Propranolol and serotonin removal in lung injury

Indicator dilution technique was used to study effects of reduced vascular volume or acute injury on removal of low doses of [3H]propranolol and [14C]serotonin (5-hydroxytryptamine, 5-HT) by perfused rabbit lung. Glass-bead (500 micron) embolization doubled pulmonary arterial pressure (Ppa) at flow rates of 20, 50, and 100 ml/min, decreased volume of distribution by approximately 50%, and increased pulmonary vascular resistance by at least 60%. Before embolization, (flow rate 20 ml/min) removal of [3H]propranolol and [14C] 5-HT was 89 +/- 2 and 75 +/- 5%, respectively, and was unaltered by changes in flow rate. However, after embolization, [3H]propranolol and [14C]5-HT removal decreased in a flow-dependent manner, reaching 28 +/- 4 and 1 +/- 3% (P less than 0.05), respectively, at a flow rate of 100 ml/min. When phorbol myristate acetate (PMA, 200 nM) was perfused (50 ml/min) through the lungs for 15 min, Ppa increased from 13 +/- 1 to 25 +/- 2 cmH2O (P less than 0.05), whereas [3H]propranolol removal decreased from 92 +/- 1 to 75 +/- 5% (P less than 0.05) and [14C]5-HT removal decreased from 73 +/- 3 to 46 +/- 8% (P less than 0.05). The PMA also caused vasoconstriction, which could be partially blocked by adding papaverine (500 microM) to the perfusion medium. Under the latter conditions, Ppa increased to 19 +/- 1 cmH2O and [3H]propranolol removal was unaffected. However, the combination of PMA and papaverine reduced [14C]5-HT removal from 64 +/- 4 to 19 +/- 3%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary metabolic function in the awake lamb: effect of development and hypoxia

The effect of postnatal development and acute alveolar hypoxia on pulmonary metabolic function was studied in conscious newborn lambs. Measurements of the ability of the lungs of these animals to metabolize [3H]benzoyl-L-phenyl-alanyl-L-alanyl-L-proline ([3H]BPAP; a synthetic substrate for angiotensin-converting enzyme, ACE) and to remove 5-hydroxy-[14C]tryptamine (5-[14C]HT) were made by modified indicator-dilution techniques during normoxic and hypoxic (fraction of inspired O2 = 0.10) conditions at 1 day, 1 wk, and 1 mo of age. Six additional sheep (8-23 wk old) were studied acutely as "adult" controls. BPAP metabolism in the 1-day-old group was 48 +/- 3% and increased slowly to 57 +/- 1% (P less than 0.05) at 1 mo of age and to 79 +/- 3% (P less than 0.01) by 23 wk of age. Pulmonary 5-[14C]HT removal was adultlike at birth (69 +/- 2%). Alveolar hypoxia significantly decreased BPAP only in the 1-day-old group (41 +/- 3%; P less than 0.05) and had no significant effect on 5-[14C]HT removal over the range of ages studied. These data demonstrate a selective and gradual postnatal development of pulmonary ACE which could be due to alterations in either the affinity or maximal capacity of pulmonary ACE, or increased endothelial cell surface area secondary to rapid growth of small blood vessels in this period. Alveolar hypoxia does not appear to closely regulate either ACE activity or 5-HT removal in conscious lambs greater than 1 day old when trace amounts of substrate are used.     

Effects of 2-substituted-4-phenylquinolines on uptake of serotonin and norepinephrine by isolated brain synaptosomes

In this present communication, the in vitro inhibition of the uptake of [3H]-L-norepinephrine ([3H] NE) and [3H]-Serotonin ([3H] 5-HT) by eleven synthesized 2-substituted-4-phenyl quinolines were studied using rat brain synaptosomal preparations. Compounds with an open side chain were relatively weak inhibitors of the synaptosomal uptake of [3H] NE and [3H] 5HT. Compounds having a distance of three atoms between the terminal basic nitrogen of the side chain and the quinoline ring were better inhibitors of serotonin uptake than those compounds having a four-atom distance. The replacement of the side chain with a piperazine ring produced compounds which were more potent and selective inhibitors of the uptake of either [3H] 5-HT or [3H] NE. Further structure-activity relationships are also discussed.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective effect of phorbol ester on serotonin removal and ACE activity in rabbit lungs

