Electrophysiological actions of the plant alkaloid 6-benzoylheteratisine in rat hippocampal slices

The effects of the Aconitum alkaloid 6-benzoylheteratisine on neuronal activity was investigated in the in vitro slice preparation of rat hippocampus by extracellular recording of the stimulus-evoked population spike. 6-Benzoylheteratisine (0.01-10 μΜ) depressed the orthodromic and antidromic population spike in a concentration-dependent manner.The action of the drug was activity-dependent. The latency of onset of the inhibition was accelerated when the frequency of electrical stimulation had been increased. Furthermore, the effect of 6-benzoylheteratisine was evaluated in two different models of epileptiform activity induced either by blockade of GABA receptors by bicuculline (10 μΜ) or by a nominal Mg²⁺-free bathing medium. Due to the activity-dependent mode of action, this drug effectively reduced the number and the size of the synaptically evoked population spikes in the presence of bicuculline or nominal Mg²⁺-free bathing medium, respectively. Received: 23 September 1996 / Accepted: 2 January 1997     

Structure-dependent inhibitory action of the Aconitum alkaloids 14-benzoyltalitasamine and talitasamine in rat hippocampal slices

In the present study the effects of the two Aconitum alkaloids 14-benzoyltalitasamine and talitasamine on neuronal activity were investigated in order to obtain further insight into structure-dependent effects of this group of alkaloids on central nervous activity. Both alkaloids are closely related to aconitine, the main alkaloid of plants of Aconitum species. However, they have shortened side chains at position C3 and C8 of the molecule. The experiments were performed as extracellular recordings of orthodromically and antidromically evoked population spikes as well as of field excitatory potentials (EPSPs) from the CA1 region of rat hippocampal slices. 14-Benzoyltalitasamine exerted a reversible inhibition of the field potentials in a concentration-dependent manner. The orthodromic population spike was attenuated at concentrations higher than 1 μM, while the field EPSP was already affected at a concentration of at least 0.3 μM. Both responses were completely blocked at a concentration of 30 μM. The alkaloid failed to affect the presynaptic fiber spike at concentrations less than 10 μM. There was only a up to 30% decrease in the antidromic population spike (10–100 μM). The inhibition of the antidromic spike was increased by using a higher stimulus frequency. In contrast to 14-benzoyltalitasamine, the alkaloid talitasamine which is lacking the benzoylester side chain was a less effective inhibitor of the orthodromic population spike and even failed to affect the antidromic spike. Furthermore, the effects of the alkaloids on experimentally induced epileptiform activity was examined. While talitasamine was lacking any significant effect at concentrations less than 100 μM, 14-benzoyltalitasamine reversibly reduced both stimulus-triggered epileptiform activity in area CA1 elicited by omission of Mg²⁺ from the bathing medium as well as spontaneously occurring epileptiform activity in CA3 elicited by omission of Mg²⁺ and elevation of K⁺ to 5 or 8 mM. The antiepileptiform efficacy of this compound was concentration-dependent (0.3–10 μM) and manifested itself as a decrease in burst frequency as well as in burst amplitude and was significantly increased by the higher K⁺ concentration. Received: 25 August 1997 / Accepted: 15 February 1998     

Electrophysiological properties of the propafenone-analogue GE 68 (1-[3-(phenylethyl)-2-benzofuryl]-2-(propylamino)-ethanol) in isolated preparations and ventricular myocytes of guinea-pig hearts

