Effects of compound 48/80 on mast cells,histamine and cyclic AMP in isolated superior cervical ganglia

Summary Superior cervical ganglia of the rat contain mast cells which are sensitive to degranulation by compound 48/80. The granulation process is shown to the independent of the ATP content of the ganglion. Compound 48/80 released histamine into the incubation medium, thereby decreasing the histamine content of the ganglia. Moreover, the release of ³H-noradrenaline was accelerated by the compound. Histamine and adrenaline induced a rapid accumulation of cyclic AMP in the ganglia. This effect of the amines was specifically blocked by diphenhydramine or propranolol with an ID₅₀ of 1.5×10^–9 M and 2.2×10^–7 M, respectively.In contrast to other findings with isolated mast cell preparations, compound 48/80 induced a rapid and marked accumulation of cyclic AMP in intact ganglia and an enhanced release of cyclic AMP into the incubation fluid. Diphenhydramine prevented the accumulation in the tissue but only partly inhibited the enhanced appearance of cyclic AMP in the medium. The accumulation of the cyclic nucleotide in the tissue was partly blocked by propranolol, suggesting an additional action of compound 48/80 on cyclic AMP through catecholamines.The cyclic nucleotide phosphodiesterase activity in homogenates of superior cervical ganglia was completely inhibited by compound 48/80 at 7 g/ml when low cyclic AMP concentrations were used.In addition to cyclic AMP release, rapid and marked efflux of ATP into the medium was observed during incubations with compound 48/80. The lactate dehydrogenase activity in the incubation medium was significantly enhanced with incubation periods of 40 to 60 min indicating rather slowly occurring toxic damage to cell membranes by compound 48/80.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70).     

The in vitro uptake of biogenic amines by snail (Heliz pomatia) nervous tissue.

The uptake of [³H]5-HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the suboesophageal ganglia of the snail, Helix pomatia, was studied. When tissues were incubated at 25° in mediums containing radioactive amines, tissue: medium ratios of about 30:1, 18: l, 4:1 and 5:1 for 5-HT, dopamine, noradrenaline and octopamine respectively were obtained after 20–30 min incubation. Tissues incubated at 25° in mediums containing radioactive amines for 20–30 min showed that 90% of the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]noradrenaline or [³H]octopamine. The high tissue : medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, demonstrated saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver-Burk plots, two uptake mechanisms for 5-HT at 25° were resolved, the high affinity uptake process having a K_m1valueof 8.48 × 10^?8 M and V_max1 value of 0.077 nmole/g per min, while the lower affinity process had a K_m2 value of 1.8 × 10^?6 M and a V_max2 value of 0.66 mole/g per min. At 0° a single uptake mechanism for 5-HT occurred which gave a K_m value of 0.152 × 10^?8 M and a V_m value of 0.0203 nmole/g per min. In the case of dopamine, the Lineweaver-Burk plot of 25° showed a single uptake process with values for K_m and V_max of 1.02 × 10^?7 M and 0.0673 nmole/g per min respectively. This process did not function at 0°. The effects of various agents and ions upon the accumulation processes for all amines were also studied, and the findings indicate that the same neurons may well accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the snail ganglia is a mechanism for inactivating these substances at 25° and that an uptake mechanism for 5-HT also functions at 0°. The present results are discussed from the point of view of the monoamines having transmitter functions in the snail CNS.     

Lidocaine uptake in isolated rat hepatocytes and effects of dl-propranolol

Isolated hepatocytes were prepared from adult male rats and were incubated at 37°C in Krebs' bicarbonate buffer (pH 7.4) containing trace amounts of [¹⁴C]lidocaine. Accumulation of lidocaine was demonstrable within 2 min following the addition of the labeled material. Prolongation of the incubation period did not result in an increase in tissue to medium ($\text{T}\text{M}$) ratio. The uptake was not affected by a reduced temperature (27°C). There was no saturation of uptake over a wide range of lidocaine concentration. Uptake of lidocaine was not inhibited by metabolic inhibitors. Thin-layer chromatography indicated that a portion of lidocaine was metabolized during the experimental period. Gel permeation chromatography showed that the majority of lidocaine was reversibly bound to cellular components, while one-third of the radioactivity was irreversibly associated with macromolecules. These results suggest that hepatic uptake of lidocaine is not mediated through a carrier system; binding to hepatic components may account for the observed accumulation. In addition, inclusion of dl-propranolol in the incubation medium caused a concentration-related decrease in lidocaine uptake and indicated a potential for clinically important interaction between lidocaine and propranolol.     

