Naftopidil inhibits 5-hydroxytryptamine-induced platelet aggregation and 5-hydroxytryptamine uptake in platelets of healthy volunteers

Naftopidil exerts its antihypertensive action via ₁-adrenoceptor blockage and Ca²⁺ antagonism in vascular smooth muscle. Since the chemically similar 1-(1-naphthyl) piperazine is known to be a 5-hydroxytryptamine₂ receptor antagonist, the 5-hydroxytryptamine (5-HT) antagonistic properties of naftopidil were tested by examining 5-HT-induced aggregation and 5-HT uptake in platelets from 12 healthy volunteers after oral administration of 60 mg naftopidil or placebo.Platelet aggregation in vitro was inhibited by naftopidil with a K_i value of 1.1 M, the pIC₅₀ was 5.09 with induction of aggregation by 1 M 5-HT. After oral administration of naftopidil, 5-HT-induced aggregation was significantly inhibited by 36%. 4 h after naftopidil administration, 5-HT uptake velocity was reduced by 33%. Naftopidil not only cancelled the circadian increase in 5-HT-induced aggregation velocity observed during placebo application, but also caused a decrease in aggregation velocity directly after peak plasma naftopidil levels. 5-HT uptake in platelets was also reduced following peak naftopidil plasma concentrations. The 5-HT inhibitory action of naftopidil adds a third possible antihypertensive property to naftopidil's ₁-adrenoceptor blocking and Ca²⁺ antagonistic properties.     

The active uptake of serotonin by platelets of schizophrenic patients and their families: Possibility of a genetic marker

The active uptake of serotonin (5-HT) by blood platelets of 20 schizophrenic patients and their families was studied. The uptake was studied over a wide range of 5-HT concentrations (0.1–20M), and K _m and V _max of the uptake process were calculated. Of 20 patients, 18 exhibited a lower rate of uptake than the family average at 5-HT concentrations lower than the K _m value. At a 5-HT concentration of 0.1 M, the average 5-HT uptake of patients was 2.15 pmol/10⁸ platelets/min, while that of families was 2.99 pmol/10⁸ platelets/min (33% difference). At a high 5-HT concentration, only the drug-treated patients had lower V _max than the family average, and this might be attributed to the effect of the drugs. K _m of patients and families were very similar. In eight families, one or more of the family members showed 5-HT uptake patterns very similar to that of the patient. We termed these healthy family members schizophrenic risks. Our findings indicate the involvement of some genetic factors in this disease.     

Uptake,accumulation and release of ouabain by isolated rat hepatocytes

Summary We investigated uptake of ouabain into isolated rat hepatocytes and release of ouabain from preloaded hepatocytes, thus assessing separately the two membrane transport steps, involved in biliary elimination of the drug. The following results were obtained:Uptake of ouabain was saturable (K _m=263±61M, V=3.3±1.0 nmol/mg prot.×min), energy-dependent, sensitive to dinitrofluorobenzene and temperature-dependent (E=80–96 kJ/mol). It had no pH-optimum in the physiological pH-range and was independent of the extracellular cation composition.Uptake of ouabain was competitively inhibited by the cardiac glycoside digitoxin (K _i=0.3 M).Uptake was not inhibited in the presence of the glycosidic sugar l-rhamnose, but it was competitively inhibited by the steroid taurocholate (K _i=6.3 M).Ouabain was accumulated within hepatocytes 170-fold. The predominant fraction of intracellular ouabain beeing unbound.Release of ouabain from preloaded cells was energyindependent, independent of the Na⁺-concentration and not susceptible to inhibition by dinitrofluorobenzene or taurocholate.It is concluded, first that hepatocellular uptake of ouabain is mediated by a carrier for steroids and second that the pathway of release is distinct from that of uptake. We assume, that the high bile/plasma concentration-gradient of ouabain in vivo is generated during active uptake into the cell and not during release into bile.     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus

Summary The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [³H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [³H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 mol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 mol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK_B value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT₄ receptors. 5-HT caused an increase in basal outflow of [³H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC₅₀: 6.9) was less potent than 5-HT (-log EC₅₀ of the high affinity component: 7.9). ICS 205-930 (0.01–1.0 mol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA₂ value of 8.0. It is concluded that stimulation of both 5-HT₄ receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT₃ receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [³H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 mol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 mol/1) 5-methoxytryptamine enhanced the evoked outflow of [³H]acetylcholine, an effect which was attenuated by 3 mol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT₁ receptors) and facilitate (via 5-HT₄ receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT₁ receptor is blocked by methiothepine. Send offprint requests to H. Kilbinger at the above address     

5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well.Antagonists to block the contractile response to 5-HT via 5-HT₂, 5-HT₃ or 5-HT₄ receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) strongly inhibited the relaxations to ATP and VIP. l-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of l-NNA (100 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70–100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -Chymotrypsin (0.015 mg·ml^–1) and trypsin (0.005 mg·ml^–1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-d-Phe⁶, Leu¹⁷]VIP (1 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

[3H]-5-Methoxytryptoline is not actively accumulated by rabbit platelets

Summary 5-Methoxytryptoline (5-MeO-TLN, 6-methoxytetrahydro--carboline) inhibits with high affinity [³H]-imipramine binding to the serotonin transporter in platelets. To evaluate whether 5-MeO-TLN is a substrate for the serotonin transporter, the accumulation of [³H]-5-MeO-TLN into rabbit platelets was studied in vitro. At short incubation times (5 min), [³H]-5-MeO-TLN accumulation was temperature-sensitive, but not saturable over a concentration range from 0.06 mol/l to 10 mol/l Moreover, [³H]-5-MeO-TLN uptake was not affected by 100 mol/1 ouabain, its structural analogs tryptoline and 5-hydroxytryptoline, nor by the serotonin uptake inhibitors imipramine and citalopram. After longer incubation times (60 min), [³H]-5-MeO-TLN accumulation at O°C approached that seen at 37°C and temperature-sensitive [³H]-5-MeO-TLN uptake could no longer be observed. It is concluded that temperature-sensitive accumulation of [³H]-5-MeO-TLN is not mediated by the serotonin transporter and most likely represents a passive, diffusional process, the rate of which is temperature-dependent. The present studies thus confirm the hypothesis that 5-MeO-TLN affects [³H]-imipramine binding in platelets through a competitive mechanism and not via an allosteric interaction mediated through the substrate recognition site of the macromolecular complex of the serotonin transporter. Send offprint requests to S. Z. Langer at the above address     

Der Einfluß des Prednison- und Prednisolonbisguanylhydrazons auf die Na+ + K+-stimulierte Membran-ATPase des Meerschweinchenherzens

Zusammenfassung Prednison- und Prednisolonbisguanylhydrazon hemmen ebenso wie k-Strophanthin eine aus Meerschweinchenherzen gewonnene durch Na⁺ + K⁺ stimulierte Membran-ATPase. Eine 50%ige Hemmung erfolgt bei Konzentrationen von 3,8 M Prednisonbisguanylhydrazon, 0,28 M Prednisolonbisguanylhydrazon bzw. 1,3 M k-Strophanthin. Dieses Wirksamkeitsverhältnis der drei Verbindungen entspricht etwa der Hemmung des aktiven Ionentransportes an Meerschweinchenerythrocyten und dem positiv inotropen Effekt am isoliert durchströmten Meerschweinchenherzen.

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Summary Prednison- and Prednisolonbisguanylhydrazon inhibit like k-Strophanthin the Na⁺ + K⁺-stimulated ATPase from guinea-pig hearts. 50% inhibition was stated with concentrations of 3,8 M Prednisonbisguanylhydrazon, 0,28 M Prednisolonbisguanylhydrazon or 1,3 M k-Strophanthin. This difference in effectiveness of the compounds corresponds to the inhibition of the active ion-transport in erythrocytes of guinea-pigs and to the positive inotropic effect in isolated perfused guinea-pig hearts.

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Mit 1 TextabbildungDie Ergebnisse wurden auf der 5. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft am 28. April 1964 in Mainz vorgetragen. [Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 247, 341 (1964).]     

Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines

1-Methyl-4-phenylpyridinium (MPP⁺), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP⁺ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of ³H-MPP⁺, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP¹ transport.The accumulation of ³H-MPP⁺ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of ³H-MPP⁺ uptake was as follows: V_max = 181.3 ± 11.1 pmol mg protein^–1 min^–1 and K_m = 47.1 M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein^–1 min^–1) for the non-saturable uptake of ³H-MPP⁺ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of ³H-MPP⁺ accumulation at a substrate concentration of 100 nM ³H-MPP⁺, it was found that the rate constant of inward transport of ³H-MPP⁺ into hepatocytes (k_in) was 15.7 ± 3.8 l mg protein^–1 min^–1, the rate constant of outward transport of ³H-MPP⁺ from hepatocytes (k_out) was 0.077 ± 0.023 min^–1 and the equilibrium accumulation (A_max) of ³H-MPP⁺ was 20.2 ± 2.0 pmol mg protein^–1 (n = 36). Decynium22 (1,1-diethyl-2,2-cyanide; 1 M) significantly reduced k_in to 6.1 ± 1.8 l mg protein^–1 min^–1 (P < 0.05) and the equilibrium accumulation (A_max) of ³H-MPP⁺ to 9.6 ± 1.3 pmol mg protein^–1 (P < 0.005) (n = 36). ³H-MPP⁺ accumulation (in cells incubated with 200 nM ³H-MPP⁺) was sensitive to (–)-adrenaline, (–)-isoprenaline, (–)-dopamine, (±)-adrenaline and (–)-noradrenaline. The most potent catecholamine in inhibiting ³H-MPP⁺ uptake was (–)-adrenaline, with an IC₅₀ of 99 (22, 449) M (n = 6). (–)-Adrenaline competitively inhibited ³H-MPP⁺ uptake, as it significantly increased the K_m value of ³H-MPP⁺ uptake (to 125.4 M (63.6; 187.1); P < 0.02; n = 3) but did not change the V_max value. The cyanide-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene]methyl)quinolinium), which inhibit uptake₂ as well as the apical type of the renal transporter for organic cations, potently inhibited ³H-MPP⁺ uptake with IC₅₀'s of 1.4 (0.4–5.3) (n = 6) and 6.5 (2.6–16) (n = 4) M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition with either pargyline (500 M + Ro01-2812) (3,5-dinitropyrocatechol; 2 M) or pargyline (500 M) + U-0521(3,4-dihidroxy-2-methyl-propiophenone; l2 M)), (–)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of ³H-MPP⁺. Moreover, the uptake of ³H-MPP⁺ in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 ± 30.4%; P < 0.05; n = 4) than in the absence of these compounds. Therefore, the effect of these MAO and COMT inhibitors on ³H-MPP⁺ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of ³H-MPP⁺ (in cells incubated with 200 nM ³H-MPP⁺): 500 M pargyline, 2 M Ro 012812 and 100 M U-0521 decreased the accumulation of ³H-MPP⁺ to 38.1 ± 6.8 (n = 5), 60.5 ± 10.1(n = 7) and 71.3 ± 14.5 (n = 7) % of control, respectively.It is concluded that ³H-MPP⁺ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cy anine863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.     

Transient Expression of a Purine-Selective Nucleoside Transporter (SPNTint) in a Human Cell Line (HeLa)

Purpose. The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na⁺-dependent, purine-selective nucleoside transporter (SPNT_int) and to study the interactions of nucleosides and nucleoside analogs with this transporter. Methods. Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNT_int. Nucleoside transport activity was measured using [³H] inosine, [³H]uridine, [³H]-dideoxyinosine (ddl), and [³H]-2-chloro-2-deoxyadenosine (2CdA) as model substrates. Results. Expression of SPNT_int was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na⁺-stimulated uptake of [³H]inosine in cells transiently transfected with SPNT_int was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na⁺-stimulated uptake of both inosine and uridine was saturable (K_m = 28.1 ± 7.1 M and 20.6 ± 5.6 M, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddl (IC₅₀ = 46 M) and 2CdA (IC₅₀ =.13 M) also significantly inhibited the Na⁺-stimulated uptake of [³H]inosine. A Na⁺-stimulated uptake of [³H]2CdA was observed suggesting that 2CdA is also a permeant of SPNT_int. Conclusions. HeLa cells transiently transfected with SPNT_int represent a useful tool to study the kinetics and interactions of drugs with SPNT_int.     