GE 68 ((Rac.)-1-[3-(Phenylethyl)-2-benzofuryl]-2-(propylamino)-ethanol hydrochloride) is structurally related to propafenone, and exerts negative inotropic and negative chronotropic effects similar to the parent drug, but lacks any β-adrenoceptor blocking activity contrary to propafenone. Thus, the electrophysiological effects of GE 68 were studied in papillary muscles, left atria, Purkinje fibres, sinoatrial nodes and ventricular myocytes of the guinea-pig heart with the intracellular microelectrode technique and the patch-clamp technique in the cell-attached mode. The decrease of the maximum upstroke velocity (V˙_max) by GE 68 (1 to 10 μM) was use- and frequency-dependent. V˙_max recovered from the use-dependent block with a time constant of 4.1 ± 0.6 s. In papillary muscles and Purkinje fibres action potential duration was shortened, while it was prolonged in left atria and sinoatrial nodes. Half-maximal steady-state inactivation of the sodium channels was shifted to more negative membrane potentials (control: –91.5 ± 0.8 mV, 10 μM GE 68: –97.9 ± 2.5 mV). The peak of the current-voltage relationship and the reversal potential were not changed by GE 68. The amplitude of the unitary current remained unaltered, while open state probability was decreased. The most striking effect of GE 68 was an increase of the number of sweeps without single channel openings (1 μM: 2 fold, 10 μM: 6 fold). GE 68 also caused a decrease of the mean open times, and an increase of the mean closed times in unmodified and pronase-modified sodium channels. Besides the lack of β-adrenoceptor blocking activity, data present a faster recovery from the use-dependent block by GE 68 and a lower affinity to inactivated sodium channels compared to the reference drug propafenone, as well as differences in the effect on single channel kinetics. Received: 25 July 1996 / Accepted: 14 October 1996     

Pharmacological differentiation of synergistic contribution of L-type Ca2+ channels and Na+/H+ exchange to the positive inotropic effect of phenylephrine,endothelin-3 and angiotensin II in rabbit ventricular myocardium

The present study was designed to delineate pharmacologically the role of sarcolemmal L-type Ca²⁺ channels and Na⁺/H⁺ exchange in the positive inotropic effect (PIE) of phenylephrine mediated by alpha-1 adrenoceptors, endothelin (ET) and angiotensin II (Ang II) that stimulate phosphoinositide (PI) hydrolysis in the rabbit ventricular muscle. The PIE of these receptor agonists was compared with the PIE of isoprenaline that accumulates cyclic AMP. For this purpose, we investigated the influence of a Ca²⁺ antagonist, verapamil, and of an inhibitor of Na⁺/H⁺ exchange, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), alone or in combination, on the cumulative concentration-response curve (CRC) for phenylephrine (with 0.3 μM bupranolol), ET-3 and Ang II in isolated right ventricular papillary muscles of the rabbit, which were electrically stimulated at 1 Hz in Krebs-Henseleit solution at 37°C. Verapamil at 0.3 and 1 μM decreased the basal force of contraction to 37.0 ± 4.0% and 13.2 ± 1.1% of the control, respectively, while EIPA even at 10 μM affected the basal force to much less extent and decreased it to 87.0 ± 1.4%. Verapamil (0.3 and 1 μM) and EIPA (1 and 10 μM), when used alone, each significantly attenuated but did not abolish the PIEs induced by phenylephrine, ET-3 and Ang II, while the simultaneous administration of verapamil (1 μM) and EIPA (10 μM) consistently and almost completely inhibited the PIE induced by these receptor agonists. By contrast, the PIE of isoprenaline was retained even in the presence of verapamil and EIPA. These results indicate that both the influx of Ca²⁺ ions through L-type Ca²⁺ channels and activation of Na⁺/H⁺ exchange contribute synergistically to the PIE that is mediated by alpha-1 adrenergic, ET and Ang II receptor agonists, while these mechanisms are not essential for the beta-adrenoceptor-mediated PIE. Received: 20 February 1996 / Accepted: 20 August 1996     

Levetiracetam (ucb LO59) affects in vitro models of epilepsy in CA3 pyramidal neurons without altering normal synaptic transmission

