Inhibition of superoxide anion production in non-stimulated guinea pig peritoneal exudate cells by anti-inflammatory drugs.

Inhibitory effects of anti-inflammatory drugs on the production of superoxide anion (·O₂^? by isolated non-treated guinea pig peritoneal exudate cells (PEC) was studied spectro-photometrically using NADH and lactate dehydrogenase (LDH). Values of ID₅₀ were; diclofenac sodium (2 × 10^?5M), indomethacin (3 × 10^?5M), oxyphenbutazone (8 × 10^?5M), fenamic acid (1 × 10^?4M), ibuprofen (1 × 10^?4M), benzydamine (3 × 10^?4M), aspirin (10^?3M<) and dexamethasone (10^?3M<). The mechanism of inhibition seemed to block plasma membrane associated NAD(P)H oxidase(s) activity which produces ·O₂^? ID₅₀ values of other drugs; superoxide dismutase (SOD, 2 × 10^?8M), cytochalasin B(1 × 10^?7M) and NEM (6 × 10^?6M). d-Mannitol radical scavenger), 1,3-diphenyl-isobenzofuran (singlet oxygen scavenger) and sodium azide (mitochondrial electron transport inhibitor and singlet oxygen scavenger) were negative.Superoxide radical itself or oxygen-centered radical(s) derived from ·O₂^? is supposed recently as a rate-limiting factor for prostaglandin (PG) synthetase. Whether the inhibition of non-steroidal anti-inflammatory drug (NSAID) on ·O₂^? production is linked directly to PG biosynthesis or not, ·O₂^? was already demonstrated in our laboratory to make a role for the development of rat carageenan foot oedema. It may serve as a new in vitro sceening method of NSAID, to check the inhibitory potency of a compound on ·O₂^? production by guinea pig PEC.     

Effects of anti-inflammatory and other compounds on the release of lysosomal enzymes from macrophages.

The effects of anti-inflammatory drugs and other compounds on the selective release of lysosomal enzymes from cultured peritoneal-macrophages as a result of phagocytic uptake of zymosan particles were investigated. Two types of inhibitory effect were observed: (i) a biphasic effect which produced maximum inhibition at 3 × 10^?7?6 × 10^?6 M for steroids, 3 × 10^?5 M for guaiol and 10^?5 M for phenylbutazone; and (ii) a linear log-dose effect with ed₅₀'s of 10^?4 M for flufenamic acid and 3 × 10^?7 M for concanavalin. Stimulation of zymosan-induced enzyme release was observed with chloroquine and indomethacin at concentrations of 10^?4 M and greater. This effect was associated with cytotoxicity. Guaiol also stimulated hydrolase-release at 10^?4 M, but only during short incubation times and the effect was not cytotoxic. All other non-steroidal anti-inflammatory compounds were without significant activity.     

Stimulatory effect of platinum (IV) ion on the production of superoxide radical from xanthine oxidase and macrophages.

Superoxide radical (^.O^?₂) production was measured spectrophotometrically using NADH and lactate dehydrogenase (LDH) in a xanthine oxidase(XOD) plus hypoxanthine (HX) system and in an isolated guinea pig macrophages system. Sodium platinum (IV) chloride (Na₂PtCl₆: 2.5 × 10^?4?1 × 10^?3M) enhanced the production of ^.O₂^? in both systems (2–10 times). The degree of the enhancement was dependent on incubation time, basal level of ^.O₂^? production and concentration of Na₂PtCl₆. The stimulated ^.O₂^? production in the XOD system was inhibited by luminol (O-aminophtalhydrazide) and that in the macrophages was inhibited by an anti-inflammatory drug, Diclofenac sodium (Dc). These results show that platinum (IV) ion is either a potent stabilizer of ^.O₂^? or a stimulator of ^.O₂^? production as are paraquat or streptonigrin. This specific character of platinum (IV) ion may explain its bactericidal and inflammation-inducing properties.     