Frequency- and train length-dependent variation in the roles of postjunctional α1- and α2-adrenoceptors for the field stimulation-induced neurogenic contraction of rat tail artery

Summary The present paper examines the roles of postjunctional ₁- and ₂-adrenoceptors for the noradrenaline (NA)-induced neurogenic contractile response to field stimulation mainly with 1–100 pulses at 2 or 20 Hz, in the tail artery of adult normotensive rats. Pharmacological tools were employed to isolate and characterize the ₁- and ₂-adrenoceptor-mediated components of this response. The degree to which the drugs influenced NA release or reuptake was assessed by their effects on the electrochemically determined, stimulation-induced rise in the NA concentration at the innervated outer surface of the media. This response was unaffected by ,-methylene ATP (10 M) or suramin (500 M), added to desensitize or block P₂-purinoceptors, respectively prazosin (0.1 M) or SK&amp;F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxyl]-3-methyl-1H-2, 3, 4, 5-tetrohydro-3-benzazepine, 0.1 M), used to block postjunctional ₁- and ₂-adrenoceptors respectively, nifedipine (10 M), blocker of Ca²⁺ influx through L-type channels, and ryanodine (10 M), which blocks mobilization of Ca²⁺ from intracellular stores; it was moderately enhanced by yohimbine (0.1 M), blocker of pre- and postjunctional ₂-adrenoceptors, and strongly enhanced by cocaine (3 M) or desipramine (1 M), blockers of NA reuptake. Judging from their inhibitory effects on the contractile responses to the ₁- and ₂-adrenoceptor agonists, phenylephrine andxylazine, prazosin (0.1 M)and SK & F 104078 (0.1 M) could be used to selectively block ₁- and ₂-adrenoceptors respectively, while yohimbine (0.1 M) was less selective, strongly depressing ₂- and slightly depressing ₁-adrenoceptor-mediated responses. The ₁-adrenoceptor-mediated component of the contractile response to short trains at 20 Hz was fast in onset, brief in duration and abolished by ryanodine; that mediated by ₂-adrenoceptors was more delayed, prolonged and insensitive to ryanodine. Both components were dose-dependently depressed by nifedipine (0.1–10 M). The small contractile responses to single pulses, or up to 50 pulses at 2 Hz, or short train (< 4 pulses) at 20 Hz, were more markedly depressed by 0.1 M yohimbine or SK & F 104078 than by 0.1 M prazosin and, hence, mediated mainly by ₂-adrenoceptors. The reverse was true of the much larger response to longer trains at 20 Hz, which thus probably was mediated mainly by ₁-adrenoceptors. Cocaine or desipramine, as well as ,-methylene ATP or suramin, amplified both components of the NA induced contractile response especially that mediated via a₁-adrenoceptors and caused by single pulses or short trains.The main conclusions are (i) that the small NA-induced contractile responses of this artery to single pulses, or pulses at low frequency, or in short trains at high frequency, are mediated mainly via ₂-, and the larger responses to longer trains at high frequency increasingly via ₁-adrenoceptors, (ii) that the ₁- and ₂-adrenoceptor-mediated components interact cooperatively, probably at least in part by utilizing two different pathways to increase the intracellular Ca²⁺, (iii) that neuronal reuptake of NA strongly restricts both components of the NA-induced contraction, especially the ₁-adrenoceptor-mediated response to single pulses or short trains, and (iv) that both components of the NA-induced contraction, especially that mediated by ₁-adrenoceptors, may be depressed by ATP released by field stimulation and acting via P_2x-purinoceptors on smooth muscle. Based on these results a novel working hypothesis is proposed, in which it is assumed that the geometry of NA-mediated neuromuscular transmission in this vessel varies with the frequency and number of impulses in a stimulus train.Correspondence to J.-X. Bao at the above address     

Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin

This study investigated the vasorelaxant activity, superoxide radicals (O₂^–)-scavenging capacity and cyclic nucleotide phosphodiesterase (PDE)-inhibitory effects of hesperidin and hesperetin, two flavonoids mainly isolated from citrus fruits. Hesperetin concentration-dependently relaxed the isometric contractions induced by noradrenaline (NA, 1 M) or by a high extracellular KCl concentration (60 mM) in intact rat isolated thoracic aorta rings. However, hesperetin (10 M–0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 M). Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 M), tetraethylammonium (TEA, 2 mM) or nifedipine (0.1 M) did not significantly modify the vasorelaxant effects of this flavonoid. Hesperetin (10 M–0.1 mM) did not affect the basal uptake of ⁴⁵Ca²⁺ but decreased the influx of ⁴⁵Ca²⁺ induced by NA and KCl in endothelium-containing and endothelium-denuded rat aorta. Hesperetin (10 M–0.1 mM) did not scavenge O₂^– generated by the phenazine methosulfate (PMS)-reduced -nicotinamide adenine dinucleotide (NADH) system. Hesperetin (0.1 mM) significantly reversed the inhibitory effects of NA (1 M) and high KCl (60 mM) on cyclic nucleotide (cAMP and cGMP) production in cultured rat aortic myocytes. Hesperetin preferentially inhibited calmodulin (CaM)-activated PDE1 and PDE4 isolated from bovine aorta with IC₅₀ values of about 74 M and 70 M respectively. In contrast, the 7-rhamnoglucoside of hesperetin, hesperidin (10 M–0.1 mM), was inactive in practically all experiments, although it inhibited basal and cGMP-activated PDE2 isolated from platelets (IC₅₀ values of 32±4 M and 137±34 M respectively). These results suggest that the vasorelaxant effects of hesperetin are basically due to the inhibition of PDE1 and PDE4 activities.     

Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: Involvement of 5-HT3 receptors

Summary Isolated segments of the guinea-pig small intestine were vascularly perfused and the release of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) into the portal venous effluent determined by high pressure liquid chromatography with electrochemical detection. Release of acetylcholine from isolated superfused intestinal segments was determined as outflow of [³H]radioactivity from preparations preincubated with [³H]choline. Cisplatin (3 M) increased the outflow of 5-HT and 5-HIAA by about 90%. At 30 and 100 M cisplatin decreased the outflow of 5-HT and its metabolite by 40%–50%. The stimulatory effect of cisplatin was consistently observed only when the bicarbonate-phosphate buffer of the Tyrode's solution was replaced by HEPES-buffer. The stimulatory effect of cisplatin was abolished in the absence of extracellular calcium or presence of tetrodotoxin (1 M). The stimulatory effect of cisplatin was also prevented by hexamethonium (100 M) or scopolamine (100 nM). The 5-HT₃ receptor antagonists ondansetron and ICS 205-930 in concentrations as low as 1 pM also abolished the stimulatory effect of cisplatin. The 5-HT₃ receptor antagonist MDL 72222 prevented the stimulatory effect of cisplatin only at a concentration of 1 M. None of the 5-HT₃ receptor antagonists alone significantly altered the outflow of 5-HT and 5-HIAA.Cisplatin (3 M) enhanced the outflow of [³H]radioactivity from intestinal segments and caused longitudinal muscle contractions that were abolished by 100 nM scopolamine.In conclusion, cisplatin, at concentrations which occur during anti-cancer therapy in humans and induce emesis, increases the release of 5-HT from the enterochromaffin cells of the small intestine of the guinea-pig. This effect of cisplatin is mediated by a cascade of events which involves release of acetylcholine and stimulation of 5-HT₃ receptors. Send offprint requests to H. Schwörer at his present address     

Further studies on the extraneuronal uptake and metabolism of isoprenaline in the perfused rat heart