Previous behavioural and electrophysiological studies have indicated that levetiracetam (ucb LO59) acts as an anticonvulsant drug in vivo. The purpose of the present study was to investigate the effects of levetiracetam on normal synaptic transmission and epileptiform activity in vitro. Intracellular recordings were obtained from the CA3 subfield of the rat hippocampal slice preparation. Levetiracetam in a concentration of 10 μM did not influence basic cell properties or normal synaptic transmission evoked by subthreshold and suprathreshold stimuli to the commissural pathway. However, it strongly inhibited the development of epileptiform bursting by the γ-aminobutyric acid (GABA)_A-receptor antagonist bicuculline (1– 30 μM). Levetiracetam also decreased the size of bursts previously established by bicuculline. In experiments in which the glutamate-receptor agonist N-Methyl-D-Aspartate (NMDA) was used to generate spontaneous bursting, levetiracetam had no effect on the size of the bursts but decreased bursting frequency. The difference in effects of levetiracetam on bicuculline- and NMDA-induced bursting appeared to be dependent on the convulsant used, since in the presence of 10 μM bicuculline, levetiracetam decreased the size of NMDA-bursts to the same extent as the size of synaptically evoked bicuculline-bursts but had little effect on bursting frequency. The results show that under our experimental conditions, levetiracetam did not alter the components of normal synaptic transmission. However, levetiracetam at the concentrations studied inhibited epileptiform bursting induced by bicuculline and NMDA in vitro in a manner consistent with the profile of an antiepileptogenic drug. Received: 27 December 1996 / Accepted: 5 June 1997     

Inhibition of nitric oxide synthase prevents magnesium-free-induced epileptiform activity in guinea-pig piriform cortex neurones in vitro

The effects of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, were examined on Mg²⁺-free-induced epileptiform activity, in guinea-pig piriform cortex slices in vitro. L-NAME (0.1-1mM) had no effect on neuronal membrane properties or electrically-evoked postsynaptic potentials (PSPs). In contrast, during superfusion of the slices with Mg²⁺-free solution neurones exhibited spontaneous and stimulus-evoked epileptiform potentials that were suppressed in the presence of L-NAME (100 μΜ) or the selective NMDA receptor antagonist DL-APV (100 μΜ). The inhibitory effects induced by L-NAME were reversibly reduced by L-arginine (1mM), but not D-arginine (1mM), the latter drug not being a substrate for NO formation. It was concluded that L-NAME can suppress epileptiform activity induced by Mg2⁺-free exposure primarily through a decrease in presynaptic transmitter release, although additional actions on the NMDA-receptor complex were also considered. Received: 24 October 1996 / Accepted: 7 January 1997     

Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (l-aspartyl-l-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10–100 μM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC₅₀, was c.␣14.5 μM). Aspartame (A₁₉), at tenfold higher concentrations than OTA (100–1000 μM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC₅₀ 34 μM) than together (IC₅₀ 22 μM). As expected A₁₉(250 μM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, γ-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 μM)-treated cells. In order to investigate the effect of aspartame (A₁₉) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A₁₉ (34 μM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 μM) in the lower one, OTA binding was largely prevented (95–98%). When A19 (34 μM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 μM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A₁₉ seemed less efficient. Received: 9 July 1996 / Accepted: 1 October 1996     

Relationship between acute toxicity of (bis)aziridinylbenzoquinones and cellular pyridine nucleotides

The toxicity of aziridinylbenzoquinones may occur by a number of mechanisms, including oxidative stress caused by redox cycling and the activation of the aziridine groups. Isolated hepatocytes were used to assess the relationship between the redox status of NADP(H) associated with oxidative stress, the level of NAD(H) closely linked with DNA repair and the cytotoxicity of three 2,5-bis(aziridinyl)-1,4-benzoquinones (BABQ). Exposure of hepatocytes to the BABQ TW13 (200 μM) and TW25 (100 μM), which are able to arylate and to redox cycle, resulted in increased intracellular NADP⁺ from <0.3 nmol/mg protein to 1.5 nmol/mg protein within 60 min. The increase in intracellular NADP⁺ was followed by the onset of cell death by 180 min. In contrast, exposure to lower concentrations of TW13 (100 μM), TW25 (50 μM) and carboquone (100–200 μM) (which neither arylates nor redox cycles via a one-electron reduction) resulted in a less pronounced (<1.0 nmol/mg) increase in NADP⁺ and there was no evidence of cell death within the 180 min incubation. BABQ had a concentration dependent effect on intracellular NAD⁺. Exposure of hepatocytes to TW13 (200 μM) and TW25 (100 μM) resulted in a decrease in intracellular NAD⁺ from >2.7 to <1.0 nmol/mg protein within 60 min. At concentrations of the BABQ where the level of NAD⁺ remained  >1.0 nmol/mg protein after 30 min, the hepatocytes remained viable at 180 min. These changes in intracellular pyridine nucleotides suggests two mechanisms may be involved in BABQ cytotoxicity. At high concentrations, aziridinylbenzoquinones may cause cytotoxicity via oxidative stress following redox cycling. At lower concentrations, however, the predominant pyridine nucleotide change is a prolonged depletion of NAD⁺, suggesting extensive DNA damage which may lead to delayed cell death. Received: 30 October 1996 / Accepted: 13 January 1997     