Inhibition by (−)-Deprenyl of Agonist-Evoked Contractions in Rabbit Aorta

Abstract: The effect of (?)-deprenyl, a relatively selective MAO-B inhibitor, was examined for its ability to inhibit the contractions of rabbit isolated aorta evoked by various agonists and potassium. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by noradrenaline (10^?8?3 × 10^?4 M); pA₂: 5.10. The antagonism was reversible. It was attenuated by cocaine (3x M); pA₂: 4.38, unchanged by corticosterone (4 × 10^?5 M); pA₂ 4.79 and enhanced by cocaine (3 × 10^?5 M) plus corticosterone (4 × 10^?5 M); pA₂: 5.48. (+)-Deprenyl (10^?6?10^?4 M) did not alter the contractions evoked by noradrenaline (3 × 10^?9?10^?4 M). Clorgyline (10^?5 and 10^?4 M) antagonized the noradrenaline-evoked contractions. Pargyline (10^?4 and 3 × 10^?4 M) had no effect. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by phenylephrine (10^?8?10^?4 M); pA₂: 5.10. Removal of the endothelium did not alter the antagonism; pA₂: 5.35. (?)-Deprenyl (10^?5?3 × 10^?4 M) antagonized the contractions evoked by either 5-hydroxytryptamine (3 × 10^?8?3 × 10^?4 M); pA₂: 4.61 or by histamine (10^?6?3 × 10^?2 M); pA₂: 4.84. (?)-Deprenyl (3 × 10^?4 M) caused a non-competitive antagonism of the contractions evoked by potassium (1.5-5.5 × 10^?2 M). It is concluded that (?)-deprenyl is a weak inhibitor of postjunctional α-adrenoceptors, 5-hydroxytryptamine (5-HT₂) receptors, and histamine (H₁) receptors.     

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.     

Dual Inhibitory Action of ATP on Adrenergic Neuroeffector Transmission in Rabbit Pulmonary Artery

The aim of this study was to determine whether the inhibitory action of ATP on sympathetic neuroeffector transmission in the isolated pulmonary artery is due to ATP itself or one of its dephosphorylated breakdown products, ADP, AMP or adenosine. Furthermore, the mechanism of the inhibitory action was investigated. ATP (10^?6?3 × 10^?4 M), the degradation-resistant ATP-analogue, β, γ-methylene-5′-triphosphate (10^?5?3 × 10^?4 M), ADP (10^?6?3 × 10^?4 M), AMP (10^?5?3 × 10^?4 M), adenosine (10^?5?3 × 10^?4 M) and 2-chloroadenosine (10^?7?3 × 10^?4 M) reduced the contractions evoked by field-stimulation. This was also the case for prostaglandin E₂ (3 × 10^?9?3 × 10^?7 M), while prostaglandin F_2α(1.4 × 10^?8 M) slightly augmented the neurogenic response. The time course of the inhibitory effect of purinergic compounds on the stimulation evoked contractions was studied. In the case of ATP and ADP the inhibition was biphasic: an initial marked block (1 min. after drug addition) which in the continued presence of either compound recovered partially 10 min. later and then remained almost constant for another 90 min. The other purinergic agents caused a monophasic reduction. In the presence of indomethacin (5 × 10^?5 M), ATP and ADP also reduced the neurogenic contractions in a monophasic manner. Indomethacin did not alter the β,γ-methylene-5′-triphosphate-induced inhibition. Dilazep (3 × 10^?6 M) plus deoxycoformycin (3.6 × 10^?6 M), augmented the inhibitory effect of ATP. In contrast, theophylline (5 × 10^?5 M) did not alter the effect of ATP. The inhibitory effect of ATP (10^?4 M) on stimulation-evoked contractions was inversely proportional to the extracellular Ca²⁺ concentration (0.3–5.2 mM) and to frequency of stimulation (3–15 Hz). These results suggest that ATP initially causes a presynaptic inhibition of noradrenaline release evoked by field-stimulation. This phase I block is probably mainly due to an ADP-mediated short-lasting release of prostaglandins of the E type. The continuous inhibition (phase II) is probably due to ATP and its metabolites, possibly mainly adenosine. The phase II inhibition may possibly involve a decreased entry of Ca²⁺ into adrenergic nerve terminals during depolarization.     