Selective inhibition of collagen synthesis by D-penicillamine in carrageenin-induced inflammation in rats.

The effect of d-penicillamine on syntheses of collagen and noncollagen protein in inflammed tissued was studied by incubating minced granulation tissue with [³H]proline and various amounts of d-penicillamine. d-Penicillamine selectively inhibited the incorporation of [³H]proline into collagen hydroxy-proline when 30 mM d-penicillamine was present in the incubation medium, and the accumulation of [³H]proline-labeled protocollagen was found in the minced granulation tissue incubated with 30 mM d-penicillamine. In addition, administration of d-penicillamine (800 mg/kg) into granuloma pouch every 12 hr for 4 days, selectively inhibited the collagen synthesis and increased the collagen solubility in granulation tissue. When partially purified prolyl hydroxylase was incubated with [3,4-³H]proline-labeled protocollagen substrate in the presence of d-penicillamine, the enzyme activity was decreased in proportion to the concentration of d-penicillamine. This inhibition was restored by increasing the amount of ferrous iron in the incubation medium. These results suggest that the selective inhibition of collagen synthesis in vivo and in vitro may be mainly due to the ability of d-penicillamine to chelate ferrous iron, a cofactor of prolyl hydroxylase.     

Compartmentation of an ATP substrate pool for histamine and adrenaline sensitive adenylate cyclase in rat superior cervical ganglia.

Incubation with adrenaline or histamine induces a rapid accumulation of adenosine 3′,5′-monophosthate (cyclic AMP) in the isolated superior cervical ganglion of the rat. The concentration of cyclic AMP is significantly elevated in the incubation medium after 4 min incubation with either amine. When ganglia are incubated with [¹⁴C]adenine ATP and cyclic AMP are labelled. The formation of [¹⁴C]cyclic AMP is enhanced by a subsequent incubation with histamine or adrenaline. Within the stimulation period of 10 min about 40 per cent of the radioactive cyclic AMP is released into the incubation medium. Endogenous ATP levels are not altered by incubation with adrenaline or histamine. Cultivation of ganglia for 4 hr increases their total ATP content, but the level of ATP falls to about 5 per cent when ganglia are incubated in a glucose-free medium. Liberation of ATP into the incubation medium is not observed under normal conditions, but after transfer of ganglia from cold medium into 37° Krebs-Henseleit medium considerable amounts of ATP are released into the incubation fluid within the first hour. This leakage of ATP is less pronounced when ganglia are incubated in the presence of adrenaline or histamine. Antimycin A₁ inhibits the formation of [¹⁴C]ATP from [¹⁴C]adenine, while the conversion rate of [¹⁴C]ATP to [¹⁴C]cyclic AMP is unaffected. Compartmentation of the ATP precursor pool for cyclic AMP is suggested by the difference in ATP/cyclic AMP ratios for endogenous and labelled compounds, and by the ineffectiveness of antimycin A₁ in decreasing the conversion rate of labelled adenine nucleotides to [¹⁴C]cyclic AMP.     

Histamine catabolism in guinea pigs, rats and mice

Histamine catabolism in vivo was studied in unanesthetized rats and guinea pigs; tissues from animals sacrificed 10 min after s.c. injection of ¹⁴C-histamine were assayed for ¹⁴C-histamine and total ¹⁴C. Groups pretreated with aminoguanidine, a diamine oxidase inhibitor, and methylhistamine, an inhibitor of the histamine-methylating enzyme, were used to evaluate roles of these enzymes in individual tissues. For rats the data suggested presence of both enzymes in intestine, diamine oxidase in lung and liver, methylation in kidney, and marked ability of heart to extract ¹⁴C-histamine from blood; these results closely check earlier data from tissues of anesthetized rats sacrificed 2.5 min after i.v. injection of ¹⁴C-histamine. For guinea pigs intestine showed evidence for both enzymes, liver and kidney for diamine oxidase, and lung and heart, for methylation. Since in animals injected with ¹⁴C-histamine, aminoguanidine pretreatment sharply reduced total ¹⁴C in mouse intestine and rat liver, another diamine oxidase inhibitor was tested; similar results were obtained. Amodiaquine, a strong inhibitor of histamine methylation in vitro, was found to inhibit in vivo. Catabolism of endogenously formed ¹⁴C-histamine was tested in the female rat, an animal which destroys exogenous histamine largely through the diamine oxidase pathway. Rats pretreated with aminoguanidine and then injected with ¹⁴C-L-histidine, excreted only slightly more ¹⁴C-histamine in urine than did controls. The ineffectiveness of aminoguanidine suggests that even in the female rat, diamine oxidase may have little significance in local catabolism of newly formed histamine. Possible reasons are discussed.     