The effect of phorbol myristate acetate (PMA) on pulmonary removal of [14C]serotonin (5-[14C]HT) and metabolism of [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), a synthetic substrate for angiotensin-converting enzyme (ACE), was evaluated in isolated rabbit lungs perfused in situ with Krebs-albumin. Metabolic functions were assessed before, during, and after perfusion with 80 nM PMA (n = 11), or PMA plus 133 microM papaverine (n = 10) or PMA diluent (dimethyl sulfoxide, n = 11). Organ kinetic parameters (apparent Vmax, Km) were calculated by use of indicator-dilution techniques and by a mathematical model of whole-organ metabolism. PMA treatment resulted in a significant decline in Vmax for BPAP metabolism (from 52 +/- 4 to 30 +/- 4 nmol/s) and 5-HT removal (from 2.1 +/- 0.2 to 1.1 +/- 0.1 nmol/s). Km for BPAP was not significantly altered, whereas Km for 5-HT removal was higher after treatment (before treatment, 1.1 +/- 0.1 microM; after treatment, 2.3 +/- 0.6 microM). Coperfusion with papaverine, which attenuated the pressor response to PMA, abolished PMA-induced changes in Vmax for BPAP metabolism and in Km for 5-HT removal but left PMA-induced changes in Vmax for 5-HT removal intact. We conclude that PMA alters endothelial metabolic function by both hemodynamic and biochemical mechanisms that are independent of circulating blood cells. Pulmonary capacity for BPAP metabolism may largely reflect perfused surface area, and capacity for 5-HT removal may be more sensitive to frank endothelial cell dysfunction in this model.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic hypoxia on pulmonary vascular responses to biogenic amines

The effects of chronic hypoxia on pulmonary vascular resistance changes (% delta Rpv) to histamine, 5-hydroxytryptamine (5-HT), norepinephrine (NE), and KCl were studied in isolated perfused lungs from control rats and rats exposed to 7, 14, and 28 days of hypoxia. Histamine, which produced linear increases in % delta Rpv with increasing doses in the control, was reversed to vasodilation by chronic hypoxia of 7 and 14 days and at 28 days, vasodilation to this amine still predominated (7 out of 10). Control responses to 5-HT were unaltered by 7 days of hypoxia but enhanced at 14 and 28 days. Control responses to NE showed either vasoconstriction or vasodilation; at 7 days of hypoxia, NE had no significant vasoactivity; however, at 14 days, vasoconstriction and vasodilation were both observed, with vasodilation being more effective. Lastly, the pressor responses to KCl were not affected by chronic hypoxia of any duration. These results suggest that chronic hypoxia: 1) does not alter pulmonary vascular contractility (KCl); 2) reduces H1 and alpha-receptor activity while enhancing H2- and beta-receptor activity; and 3) enhances the pressor responses to 5-HT by increasing either the efficacy of this amine or the number of 5-HT vasoconstrictor receptors.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Compartmental analysis of the efflux of l-[3H]norepinephrine from isolated perfused rat lungs

Isolated rat lungs, pretreated with 100 microM pargyline and 100 microM U-0521 (3',4'-dihydroxy-2-methylpropriophenone) to block metabolism of norepinephrine (NE), were perfused with 0.3 microM 3H-labeled l-norepinephrine (1-[3H]-NE) for 30 min. Efflux samples were then collected for 30 min during washout of the tissue with amine-free Krebs solution. Compartmental analysis (nonlinear least-squares regression) of the efflux of tissue l-[3H]NE content vs. time indicates that NE is accumulated in a large slowly equilibrating compartment (t 1/2 = 58.15 +/- 6.84 min) in addition to distribution in the vascular (blue dextran tracer) and extracellular ([3H]sorbitol tracer) fluid compartments of the lung. Pretreatment of the lungs with 100 microM cocaine hydrochloride reduces the total l-[3H]NE space from 7.44 +/- 1.91 to 2.48 +/- 0.23 ml/g (P less than 0.05) by selectively decreasing the size of the slow NE compartment from 6.99 +/- 1.97 to 1.67 +/- 0.14 ml/g (P less than 0.05). The large size, cocaine sensitivity, and long efflux half time of this compartment suggest that neuronal uptake contributes to the pulmonary vascular inactivation of l-[3H]NE.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Dopamine Stimulates [3H]Phorbol 12,13-Dibutyrate Binding in Cultured Striatal Cells

