmacological properties of human alpha(2A)-adrenoceptors.

On the basis of data obtained in rabbits, the imidazoline receptor ligand rilmenidine has been suggested to decrease blood pressure in humans by activating central alpha(2A)-adrenoceptors. A prerequisite for this hypothesis was the unproved assumption that rabbit and human alpha(2A)-adrenoceptors are equally activated by rilmenidine. Because alpha(2A)-adrenoceptors in the brain and on cardiovascular sympathetic nerve terminals are identical, the latter were used as a model for the former to confirm or disprove this assumption. Human atrial appendages and rabbit pulmonary arteries were used to determine the potencies of alpha(2)-adrenoceptor agonists in inhibiting the electrically (2 Hz) evoked [(3)H]norepinephrine release and of antagonists in counteracting the alpha(2)-adrenoceptor-mediated inhibition induced by moxonidine. In the rabbit pulmonary artery, rilmenidine and oxymetazoline are potent full agonists, whereas in the human atrial appendages they are antagonists at the alpha(2)-autoreceptors, sharing this property with rauwolscine, phentolamine, and idazoxan. In contrast, prazosin is ineffective. In addition, a partial nucleotide and amino acid sequence of the rabbit alpha(2A)-adrenoceptor (a region known to substantially influence the pharmacological characteristics of the alpha(2)-adrenoceptor) revealed marked differences between the rabbit and the human alpha(2A)-adrenoceptor. The sympathetic nerves of both the human atrial appendages and rabbit pulmonary artery are endowed with alpha(2A)-autoreceptors, at which, however, both rilmenidine and oxymetazoline exhibit different properties (antagonism and agonism, respectively). The antagonistic property of rilmenidine at human alpha(2A)-adrenoceptors indicates that in contrast to the suggestion based on rabbit data, the hypotensive property of the drug in humans is not due to activation of alpha(2A)-adrenoceptors but other, presumably I(1)-imidazoline receptors, are probably involved.

Dual interaction of agmatine with the rat alpha(2D)-adrenoceptor: competitive antagonism and allosteric activation.

In segments of rat vena cava preincubated with [(3)H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [(3)H]-noradrenaline release, the EP(3) prostaglandin receptor-mediated and the alpha(2D)-adrenoceptor-mediated inhibition of evoked [(3)H]-noradrenaline release was investigated. Agmatine (0.1-10 microM) by itself was without effect on evoked [(3)H]-noradrenaline release. In the presence of 10 microM agmatine, the prostaglandin E(2)(PGE(2))-induced EP(3)-receptor-mediated inhibition of [(3)H]-noradrenaline release was not modified, whereas the alpha(2D)-adrenoceptor-mediated inhibition of [(3)H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [(3)H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [(3)H]-clonidine and [(3)H]-rauwolscine to rat brain cortex membranes (K(i) values 6 microM and 12 microM, respectively). In addition, 30 and 100 microM agmatine increased the rate of association and decreased the rate of dissociation of [(3)H]-clonidine resulting in an increased affinity of the radioligand for the alpha(2D)-adrenoceptors. [(14)C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [(14)C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the alpha(2D)-adrenoceptor and enhances the effects of alpha(2)-adrenoceptor agonists probably by binding to an allosteric binding site of the alpha(2D)-adrenoceptor which seems to be labelled by [(14)C]-agmatine.

In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids L-arginine and L-ornithine.