In vitro analysis of 2,2-bis(p-chlorophenyl) acetic acid (DDA) handling by rat kidney and liver

On exposure of renal cortical slices or liver slices to [¹⁴C]DDA (2,2-bis[p-chlorophenyl] acetic acid), both tissues accumulate DDA in a concentration- and time-dependent manner. At 10 μm DDA maximum tissue to medium ratios were approximately 20 in both tissues. Incubation under N₂ or addition of metabolic inhibitors reduced uptake by 50%. Other organic acids depressed energy-dependent DDA uptake, but did not influence uptake in the presence of metabolic inhibitors. Neither the parent pesticide, DDT (1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane), nor organic bases significantly inhibited DDA accumulation. Accumulation of a second organic acid, p-aminohippurate (PAH), was depressed by DDA but not by DDT. Thus, DDA is apparently accumulated by an energy-dependent carrier-mediated process in both liver and kidney. The specificity of this process indicates that it is the organic acid transport system.     

Dissipation by tetraethylammonium of verapamil-stimulated p-aminohippurate accumulation in rat kidney cortical slices

The stimulatory effect of verapamil on p-aminohippurate (PAH) accumulation was studied in rat kidney cortical slices. PAH accumulation was stimulated by the presence of 0.3 mM verapamil in the incubation medium, but this stimulatory action of verapamil was dissipated by adding tetraethylammonium (TEA) to the medium. TEA accumulation by the kidney cortical slices was inhibited by verapamil in a concentration-dependent fashion. In addition, kinetic studies revealed that verapamil resulted in an increase in the Michaelis constant (K_m) of TEA transport, with the V_max remaining constant, suggesting that verapamil competitively inhibited TEA transport. These results suggest that the transport system for verapamil is the same as that for TEA and that verapamil taken up within the cells may stimulate PAH transport.     

In vitro analysis of the renal handling of paraquat.

Accumulation of paraquat by mouse renal cortical slices was related to the concentration of paraquat in the medium and the duration of incubation. Paraquat accumulation was depressed by incubation of slices under nitrogen or by addition of metabolic inhibitors. Accumulation of a second organic base, N-methylnicotinamide (NMN), was depressed by a concentration of paraquat which failed to influence accumulation of the organic acid, p-aminohippurate (PAH). The uptake component of NMN accumulation was inhibited by paraquat. The data suggest that paraquat is accumulated by an energy-requiring process and that this accumulation occurs via the organic base transport system. In addition, an apparently toxic effect of paraquat on cortical slice function was observed when the incubation temperature was raised from 25 to 37°C. At this temperature, 10^?3m paraquat depressed not only NMN accumulation but PAH accumulation and slice oxygen consumption as well. Thus, paraquat can be toxic to the function of kidney slices and this effect appears to be temperature-dependent.     

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells.

The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F₃) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations (< 1 × 10^?5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity (K_m < 1 × 10^?6 M) and low affinity (K_m > 1 × 10^?5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K_m) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the other's high affinity transport. Hemicholinium (1 × 10^?5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10^?6?1 × 10^?4 M) did not. Choline transport was also found to be dependent on the pH and pCO₂ of the medium.     

Effects of acute and chronic ethanol administration on whole mouse brain synaptosomal calcium influx