The effects of A 5-HT1A receptor agonist and antagonist on the 5-hydroxytryptamine release in the central nucleus of the amygdala: a microdialysis study with flesinoxan and WAY 100635

The modulation of extracellular 5-hydroxytryptamine (5-HT) in the central nucleus of the amygdala (CeA) by 5-HT_1A receptors was studied by intracerebral microdialysis in awake and freely moving rats. Local administration of 1 μM tetrodotoxin (TTX), 60 mM K⁺ and perfusion with Ca²⁺-free Ringer containing EGTA confirmed that the major part of dialysate 5-HT levels from the CeA is of neuronal origin. Administration of 300 nM of RU 24969, a 5-HT_1B receptor agonist, through the probe into the CeA decreased dialysate 5-HT levels to 67.2% of the baseline value. Systemic administration of the 5-HT_1A receptor agonists 8-OH-DPAT and flesinoxan dose-dependently decreased 5-HT levels in the CeA. The effect of 0.3 mg/kg of flesinoxan could be completely antagonized by systemic administration of 0.05 mg/kg WAY 100635, a 5-HT_1A receptor antagonist. WAY 100635 alone had only minimal effects at this dose. These data show that a major part of the extracellular 5-HT in the CeA stems from 5-HT neurons and that the amount of 5-HT released into this brain region can be modulated by 5-HT_1A receptors. Received: 11 September 1996 / Accepted: 25 November 1996     

D-serine modulates the NMDA receptor/nitric oxide/cGMP pathway in the rat cerebellum during in vivo microdialysis

Several lines of investigation indicate that D-serine may be an endogenous ligand for the glycine site of N-methyl-D-aspartate (NMDA) receptors in some CNS regions. We here studied the in vivo effects of D-serine on the NMDA receptor/nitric oxide/cGMP pathway by monitoring extracellular cGMP in the cerebellum of freely-moving rats subjected to transcerebral microdialysis. Local application of NMDA (200, 500 μM) through the dialysis probe for 20 min evoked transient, concentration-dependent cGMP responses which peaked in the fraction of drug administration, the nucleotide levels returning to basal values after 40 min. The NMDA-induced elevation of the extracellular nucleotide was completely inhibited by the selective receptor channel blocker dizocilpine (MK-801) locally co-perfused at the concentration of 10 μM. The non-competitive antagonist had no effect on its own suggesting that endogenous glutamic acid does not tonically activate NMDA receptors. The effect of 200 μM NMDA was largely attenuated by 30 μM 7-chloro-kynurenic acid and completely abrogated when the concentration of the strychnine-insensitive glycine receptor antagonist was raised to 100 μM. D-serine (300 μM), perfused in the presence of 7-chloro-kynurenate (30 μM), was able to fully restore the NMDA (200 μM)-induced increase of cGMP extracellular levels. On the other hand, the D-amino acid directly potentiated in a concentration-dependent manner (0.3, 1 and 10 mM) the NMDA (200 μM)-evoked cGMP production whereas it was inactive on its own. These data show that in vivo the activation of the strychnine-insensitive glycine site is essential for the functioning of the NMDA receptor complex and can be activated by the selective agonist D-serine. They also confirm that cerebellar NMDA receptors do not have their glycine sites saturated. Received: 25 June 1996 / Accepted: 23 September 1996     