Anti-convulsants and brain aldehyde metabolism: Inhibitory characteristics of ox brain aldehyde reductase

The major isoenzyme of aldehyde reductase has been purified from ox brain by affinity chromatography. Carbamazepine (K_i = 7.3 × 10^?4 M) and phenacemide (K_i = 2.5 × 10^?4 M), in common with all other established anti-convulsant drugs tested, have been shown to inhibit the activity of this enzyme. A selection of structural analogues of the anti-convulsant sodium valproate were found to be potent inhibitors of the reductase (K_ivalues in the range 10^?3 M ?5 × 10^?5 M) and these analogues also showed anti-convulsant activity in the mouse maximal electroshock test. A third group of compounds, the flavonoids, constitute the most potent group of aldehyde reductase inhibitors yet reported. Quercetin and morin exhibited K_i values less than 1 μM. The possible relationship between aldehyde metabolism and anti-convulsant action is discussed and structural characteristics pre-disposing to potent inhibition of aldehyde reductase are described.     

Effects of sodium butyrate on mouse neuroblastoma cells in culture

Uncloned and cloned mouse neuroblastoma cells in vitro developed neurite-like processes upon treatment for 48 hr with 10^?6 to 3 × 10^?4 M sodium butyrate. Although treatment with 5 × 10^?4M sodium butyrate caused neurite formation after 18 hr, the per cent of the cells having neurities returned to control level by 48 hr of treatment. Cell division decreased and acetylcholinesterase activity increased with 5 × 10^?4M sodium butyrate; these parameters were unaltered in the presence of 10^?6 or 10^?5M sodium butyrate. Sodium butyrate was more effective than sodium propionate or sodium valerate in causing a decrease in cell division and an increase in acetylcholinesterase activity, and sodium isobutyrate or gamma aminobutyric acid was ineffective. The effects of sodium butyrate on cell division and acetylcholinesterase activity were reversible after treatment was discontinued. It is concluded that the ability of butyrate to stimulate neurite formation at low concentrations is opposed by additional actions on the cell as its concentration is increased.     

Further studies on the actions of salicylates on nerve membranes

The effects of sodium salicylate, sodium benzoate and 5-bromo salicylate on nerve fibres of the rabbit cervical vagus and of the frog sciatic nerve ‘in vitro’ were studied using the single sucrose gap technique. The application of 5-bromo salicylate in concentrations between 2 × 10^?3 to 5 × 10^?3 M induced a slow fall in spike height accompanied by a small hyperpolarization (0.5?2.0 mV). Higher concentrations (1 × 10^?2 ? 2 × 10^?2 M) produced depolarization and a rapid block of nervous conduction. Concentrations of 1 × 10^?2?2 × 10^?2 M of sodium salicylate were needed to induce the same hyperpolarizing and slow blocking effects in non-myelinated nerve fibres. Myelinated fibres of the frog sciatic nerve were also blocked by concentrations greater than 1 × 10^?2 M of 5-bromo salicylate, but showed an increase in the amplitude of the compound action potential and a prolonged falling phase when superfused with concentrations around 5 × 10^?3 M. A frequency-dependent decrease in the action potential amplitude was observed with sodium salicylate and 5-bromo salicylate and was shown to be more easily induced in non-myelinated than in myelinated fibres. 5-Bromo salicylate (2 × 10^?2 M) and 2,4-dinitrophenol (1 × 10^?4 M) reduced the amplitude and the rate constant of decay of the hyperpolarization produced by tetanic stimulation of non-myelinated nerve fibres. Sodium salicylate (2 × 10^?2 M) increased the time constant of decay of the hyperpolarization of the potassium-activated response and slightly decreased its amplitude. Sodium salicylate was 3–5 times less potent than 5-bromo salicylate; while sodium benzoate was practically ineffective in producing the phenomena under study.     