Whole brain synaptosomes, isolated from pair-fed acute in vitro (sucrose-Sustacal diet for 10 days), acute in vivo [4.5g/kg, 20% (w/v) i.p. ethanol pretreatment] and chronic in vivo (ethanol-Sustacal diet for 10 days) female, Swiss-Webster mice, were challenged in vitro with ethanol (80 mM, final concentration) in either an incubation medium (12-min exposure), a depolarizing medium (2-min exposure with 74 mM KCI) or a nondepolarizing medium (2-min exposure with 5 mM KCl). Depolarizing and nondepolarizing media also contained ⁴⁵Ca²⁺ (2μCi/μmole). The results showed (1) a significant enhancement of ⁴⁵Ca²⁺ influx when synaptosomes isolated from acute in vitro and acute in vivo mice groups were challenged in vitro by ethanol (80 mM) in the depolarizing medium (74 mM KCl), (2) a significant enhancement by 80 mM ethanol of ⁴⁵Ca²⁺ accumulation by nondepolarized synaptosomes isolated from the acute in vitro mouse group, (3) a significant increase in ⁴⁵Ca²⁺ accumulation in synaptosomes from acute in vivo mice as compared with acute in vitro mice without an in vitro ethanol challenge, and (4) a significant decrease in ⁴⁵Ca²⁺ accumulation by synaptosomes isolated from chronic in vivo mice as compared to acute in vitro synaptosomes. The results presented here demonstrate the ability of ethanol to significantly increase calcium accumulation into whole brain synaptosomes and that tolerance to this phenomenon occurs in parallel with behavioral tolerance to the sedative action of ethanol. These ethanol-induced changes in calcium accumulation may be involved in the production of sedation and tolerance to sedation.     

Chlorphentermine uptake by rabbit lung slices

Slices of rabbit lung tissue (～ 150 mg; 0.5 mm) were incubated in 5 ml of Krebs-Ringer phosphate buffer, in the presence of 0.25 mM [¹⁴C] chlorphentermine (CP) with shaking at 37°C and under an atmosphere of an O₂/CO₂ mixture (95 : 5). Incubation medium (M) and tissue (T)_were analyzed for radioactivity. Uptake of CP reached a plateau after 30 min at a T/M ratio of 20. Upon varying the concentration of [¹⁴C] CP from 0.125 mM to 2 mM, the concentration-response curve was seen to saturate and the T/M ratio decreased with increasing concentration. Substituting LiCl for NaCl or increasing the K⁺ content in the medium decreased CP uptake. Incubation of slices with Na⁺-pump inhibitors, harmaline and iodoacetate, significantly decreased CP uptake. Chloroamphetamine, desimipramine, imipramine, morphine, chlorpromazine, dieldrin, methadone, amphetamine (each at 1 mM) and incubation at 10°C inhibited CP uptake. Imipramine and amphetamine were both effective in displacing previously accumulated CP from the tissue slices. Efflux of CP from the lung slices was biphasic and was not affected by removal of Na⁺ from the medium. Binding of CP to lung homogenate was unaffected by substituting LiCl for NaCl or by the presence of 1 mM iodoacetate. However, 1 mM harmaline or 1 mM imipramine decreased CP binding. These studies offer evidence for a partially Na⁺-dependent, active uptake process for pulmonary sequestration of CP compatible with earlier findings obtained with perfused intact lung preparations.     

Uptake of serotonin,gamma-aminobutyric acid and histamine into synaptic vesicles of the pig caudate nucleus

Summary Dopamine-containing vesicles were isolated from the caudate nucleus of the pig by differential centrifugation and incubated with ¹⁴C-serotonin (¹⁴C-5-HT), ¹⁴C-gamma-aminobutyric acid (¹⁴C-GABA), ¹⁴C-dopamine or ¹⁴C-histamine. At 25°C the uptake of ¹⁴C-5-HT and ¹⁴C-GABA was enhanced by ATP and magnesium. The Km for the uptake of ¹⁴C-5-HT in the presence of ATP and magnesium was 0.67×10^–6 M, that of ¹⁴C-GABA 1.33×10^–4 M. ¹⁴C-Histamine was also taken up into the vesicles but its uptake was not influenced by ATP and magnesim. The ATP-magnesium-dependent uptake of ¹⁴C-5-HT and ¹⁴C-GABA was abolished at 0°C. At 37°C the accumulation of ¹⁴C-GABA in the presence of ATP and magnesium reached a steady state after 20 min, while the accumulation of ¹⁴C-5-HT reached a maximum after 2.5 min of incubation and then gradually declined. Osmotic lysis of the vesicles followed by sonication abolished the enhanced uptake of ¹⁴C-GABA, ¹⁴C-5-HT and ¹⁴C-dopamine in the presence of ATP and magnesium. Determination of the endogenous contents of the vesicles revealed 14.6 pmoles per mg protein for 5-HT and 22.7 nmoles per mg protein for GABA. It is concluded that the caudate nucles preparation used in this study contains not only dopamine-storing vesicles able to take up biogenic amines but also GABA-containing vesicles which take up GABA by an ATP-magnesium-dependent process.This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

Tyrosine hydroxylase in snail (Helix pomatia) nervous tissue.