Inhibitory H3 receptors on sympathetic nerves of the pithed rat: activation by endogenous histamine and operation in spontaneously hypertensive rats

Our previous results demonstrate the occurrence of presynaptic inhibitory histamine H₃ receptors on sympathetic neurons innervating resistance vessels of the pithed rat. The present study, in which new H₃ receptor ligands with increased potency and selectivity (imetit, clobenpropit) were used, was designed to further explore the role of H₃ receptors in the regulation of the rat cardiovascular system. In particular we were interested whether these receptors may be activated by endogenous histamine and whether they are detectable in an experimental model of hypertension. All experiments were performed on pithed and vagotomized rats treated with rauwolscine 1 μmol/kg. In normotensive Wistar rats the electrical (1 Hz, 1 ms, 50 V for 20 s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 35 mmHg. Two H₃ receptor agonists, R-(–)-α-methylhistamine and imetit, inhibited the electrically induced increase in diastolic blood pressure in a dose-dependent manner. The maximal effect (about 25%) was obtained for R-(–)-α-methylhistamine at about 10 μmol/kg and for imetit at about 1 μmol/kg. Two H₃ receptor antagonists, thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg, attenuated the inhibitory effect of imetit. The neurogenic vasopressor response was increased by about 15% by thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg and decreased by 25% by the histamine methyltransferase inhibitor metoprine 37 μmol/kg. R-(-)-α-Methylhistamine, imetit, thioperamide, clobenpropit and metoprine did not affect the vasopressor response to exogenously added noradrenaline 0.01 μmol/kg (which increased diastolic blood pressure by about 40 mmHg). Metoprine had only a very low affinity for H₃ binding sites (labelled by ³H-N^α-methylhistamine; pK_i 4.46). In pithed Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats, electrical (1 Hz, 1 ms, 50 V for 10 s) stimulation increased diastolic blood pressure by 28 and 37 mmHg, respectively. Imetit inhibited the neurogenic vasopressor response to about the same extent in WKY and SHR rats (maximal effect of about 30%). The inhibitory influence of imetit was diminished by thioperamide 1 μmol/kg to about the same degree in rats of either strain. The present study confirms the occurrence of presynaptic H₃ receptors on sympathetic nerve fibres involved in the inhibition of the neurogenic vasopressor response. Moreover, it demonstrates that these H₃ receptors are probably activated by endogenous histamine and appear to be operative in hypertension. Received: 25 July 1996 / Accepted: 24 October 1996     

Congener specific effects by polychlorinated biphenyls on catecholamine content and release in chromaffin cells

The effects of the non-planar polychlorinated biphenyl (PCB) congener 2,2′,4,4′-tetrachlorobiphenyl (2,4-TCB) and of the coplanar PCB congener 3,3′,4,4′-tetrachlorobiphenyl (3,4-TCB) were investigated on the catecholamine content and release from bovine adrenal chromaffin cells in culture. Each congener was tested at three concentrations (20, 50 and 100 μM) and two exposure periods (24 h and 5 days). Catecholamine release induced by K⁺-stimulation as well as catecholamine content of Triton X-100 treated cell cultures were examined using high-performance liquid chromatography (HPLC). 2,4-TCB showed dose- and time-dependent effects. 2,4-TCB at 100 μM reduced the K⁺-stimulated catecholamine release after 24 h of exposure. After 5 days of exposure, 2,4 TCB at 50 and 100 μM drastically reduced the K⁺-stimulated catecholamine release. 3,4-TCB even at a concentration of 100 μM over exposure of either 24 h or 5 days had no effects on the K⁺-stimulated secretion. When chromaffin cells, exposed to 2,4-TCB, were lysed with 0.5% Triton X-100, a dose- and time-dependent reduction of the catecholamine content appeared. The 3,4-TCB did not reduce the catecholamine content. Conversely there seemed to be a trend towards an increase in catecholamine content. Spontaneous release of catecholamines was strongly increased by the non-planar 2,4 TCB, while the coplanar 3,4 TCB showed no effects on this parameter. Furthermore, the effects of 2,4 TCB appeared to be reversible after replacing the highest concentration (100 μM) of the TCB-solution with culture-medium at the end of the 24-h exposure. Thus, K⁺-stimulated catecholamine release and the catecholamine content of bovine adrenal chromaffin cells was effectively reduced by the non-planar PCB congener whereas spontaneous catecholamine release was strongly increased. The coplanar PCB congener was ineffective at the same conditions. Received: 7 January 1997 / Accepted: 11 February 1997     