Interactions between tetraethylammonium and methohexitone at the chick neuromuscular junction: Experiments with the moving fluid electrode technique

The effect of methohexitone on the depolarization and contracture responses produced by tetraethylammonium (TEA), acetylcholine (ACh) and repetitive indirect stimulation were investigated, using the moving fluid electrode technique, in the chick biventer cervicis (BVC) nerve muscle preparation. TEA (4.8 × 10^?4?4.8 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were potentiated by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M.) 6.5 ± 0.03 × 10^?3 M and 1.3 ± 0.04 × 10^?3 M (N = 6) respectively. The mean ED₅₀S for the depolarizations were (mean ± S.E.M.) 5.9 ± 0.1 × 10^?3 and 1.5 ± 0.06 × 10^?3 M (N = 6) respectively. ACh (5.5 × 10^?6?1.1 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were reduced by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M. 2.4 ± 0.21 × 10^?4 and 2.3 ± 0.1 × 10^?3 M (N = 6) respectively. The mean (± S.E.M.) ED₅₀S for the depolarizations were 8.4 ± 0.33 × 10^?4 and 3.7 ± 0.14 × 10^?3 M (N = 6), respectively. Repetitive indirect stimulation, at 1–20 Hz, produced contraction and depolarization responses which were frequency-dependent. These responses were slightly potentiated by methohexitone (8.8 × 10^?5 M). The mean (± S.E.M.) frequency₅₀S for the contractions produced in the control Krebs solution and with methohexitone were 9.2 ± 0.1 and 8.5 ± 0.2 Hz (N = 6) respectively. The mean frequency₅₀S for the depolarizations were (mean ± S.E.M.) 7.2 ± 0.1 and 5.8 ± 0.19 Hz (N = 6) respectively. It is concluded that TEA may have a direct post-synaptic action, in addition to releasing ACh from the presynaptic nerve terminals. TEA produces more contracture tension than does ACh for a given level of membrane depolarization. Methohexitone, non-competitively, reduces the responses produced by applied ACh whereas it potentiates those produced by TEA and repetitive nerve stimulation.     

Paracetamol does not potentiate the acetylsalicylate inhibition of prostaglandin release from macrophages

Prostaglandin (PGE₂ and PGI₂) synthesis can be stimulated in macrophages by the tumour promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) and inhibited by acetylsalicyclic acid (IC₅₀ = 8.2 × 10^?6 M) and paracetamol (IC₅₀ = 1.9 × 10^?4 M). When mixtures of both drugs were tested only an additive effect was observed. This result is in contrast to recent reports describing both potentiation of acetylsalicylate-induced PG synthesis inhibtion and enhancement of PG production by paracetamol.     

Influence of non-steroid anti=inflammatory drugs (NSAIDd) on hepatic tyrosine aminotransferase (TA) activity in rats in vitro and in vivo.

Indomethacin, flufenamic acid, mefenamic acid, gold sodium thiosulphate and ibuprofen were found to be inhibitors of hepatic TA in vitro. The type of inhibition was competitive relative to l-tyrosine; apparent K_i values ranged from 0.7 × 10^?4 to 1.18 × 10^?3 M. Several other NSAIDs did not, or poorly, inhibit TA at concentrations of ≤ 10^?3M. In intact rats, TA activity was stimulated 18-fold by pretreatment with mefenamic acid, 7-fold by dimethylsulfoxide and ibuprofen, 5-fold by acetylsalicylic and flufenamic acid, and doubled by benzydamine, amidopyrine, sodium salicylate and gold sodium thiosulphate. Indomethacin was somewhat less effective. Drug dosage was 20 per cent of the acute p.o. LD₅₀, three times with 12-hourly intervals. Phenylbutazone, chloroquine and cyclophosphamide significantly increased TA activity also, though the dose was smaller relative to LD₅₀. Adrenalectomy decreased the drug effects in almost all cases. Equi-toxic i.p. or p.o. doses of acetylsalicylic acid were equi-effective in intact rats. The action was dose-dependent and could be observed at a therapeutic dose. Elevated TA activities were determined up to 28 hrs after the last drug administration. Concomitant administration of glucose did not suppress the effect of mefenamic acid. The findings led to the following hypothesis: TA stimulation by NSAIDs depends on the actions of endogenous corticosteroids and, possibly, cyclic 3',5'-AMP. Similar processes might play a role in the anti-inflammatory mechanism of NSAIDs.     