Snail nervous tissue synthesizes [¹⁴]dopamine and [¹⁴]dihydroxyphenylalanine (DOPA) from [^14C]tyrosine. The K_m value for the overall conversion of [¹⁴C]dopamine was 6 × 10^?4M. The enzyme converting [¹⁴C]tyrosine to [¹⁴C]DOPA. tyrosine hydroxylase, has the following characteristics. Approximately 85–90 per cent of the enzyme is soluble, and the enzyme of the nervous tissue, isolated by ammonium sulfate fractionation, had the highest activity in the 25–40 per cent fraction. The enzyme has a pH optimum of 6.5 in Tris-HCl, sodium acetate and potassium phosphate buffers. The enzyme requires a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine (DMPH₄), 2-amino-4-hydroxy-6-methyltetrahydropteridine (6MPH₄), and 2-amino-4-hydroxy-6-(L-erythro-1, 2-dihydroxypropyl)tetrahydropteridine (l-erythro-tetrahydrobiopterin) (BH₄) are 5 × 10^?4 M. 3.2 × 10^?4 M and 1.1 × 10^?4 M respectively. The K_m values for tyrosine at 10^?3 M BH₄ or 6MPH₄ are 1.4 × 10^?4 M and 4.2 × 10^?5 respectively. The enzyme is markedly stimulated by Fe²⁺ and catalase. The activity is drastically inhibited by dopamine, 6-hydroxydopamine, 5-hydroxytryptophan (5-HTP), noradrenaline and sodium dodecyl sulphate. Analogues of tyrosine also slightly inhibit the activity while Triton X-100, homovanillic acid, dihydroxyphenylacetic acid (DOPAC), reserpine, tyramine, pargyline and sucrose have little effect. The properties of the snail tyrosine hydroxylase are compared with those of the vertebrates.     

Sodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol

Epithelial damage caused by intestinal permeation enhancers is a source of debate concerning safety. The medium chain fatty acid, sodium caprate (C₁₀), causes reversible membrane perturbation at high dose levels required for efficacy in vivo, so the aim was to model it in vitro. Exposure of Caco-2 monolayers to 8.5 mM C₁₀ for 60 min followed by incubation in fresh buffer led to (i) recovery in epithelial permeability (i.e. transepithelial electrical resistance (TEER) and apparent permeability coefficient (P_app) of [¹⁴C]-mannitol), (ii) recovery of cell viability parameters (monolayer morphology, plasma membrane potential, mitochondrial membrane potential, and intracellular calcium) and (iii) reduction in mRNA expression associated with inflammation (IL-8). Pre-incubation of monolayers with a mucosal prostaglandin cytoprotectant was attempted in order to further decipher the mechanism of C₁₀. Misoprostol (100 nM), inhibited C₁₀-induced changes in monolayer parameters, an effect that was partially attenuated by the EP₁ receptor antagonist, SC51322. In rat isolated intestinal tissue mucosae and in situ loop instillations, C₁₀-induced respective increases in the [¹⁴C]-mannitol P_app and the AUC of FITC-dextran 4000 (FD-4) were similarly inhibited by misoprostol, with accompanying morphological damage spared. These data support a temporary membrane perturbation effect of C₁₀, which is linked to its capacity to mainly increase paracellular flux, but which can be prevented by pre-exposure to misoprostol.     

Effects of phencyclidine on synaptosomal dopamine continuously appearing from phenylalanine: Sensitivity to reserpine