Summary To simultaneously determine the kinetics of removal, O-methylation and accumulation of ³H-isoprenaline, isolated rat hearts were perfused for 4 min with various concentrations of ³H-isoprenaline. The apparent K _m for the O-methylation of ³H-isoprenaline (3.3±0.5 M) was more than one order of magnitude lower than the corresponding value for the accumulation of unchanged amine (71.3±7.1 M). The apparent K _m for removal was very similar to that for accumulation (63.2±5.9 M). At perfusion concentrations higher than 25 M, i.e. when O-methylation was saturated, removal virtually equalled accumulation. However, at low substrate concentrations removal of ³H-isoprenaline was overwhelmingly followed by O-methylation; this led to a marked difference between rates of removal and those of accumulation.When initial rates of uptake of ³H-isoprenaline were determined after 1.5 min of perfusion of the hearts by the method of Graefe et al. (1978), the uptake of ³H-isoprenaline consisted of two components: a nonsaturable and a saturable (after subtraction of the nonsaturable component from the total uptake).The kinetic constants of the saturable component of uptake were higher than those obtained after 4 min perfusion (see above) (K _m : 110±19 M; V _max: 80±4 nmoles·g^–1·min^–1).Corticosterone competitively inhibited the saturable component of uptake of ³H-isoprenaline (K _m : 1.2 M).During wash out of accumulated ³H-isoprenaline, O-methylation took place predominantly in one of the two extraneuronal compartments. The efflux of 3-O-methyl-³H-isoprenaline (³H-OMI), the O-methylated metabolite of ³H-isoprenaline, was characterized by a half time of about 1.2 min. O-methylation accelerated the loss of radioactivity from the tissue during wash out.The extraneuronal uptake of ³H-isoprenaline was characterized as a pump and leak system by means of steady-state kinetics of accumulation of ³H-isoprenaline. Half saturation of the steady-state accumulation was observed at a concentration of 104.5 ±18.5 M ³H-isoprenaline; the leak component was characterized by a rate constant of 0.0359 min^–1.This study was supported by the Deutsche Forschungsge-meinschaftA preliminary account was presented at the 6th International Congress of Pharmacology (Graefe et al., 1975)     

Investigation of the 5-hydroxytryptamine receptor mediating the “transient” short-circuit current response in guinea-pig ileal mucosa

5-Hydroxytryptamine (5-HT) stimulated an increase in short-circuit current (I_sc) in guinea-pig isolated ileal mucosa over a wide concentration range (0.1 nM-0.1 mM). The concentration-response relationship was biphasic, consisting of a high potency phase (0.1 nM–1 M) and a low potency phase (3–10 M). Stimulation of Isc observed at the high potency phase tended to be sustained while responses at the low potency phase (3–10 M) contained two components, an initial transient response followed by a maintained response. Both the high potency phase (maximum stimulation 30 A cm^–2) and the low potency phase (maximum stimulation 80 A cm ^–2) 5-HT response were antagonized by tetrodotoxin (TTX, 0.3 M) and atropine (1 M). However, another low potency (3 M-0.1 mM, maximum stimulation 30 A cm^–2) component of the 5-HT response was revealed in the presence of TTX or atropine.In the presence of methysergide (1 M), the concentration-response relationship of 5-HT was still biphasic and tropisetron (0.1 and 10 M) antagonized both phases of the 5-HT response. In the presence of methysergide, the high potency phase 5-HT response was mimicked by 5-methoxytryptamine (5-MeOT) and the selective 5-HT₄ agonist SC-53116 but not by BIMU 8. The potent 5-HT₄ antagonist GR 113808 antagonized the response to 5-MeOT in a surmountable manner with an affinity estimate of 9.6 ± 0.3 (n = 4). The 5-MeOT stimulated increase in I_sc was also antagonized in an unsurmountable manner by granisetron (1 M).In the presence of methysergide, desensitization of 5-HT₃ receptors with 2-methyl-5-hydroxytryptamine (10 M) abolished both phases of the 5-HT response. Under the same condition, desensitization of 5-HT₄ receptors with 5-MeOT (10 M) abolished only the high potency 5-HT response and dextrally shifted the low potency 5-HT response.These data show that neuronal and non-neuronal 5-HT receptors are involved in the regulation of secretion in ileal mucosa. We propose the presence of a neuronal 5-HT₄ receptor located upstream of the well characterized neuronal 5-HT₃ receptors to be responsible for the high potency 5-HT response. A schematic model is proposed to explain our findings and the relationship between this 5-HT₄ receptor and other 5-HT receptor subtypes regulating secretion that have been described in the literature.     