Inhibitory effects of GABA,L-glutamic acid and nicotine on the potassium-evoked release of subsaance P in substantia nigra slices of the rat

Rat substantia nigra slices were superfused with a physiological medium containing a diluted substance P (SP) antiserum, bacitracin and serum albumin to measure SP released in superfusates. As shown by measuring the degradation of a SP-labelled derivative incubated with cerebellar slices, this medium prevented the enzymatic inactivation of SP. Potassium (K⁺, 50 mM) and veratridine (5 × 10^?5M) stimulated SP release and these effects were respectively prevented in absence of calcium and in presence of tetrodotoxin (5 × 10^?7 M). GABA (5 × 10^?5 M) nicotine (10^?6 M) and L-glutamic acid (5 × 10^?5 M) reduced the K⁺ (50 mM)-evoked release of SP. In contrast, glycine (5 × 10^?5 M), oxotremorine (5 × 10^?5 M), D-glutamic acid (5 × 10^?5 M) and serotonin (5 × 10^?5 M) were without effect. Pempidine (10^? M) prevented the inhibitory effect of nicotine (10^? M) on the K⁺-evoked release of SP. Glutamic acid diethyl ester (10^?4 M) completely abolished the L-glutamic acid-induced inhibition of the K⁺-evoked release of SP. Picrotoxin (5 × 10^?5 M) did not influence the L-glutamic acid inhibitory effect excluding the intervention of GABAergic mechanisms.     

In vitro hydroxylation of diphenylhydantoin and its inhibition by other commonly used anticonvulsant drugs

Using a crude 9,000 g rat liver musomal preparation, in vitro studies were carried out on (a) the metabolism (hydroxylation) of diphenylhydantoin (DPH), (b) the effect of other commonly used anticonvulsants on this hydroxylation. DPH hydroxylation exhibited saturation kinetics at a DPH substrate concentration of approximately 10^?4M. Mean K_m and V_m values for the reaction were 9.3 × 10^?5 M and 23.3 μg/m1 respectively. The linear Hill plot with an interaction coefficient of 1.0 suggests that there is no cooperativity between different DPH molecules during DPH receptor binding process. The anticonvulsants ethosuximide, sodium phenobarbitone, sodium valproate and sulthiame all exhibited inhibition of DPH hydroxylation to varying degrees. K_i, inhibition constants for the four anticonvulsants were respectively 1.1 × 10^?2, 9 × 10^?4, 1.8 × 10^?2 and 8.8 × 10^?4M. Inhibition of DPH hydroxylation by sodium phenobarbitone and sulthiame was strong and competitive in nature. Ethosuximide showed a weak competitive type of inhibition and sodium valproate a weak uncompetitive type of inhibition.     

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.

Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0?×?10^?5–1.0?×?10^?3 M in 2?h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10^?4 M was studied. Moreover, P_app of OMT was determined in the absence/presence of cyclosporine and surfactants at 100?μM to further confirm the relative transport mechanism.

Results The P_{app AP→BL} ranged from (3.040?±?0.23)?×?10^?6 to (3.697?±?0.19)?×?10^?6?cm/s as the concentration varied from 10^?5 to 10^?3 M. OMT showed similar P_app at 4 and 37?°C (p?>?0.05). Increasing the apical pH 7.4 and 8.0 resulted in P_app versus pH 5.0 (p?<?0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, P_app was (0.318?±?0.033)?×?10^?5, (0.464?±?0.048)?×?10^?5, (0.897?±?0.115)?×?10^?5 and (1.341?±?0.122)?×?10^?5?cm/s, respectively. In the presence of surfactants, P_app significantly increased up to 1.5–4.3-fold (p?<?0.05).

Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.     