In rat alveolar macrophages (AMphi) it was tested whether induction of iNOS by lipopolysaccharides (LPS) is accompanied by changes in L-arginine transport and whether L-ornithine, the product of arginase released from AMphi, could, via inhibition of L-arginine uptake, act as a paracrine inhibitor of NO synthesis. Rat AMphi (cultured for 20 h in the absence or presence of 1 microg/ml LPS) were incubated in Krebs-HEPES solution containing [3H]-L-arginine (0.1 microM for 2 min or 100 microM for 5 min) and the cellular radioactivity was determined as a measure of L-arginine uptake. In parallel, cells were incubated for 6 h in Krebs-HEPES solution containing 0-1 mM L-arginine and nitrite accumulation was determined. [3H]-L-arginine uptake (0.1 microM or 100 microM) occurred independently of sodium ions and was inhibited by L-ornithine (EC50: 117 and 562 microM, respectively) and with similar potencies by L-lysine. In LPS-treated AMphi the concentration inhibition curve of L-ornithine was shifted to the right by about a factor of 4, whereas that of L-lysine was only marginally shifted to the right. L-Leucine (0.1 and 1 mM) inhibited [3H]-L-arginine (0.1 microM) by 43 and 58%, respectively, and the effect of 0.1 mM L-leucine was partially sodium dependent. In LPS-treated AMphi, 0.1 mM L-leucine no longer inhibited [3H]-L-arginine and the effect of 1 mM L-leucine was attenuated. Kinetic analysis of the transport of [3H]-L-arginine and [14C]-L-ornithine revealed two components for each amino acid with Km values of 21 and 114 microM (L-arginine) and 39 and 1050 microM (L-ornithine), respectively. After LPS treatment Km2 of L-arginine transport was reduced to 63 microM and Vmax of both components was increased, whereas Km2 of L-ornithine transport was enhanced to 1392 microM and Vmax1 reduced. LPS-stimulated AMphi, incubated in amino acid-free Krebs-HEPES solution, produced about 4 nmol nitrite/10(6) cells per 6 h, and L-arginine enhanced nitrite accumulation maximally about threefold (EC50: 30 microM). L-ornithine, up to 3 mM, failed to affect significantly nitrite accumulation observed in the presence of 30 or 100 microM L-arginine. Rat AMphi express mRNA for two cationic amino acid transporters (CAT-1 and CAT-2B), and LPS markedly up-regulated mRNA for CAT-2B in parallel with mRNA for iNOS, but had no effect on that for CAT-1. In conclusion, in rat AMphi LPS up-regulates L-arginine transport and induces changes in the characteristics of the cationic amino acid transport resulting in preferential transport of L-arginine. These effects may be regarded as cellular measures to ensure a high L-arginine supply for iNOS.

Inhibition of neuronal Ca(2+) influx by gabapentin and subsequent reduction of neurotransmitter release from rat neocortical slices.

Cytosolic calcium ion concentrations ([Ca(2+)](i)) were measured in rat neocortical synaptosomes using fura-2, and depolarization of synaptosomal membranes was induced by K(+) (30 mM). The release of the endogenous excitatory amino acids glutamate and aspartate was evoked by K(+) (50 mM) and determined by HPLC. The release of [(3)H]-noradrenaline from rat neocortical synaptosomes or slices was evoked by K(+) (15 and 25 mM) and measured by liquid scintillation counting. Gabapentin produced a concentration-dependent inhibition of the K(+)-induced [Ca(2+)](i) increase in synaptosomes (IC(50)=14 microM; maximal inhibition by 36%). The inhibitory effect of gabapentin was abolished in the presence of the P/Q-type Ca(2+) channel blocker omega-agatoxin IVA, but not by the N-type Ca(2+) channel antagonist omega-conotoxin GVIA. Gabapentin (100 microM) decreased the K(+)-evoked release of endogenous aspartate and glutamate in neocortical slices by 16 and 18%, respectively. Gabapentin reduced the K(+)-evoked [(3)H]-noradrenaline release in neocortical slices (IC(50)=48 microM; maximal inhibition of 46%) but not from synaptosomes. In the presence of the AMPA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide (NBQX), gabapentin did not reduce [(3)H]-noradrenaline release. Gabapentin did, however, cause inhibition in the presence of the NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849). Gabapentin is concluded to reduce the depolarization-induced [Ca(2+)](i) increase in excitatory amino acid nerve terminals by inhibiting P/Q-type Ca(2+) channels; this decreased Ca(2+) influx subsequently attenuates K(+)-evoked excitatory amino acid release. The latter effect leads to a reduced activation of AMPA receptors which contribute to K(+)-evoked noradrenaline release from noradrenergic varicosities, resulting in an indirect inhibition of noradrenaline release.