In the present study some effects of phencyclidine on synaptosomal (P₂) synthesis and release of labelled dopamine, continuously appearing from [¹⁴C]phenylalanine, were investigated in vitro. Also examined was the sensitivity of such effects of phencyclidine to reserpine, acting either in vitro or in vivo. Synaptosomal (P₂) preparations from rat caudate nuclei were incubated with the drugs added at various concentrations in the presence of [¹⁴C]phenylalanine substrate. The particulates were quickly separated from the medium after incubation and the separated fractions were analyzed for labelled dopamine and the synaptosomal uptake of [¹⁴C]phenylalanine. Of the total labelled dopamine formed, the fraction that was present in the medium was determined and taken as a measure of the spontaneous or basal release and its enhancement by any drug addition. Phencyclidine (3.1–36.4 μM) stimulated both the total (medium plus particulates) formation and the synaptosomal release of labelled dopamine, while reserpine (0.09–1.80 μM) inhibited the formation and enhanced the release. A coaddition of reserpine (0.09–1.80 μM) and phencyclidine to the incubation medium blocked the stimulating effect of the latter drug on the labelled dopamine synthesis; the same experiments revealed furthermore, an inhibitory effect of phencyclidine (0.91–36.4 μM) on the synthesis, an effect that was additive to the inhibition due to reserpine alone. The enhancing effect of phencyclidine on labelled dopamine release was however maintained in the presence of reserpine. Phencyclidine (0.22–36.4 μM) also inhibited the formation of labelled dopamine and enhanced its release when the P₂ fraction was prepared from reserpine-pretreated rats (5.0 and 2.5 mg/kg, i.p., at 24 and 2 hr before the experiment). Reserpine (1.80 μM) itself, added in vitro to the same P₂ preparation, was without any significant effect. The phencyclidine congener Ketamine, with or without reserpine, had little effect either on the synthesis or the release. In none of the incubations did the addition of drugs affect the synaptosomal uptake of labelled phenylalanine.     

Calcium-dependent modulation of guanosine 3′,5′-monophosphate in renal cortex: Possible relationship to calcium-dependent release of fatty acid

The effects of Ca²⁺ on cGMP accumulation in rat renal cortical slices were correlated with the effects on ¹⁴C-fatty acid release in tissue prelabeled with ¹⁴C arachidonate. Ca²⁺ in the presence and absence of ionophore A23187 exerted parallel effects on the release of labeled arachidonate from slices and on slice cGMP content. Thus, Ca²⁺ stimulated both arachidonate release and tissue cGMP accumulation 2 to 3-fold when added to slices of renal cortex previously deprived of Ca²⁺ and Mg²⁺, whereas Mg²⁺ had no stimulatory effect on either arachidonate release or tissue cGMP content. In the presence of A23187, Ca²⁺ increased arachidonate release and tissue cGMP accumulation 4 to 6-fold. Tetracaine partially inhibited Ca²⁺-induced arachidonate release and completely blocked Ca²⁺-induced cGMP accumulation. Ca²⁺-induced arachidonate release was unaffected by the absence of O₂. Addition of exogenous arachidonate to slices of renal cortex increased tissue cGMP content 2-fold. Linoleate exerted a lesser effect on tissue cGMP, while palmitate and oleate had no effect. Ca²⁺- and arachidonateinduced cGMP contents in renal cortical slices were not additive, and both were abolished by exclusion of O₂. Since nitroprusside increased cGMP accumulation 10- to 15-fold in O₂-deprived slices, loss of the Ca²⁺ and arachidonate responses under these incubation conditions was selective. Ca²⁺-induced cGMP accumulation was unaffected by indomethacin (100 μg/ml), but was abolished by 200 μM 5,8,11,14-eicosatetraynoic acid (TYA). The results are consistent with the possibility that the Ca²⁺-dependent processes regulating cGMP in renal cortex include Ca²⁺-dependent acyl hydrolase activity, which limits the availability of free polyunsaturated fatty acids. A role for fatty acid oxygenation products in the stimulation of cGMP is suggested, but not established, by the O₂ dependence of the actions of both Ca²⁺ and exogenous fatty acids. The failure of exogenous arachidonate or linoleate to mimic quantitatively the actions of Ca²⁺ on cGMP may reflect the involvement of other Ca²⁺- and O₂-dependent processes in modulation of cGMP in this tissue or limited access of exogenous fatty acid to cGMP regulatory sites in the cell.     

Uptake,biotransformation, and elimination of rotenone by bluegills (Lepomis macrochirus)