Frequency-Dependence of 3H-Noradrenaline Release from Rabbit Pulmonary Artery: Effect of α-Adrenoceptor Antagonists and Inhibitors of Transmitter Inactivation

Abstract: The aim of this study was to examine the modulating role of presynaptic α₂-adrenoceptors on transmitter release from vascular sympathetic neurones. This was done by examining the influence of removal of inactivation pathways on the effect of α-adrenoceptor antagonists on the release of transmitter from noradrenergic neurones. The rabbit main pulmonary artery preloaded with ³H-noradrenaline (³H-NA) was used. The artery was stimulated with 300 pulses at various frequencies (1, 3, 10 and 30 Hz). Pargyline (3 × 10^?4M) increased the stimulation-evoked ³H-overflow at 1 and 3 Hz and decreased it at 30 Hz. U-0521 (3′,4′-dihydroxy-2-methylpropiophenone; 3 × 10^?6M) enhanced the overflow at 1 Hz and had no effect at 3–30 Hz. Corticosterone (4 × 10^?5M) did not alter the stimulation-evoked ³H-overflow at 1–30 Hz. Cocaine (3 × 10^?6M) enhanced the ³H-overflow slightly at 1–30 Hz. At 3 × 10^?5M, cocaine enhanced ³H-overflow at 1 Hz and reduced it at 30 Hz. Neither corticosterone (4 × 10^?5M) nor propranolol (10^?7M) modified this effect of cocaine. Propranolol (10^?7M) alone decreased the ³H-overflow at 30 Hz and had no effect at 1–10 Hz. Phenoxybenzamine (10^?6M) and chlorpromazine (3 × 10^?6M) potentiated the stimulation-evoked ³H-overflow at 1–30 Hz. Phentolamine (10^?6M) decreased the ³H-overflow at 1 Hz and enhanced it at 3–30 Hz. Rauwolscine (10^?6M) enhanced the stimulation-evoked ³H-overflow maximally at 10 Hz and had no effect at 1 and 30 Hz. Cocaine (3 × 10^?5M), cocaine (3 × 10^?5M) + corticosterone (4 × 10^?5M), pargyline (3 × 10^?4M) and U-0521 (3 × 10^?6M), but not corticosterone (4 × 10^?5M), increased the rauwolscine-induced enhancement at 1 and 3 Hz, but had no influence at 10 and 30 Hz. It is concluded that at a low frequency (1 Hz) of nerve stimulation, inhibition of either neuronal uptake, extraneuronal uptake, monoamine oxidase (MAO) or catechol-O-methyltransferase (COMT) causes an enhancement in transmitter overflow that is due to removal of inactivation pathways. The enhancement is not mediated via facilitatory β-adrenoceptors. At higher frequencies (3–30 Hz) inhibition of either neuronal uptake or MAO causes a sufficient amount of transmitter in the neuromuscular gap to activate the presynaptic α-adrenoceptor mediated negative feed-back system causing a decrease in transmitter release. α- Adrenoceptor antagonists enhance the stimulation-evoked release of ³H-NA only when the junctional gap concentration of transmitter is sufficient to trigger the presynaptic α-adrenoceptor auto-inhibitory system.     

Dual Effect of the Muscarinic Agonist McN-A-343 on Vascular Neuroeffector Transmission