The depolarizing action of 5-hydroxytryptamine on rabbit isolated preganglionic cervical sympathetic nerves

Summary This study describes a depolarizing action of 5-hydroxytryptamine (5-HT) on rabbit isolated preganglionic cervical sympathetic nerves using an extracellular recording technique. From cumulative concentration-response curves for 5-HT (1 mol/1-1 mmol/1), the mean maximal depolarization was shown to be 277 ± 32 V and EC₅₀ was 9.4 mol/l(6.5–13.6 mol/l, geometric mean, 95% confidence limits, n = 42). The responses to 5-HT displayed marked tachyphylaxis. When cumulative concentration-response curves to 5-HT and 2-methyl-5-HT were determined in the same preparations (n = 4), the mean maximal response to 5-HT was 519 ± 167 V, EC₅₀ 32.2 mol/l (8.8–118 mol/l) and the mean maximal response to 2-methyl-5-HT was 317 ± 63 V, EC₅₀ 35.1 mol/l (12.9–95.5 mol/l, geometric means, 95 % confidence limits). The action of selective 5-HT antagonists was tested on repeated cumulative concentration-response curves to 5-HT. Neither methiothepin (0.1–1 mol/l, _n = 3) nor ketanserin (0.1–1 mol/l, n = 3) had an action on 5-HT responses. The selective 5-HT₃ antagonists MDL 72222, ICS 205-930 and SDZ 206–830 were all potent antagonists of the 5-HT depolarizations. The action of these antagonists was quantified by determining the apparent pA₂ from the dose ratios and a Schild plot. For MDL 72222 (1 nmol/1-0.1 mol/l), the apparent pA₂ was 9.1 ± 0.1 (n = 12), Schild plot: 9.2; for ICS 205–930 (0.1 nmol/l–3 nmol/1), the apparent pA₂ was 10.4 ± 0.1 (n = 11), Schild plot 10.3, and for SDZ 206–830 (0.03 nmol/l-1 nmol/1), the apparent pA₂ was 11.2 ± 0.1 (n = 12), Schild plot 11.2. 5-HT depolarizations were unaffected by hexamethonium (0.5 mmol/1). 5-HT depolarizations were reduced by superfusion with both Na-free (42 ± 8% of controls, n = 4) and Na/Ca-free media (35 ± 7% of controls, n = 4). It is concluded that 5-HT depolarizations of rabbit preganglionic cervical sympathetic nerve are mediated by 5-HT₃ receptors. The data with selective 5-HT₃ receptor antagonists suggest that the receptor profile may be more like that for the 5-HT₃ receptor on the terminals of sympathetic nerves than that for the 5-HT₃ receptor on the soma of superior cervical ganglion cells or on vagal afferent neurones. Send offprint requests to D. I. Wallis at the above address     

The uptake of tyramine by rat platelets

The uptake of [¹⁴Cityramine by rat platelets was less efficient than the uptake of [¹⁴C]-5-HT. [¹⁴C]-Tyramine was taken up in a two phase curve which could be resolved into a rapid saturable phase and a slower non-saturable phase that was linear with concentration. The value of the non-saturable component was ～ 12 pmoles/10³ platelets/15 sec at 10μM tyramine concentration. The k_m of active transport was ～ 3 μM and the V_max was ～ 25 pmoles/10⁸ platelets/15 sec.[¹⁴C]Tyramine active uptake was inhibited by both serotonin (k_i ～ 0.8μM) and fennuramine (k₁ ～ 2.0μM). These results strongly suggest that the active uptake of tyramine by rat platelets is accomplished by means of the 5-HT transport mechanism. The affinity of tyramine for the 5-HT receptor (～ 3 μM) is substantially higher than the affinity reported for dopamine (～ 70 μM).This indicates that the 3-hydroxyl group of dopamine diminishes its affinity for the 5-HT receptor of rat platelets.