Yearling bluegills (Lepomis macrochirus) were exposed to sublethal concentrations of [¹⁴C]rotenone (5.2 μg/l) for 30 days in a continuous flow exposure system and then transferred to clean, flowing water for an additional 21-day depuration period. Rates of uptake and elimination and profile of the rotenoid metabolites in head, viscera, and carcass components were evaluated by ¹⁴C counting and by high performance liquid chromatography. Total [¹⁴C]rotenone derived activity was relatively uniform in all body components within 3 days after initial exposure and remained constant during the ensuing 27 days of exposure. Initial uptake rate coefficients were highest in viscera (K_u = 80 · h^?1) and were nearly identical for head (K_u = 14 · h^?1) and carcass (K_u = 10 · h^?1). Analyses of tissue extracts by high performance liquid chromatography confirmed the presence of at least six biotransformation products of rotenone. More than 60% of the activity extracted from viscera was present as a single peak which represented a compound that was extremely soluble in water. Rotenone composed only 0.3% of the extractable activity in viscera taken from fish exposed to rotenone for 30 days; however, rotenone accounted for 15.4% of extractable activity in the head and 20.1% in the carcass components. Rotenolone and 6′,7′-dihydro-6′,7′-dihydroxyrotenolone were tentatively identified as oxidation products in all tissue extracts.Elimination of ¹⁴C activity from all body components was biphasic; both phases followed first-order kinetics. The rate of elimination was nearly equal for all body components during the initial phase but was most rapid from viscera during the second phase of elimination. Bioconcentration factors for the head, viscera, and carcass were 165, 3,550, and 125, respectively, when calculated on the basis of total ¹⁴C activity but only 25.4, 11, and 26 when calculated as the concentration of parent material.     

The transport of (+)-amphetamine by the neuronal noradrenaline carrier

Summary PC-12 cells (a clonal line of rat phaeochromocytoma cells) take up noradrenaline by a transport system which is identical with the neuronal amine transport system (uptake₁). The uptake of ³H-noradrenaline into reserpine-pretreated PC-12 cells (monoamine oxidase inhibited) was saturable (K_m=0.6±0.1 mol/l), dependent on sodium and chloride, and competitively inhibited by (+)-amphetamine (K_i=0.18±0.04 mol/l), cocaine (K_i=0.55±0.15 mol/l) and desipramine (K_i=4.3±0.6 nmol/l). The uptake and accumulation of ³H (+)-amphetamine showed characteristics comparable to those of ³H-noradrenaline, since the uptake of ³H (+)-amphetamine (0.1 mol/l) was reduced by omission of sodium or chloride from the incubation medium. The sodium-sensitive component of uptake and accumulation of ³H (+)-amphetamine was fully inhibited by cocaine and desipramine. The IC₅₀ of desipramine for inhibition of the sodium-sensitive component of the 1-min uptake of ³H (+)-amphetamine (20 nmol/l) was about 2 nmol/l, i.e., identical with the K_i for inhibition of uptake of ³H-noradrenaline. At concentrations above 1 mol/l, desipramine additionally caused an inhibition of the sodium-independent permeation of ³H (+)-amphetamine into PC-12 cells.Hence, by using a homogeneous population of cells endowed with uptake₁, it is possible to demonstrate — besides a pronounced lipophilic entry — a carrier-mediated uptake of ³H (+)-amphetamine.Some of the results were communicated to the German Pharmacological Society (Bönisch 1981). This study was supported by the Deutsche Forschungsgemeinschaft (Bo 521)     

Comparative effects of amphetamine and fenfluramine on lipid biosynthesis and absorption in the rat

The effects of amphetamine and fenfluramine on lipid biosynthesis and intestinal absorption of triglycerides in the rat were observed independently of the anorectic activity of these drugs. Amphetamine and fenfluramine significantly reduced de novo lipogenesis and cholesterogenesis from ³H₂O and [¹⁴C] alanine in isolated hepatocytes. Fenfluramine was four times more potent an inhibitor than amphetamine. At high concentrations (above 2 mM), fenfluramine inhibited ¹⁴CO₂production from [¹⁴C] alanine in isolated hepatocytes while amphetamine stimulated ¹⁴CO₂ production at all concentrations tested (0.25 to 8 mM). Lipogenesis in liver and adipose tissue was not reduced in vivo by the acute administration of amphetamine. It, however, reduced lipogenesis in the small intestine. Fenfluramine significantly depressed in vivo lipogenesis in liver, small intestine and adipose tissue at 10 and 20 mg/kg, i.p. Hepatic cholesterogenesis was depressed significantly in vivo by both drugs. Fenfluramine increased serum free fatty acids while amphetamine produced no change. Triglycerides were increased significantly by fenfluramine only. Serum cholesterol and phospholipids were unchanged. Fenfluramine at 40 and 60 mg/kg, p.o., diminished significantly the intestinal absorption of triglycerides and depressed the accumulation of lipid in the liver. Analogous effects were not observed with amphetamine. Amphetamine was a weak inhibitor of rat pancreatic lipase (EC 3.1.1.3) (K_i = 21 mM) and fenfluramine was a stronger inhibitor (K_i = 3.3 mM).