Abstract: The site and mechanism of action of McN-A-343 (4-m-chlorophenylcarbamoyloxy)-2-butynyltrime-thylammonium chloride) on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery was studied. Low concentrations (10^?6 — 3 × 10^?5 M) of McN-A-343 and cocaine enhanced (up to 210 and 236%, respectively) the contractions evoked by electrical-field stimulation, while higher concentrations (10^?4 — 3 × 10^?4 M) inhibited them. McN-A-343 (10^?4 M) caused an initial transitory potentiation (222% of control) of the stimulation-evoked contractions followed by an inhibition. In the presence of cocaine (3 × 10^?5 M), the potentiation caused by McN-A-343 was prolonged and the secondary inhibitory phase was thus abolished. Physostigmine (10^?5 — 10^?4 M), hexamethonium (10^?5 M), atropine (3 × 10^?7 M), suprofen (10^?5 M), and 4-aminopyridine did not alter the effect of McN-A-343 (10^?4 M). Cocaine (3 × 10^?5 M)and(+)-amphetamine (10^?5 M) reversed the McN-A-343-evoked block, while they did not alter the inhibition caused by tetracaine (3.2 × 10^?5 M). Atropine (3 × 10^?7 M) had no effect on the McN-A-343-induced block, while 4-aminopyridine (10^?4 M) caused a partial and transitory reversal. On the aorta McN-A-343 (10^?4 M) did not alter the contractile concentration-response curve of (—)-noradrenaline (10^?9 — 3 × 10^?4 M), while that of serotonin (10^?8 — 3 × 10^?5 M) was moved to the right in a competitive manner. McN-A-343 (10^?4 M) did not alter the contractions evoked by noradrenaline (10^?7 M) during the period corresponding to the stimulation-evoked enhancement and subsequent inhibition. McN-A-343 (10^?4 M) slightly antagonized the contractions caused by tyramine (10^?6 — 10^?3 M) and carbachol (10^?6 — 10^?3 M). It is concluded that McN-A-343 enhances stimulation-evoked transmitter release by a presynaptic facilitatory action mediated via receptors localized on the outer surface of adrenergic neurones and to a lesser extent by inhibition of noradrenaline re-uptake. The enhancement does not involve presynaptic nicotine or muscarine receptors. Furthermore, McN-A-343 inhibits transmitter release by acting as an adrenergic neurone blocking agent at an intraneuronal site. The inhibition does not involve presynaptic muscarine inhibitory receptors and is prostaglandin-independent.     

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid

Abstract: The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N^w‐nitro‐l ‐arginine methyl esther (l ‐NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre‐contracted mesenteric artery rings, milonine (10^?10 M to 3 × 10^?4 M) caused a concentration‐dependent relaxation (EC₅₀ = 1.1 × 10^?6 M, E_max = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10^?5, p < 0.001), or after l ‐NAME 100 μM (EC₅₀ = 6.2 × 10^?5, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10^?4, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10^?4p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃^? levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K⁺ (EC₅₀ = 1.2 × 10^?5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10^?5, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10^?5 M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10^?4 M, n = 7). In addition, pre‐contraction with high extracellular potassium concentration prevented milonine‐induced vasorelaxation (EC₅₀ = 1.0 × 10^?4, p < 0.001). Milonine also reduced CaCl₂‐induced contraction in Ca²⁺‐free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide‐cGMP pathway and opening of K⁺ channels.     

Evaluating the effects of diclofenac sodium and etodolac on renal hemodynamics with contrast-enhanced ultrasonography: a pilot study

Purpose

Contrast-enhanced ultrasonography (CEUS) is a novel approach used for measuring organ perfusion changes. Studies using CEUS to assess the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on renal blood flow (RBF) have not yet been conducted. We aimed to evaluate the effects of NSAIDs on the renal hemodynamics of healthy subjects with CEUS.

Methods

We performed CEUS using the bolus injection method in a total of 10 healthy subjects. Measurements were completed over two study days in a randomized, crossover manner. On each study day, CEUS was performed twice, before and after the administration of NSAIDs. Subjects received an injection of contrast medium and images were recorded. A region-of-interest (ROI) was selected within the renal cortex, signal intensity in the ROI of the kidney was measured and a time-intensity curve (TIC) was automatically generated with attached software.

Results

The mean (±SD) peak intensity decreased significantly after an administration of diclofenac sodium (from 26.0?×?10^?4?±?17.4?×?10^?4 AU to 19.2?×?10^?4?±?12.0?×?10^?4 AU; P?=?0.022), but not significantly with etodolac (from 26.5?×?10^?4?±?9.7?×?10^?4 AU to 25.9?×?10^?4?±?20.8?×?10^?4 AU; P?=?0.474). The mean (±SD) percent reduction in intensity following diclofenac sodium administration was significantly reduced compared with etodolac administration (22.2?±?20.5 % vs. 3.4?±?8.9 %, P?=?0.037).

Conclusions

These finding suggests that diclofenac sodium (P?=?0.022), but not etodolac (P?=?0.474), affects renal hemodynamics even in healthy subjects.     

Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.