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Effect of Different Photoperiod Exposure on [3H]Imipramine Binding and Serotonin Uptake in the Rat Brain

Seasonal rhythmicity in the occurrence of acute depressive episodes and the therapeutic efficacy of light exposure suggest the possible involvement of the pineal gland or other biological oscillators in the pathophysiology of depressive illness. We have performed studies to clarify whether different light/dark (LD) cycle schedules may induce changes in the biochemical targets of antidepressants in the rat CNS. In particular, we have investigated the effect of short- (LD 8:16) or long-day (LD 14:10) photoperiods on different biochemical parameters of serotonergic neurons. A significant increase in the density of [3H]imipramine ([3H]IMI) binding and in the Vmax of 5-[3H]hydroxytryptamine (5-[3H]HT) uptake was found in the hypothalamus of LD 8:16-with respect to LD 14:10-exposed rats, whereas no difference was found in the kinetic properties of postsynaptic 5-HT receptors and in 5-HT metabolism in the hypothalami and cerebral cortices of rats exposed to the two different photoperiods. A seasonal rhythm of [3H]IMI binding sites and 5-HT uptake seems to exist only in certain brain areas, such as the hypothalamus, because no differences were found in the cerebral cortex of LD 14:10- and LD 8:16-accustomed rats. [3H]IMI binding and 5-HT uptake were significantly increased in the hypothalamus of rats accustomed to a light/dark-inverted cycle (DL 10:14) and killed 6 h after the stopping of lighting in comparison to rats exposed to normal LD 14:10 cycles and killed 6 h after the beginning of lighting. Therefore, a circadian modification of the serotonergic presynaptic sites seems to be present and related to light/dark exposure. Because the existence of endogenous compounds able to modulate [3H]IMI binding and 5-HT uptake, other than 5-HT, has been postulated in the mammalian brain, the involvement of these substances in the periodic changes observed could be suggested.     

Identification and Characterisation of 5-Hydroxytryptamine3 Recognition Sites in Human Brain Tissue

[3H]Zacopride displayed regional saturable specific binding to homogenates of human brain tissues, as defined by the inclusion of BRL43694 [endo-N-(9-methyl-9-azabicyclo[3.3.1]non-3-yl)-1-methylindazole-3- carboxamide] in the incubation media. Scatchard analysis of the saturation data obtained from amygdaloid and hippocampal tissues identified the binding as being of high affinity and to a homogeneous population of binding sites (KD = 2.64 +/- 0.75 and 2.93 +/- 0.41 nmol/L and Bmax = 55 +/- 7 and 44 +/- 9 fmol/mg of protein in the amygdala and hippocampus, respectively). 5-Hydroxytryptamine 3 (5-HT3) receptor agonists and antagonists competed for the [3H]zacopride binding site, competing with up to 40% of total binding with a similar rank order of affinity in both tissues; agents acting on various other neurotransmitter receptors failed to inhibit binding. Kinetic data revealed a fast association that was fully reversible (k+1 = 6.61 X 10(5) and 7.65 X 10(5)/mol/L/s and k-1 = 3.68 X 10(-3) and 3.45 X 10(-3)/s in the amygdala and hippocampus, respectively). It is concluded that [3H]zacopride selectively labels with high affinity 5-HT3 recognition sites in human amygdala and hippocampus and, if these binding domains represent 5-HT3 receptors, may provide the opportunity for 5-HT3 receptor antagonists to modify 5-HT function in the human brain.     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Lack of inhibition of thrombin-induced rise in intracellular Ca2+ levels and 5-hydroxytryptamine secretion by 1-oleoyl-2-acetylglycerol in human platelets

The effect of 1-oleoyl-2-acetylglycerol (OAG) on the thrombin-induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5-hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10-50 micrograms/ml) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10-50 micrograms/ml) 10 s to 5 min before or 10-60 s after addition of threshold concentrations of thrombin (less than 0.03 U/ml) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin-induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin-induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.     

Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.