Adenosine A2A receptor stimulation increases release of acetylcholine from rat hippocampus but not striatum,and does not affect catecholamine release

Rat striatal and hippocampal slices, preincubated with [³H] dopamine (DA) {or [³H] noradrenaline (NA)} and [¹⁴C] choline, were superfused continuously and stimulated electrically. 2-chloroadenosine (2-CADO 0.001–100 μM), a non-selective adenosine receptor agonist, produced a concentration-dependent inhibition of the electrically evoked DA and acetylcholine (ACh) release from the striatal slices and of the electrically evoked NA and ACh release from the hippocampal slices. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 3, 30 and 200 nM), a selective adenosine A₁ receptor antagonist, caused a concentration-dependent, parallel, rightward shift of the 2-CADO concentration-response curve, consistent with competitive antagonism. The pA₂ values ranged between 8.4 and 8.8. In the case of ACh release from the hippocampus, but in no other case, was there an increase in release of radioactivity at low concentrations of 2-CADO in the presence of DPCPX. The stimulation in the hippocampus could be blocked by a selective adenosine A_2A receptor antagonist KF 17837. By itself KF 17837 (0.1–100 μM) had no effect on electrically evoked NA release from hippocampal slices, but decreased electrically evoked ACh release. This inhibition was counteracted by DPCPX (1 μM). These results show that, under the conditions used, DA release in the striatum, and NA release in the hippocampus, as well as ACh release from the striatum are regulated by adenosine A₁ but not by adenosine A_2A receptors. By contrast, ACh release from the hippocampus is tonically regulated both by adenosine A₁ receptors, which inhibit release, and by adenosine A_2A receptors which stimulate release. Received: 25 April 1996 / Accepted: 30 August 1996     

Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs

Objective: The aim of this investigation was to study the inhibition of 11 nonsteroidal anti-inflammatory drugs (NSAIDs) on the human liver phenol sulfotransferases (HL-PST) and catechol sulfotransferase (HL-CST). Methods: The activities of HL-PST and HL-CST were measured with 4 μM 4-nitrophenol and 60 μM dopamine (the sulfate acceptors) and 0.4 μM 3′-phosphoadenosine-5′-phosphosulfate [³⁵S] (the sulfate donor). Samples of liver were obtained from five patients, aged 55–79 years, undergoing clinically indicated hepatectomy. The inhibition curves were constructed with at least five concentrations of the inhibitor. Results: With the exception of piroxicam, NSAIDs inhibited HL-PST, and the estimates of the inhibitory concentration for 50% of responses (IC₅₀; μM) were: 0.02 (mefenamic acid), 3.7 (diflunisal), 5.4 (nimesulide), 9.5 (diclofenac), 30 (salicylic acid), 41 (ketoprofen), 74 (indomethacin), 159 (ibuprofen), 245 (ketoralac) and 473 (naproxen). With 4-nitrophenol as the variable substrate, the inhibition of salicylic acid on HL-PST was non-competitive and the K_i and K_ies were 18 μM and 21 μM (n = 5; P = 0.548), respectively. HL-CST was less susceptible than HL-PST to inhibition by NSAIDs, with only five drugs inhibiting this enzyme. The IC₅₀ estimates for these drugs (μM) were 76 (mefenamic acid), 79 (diflunisal), 103 (indomethacin), 609 (salicylic acid) and 753 (diclofenac). Conclusion: The comparison of the IC₅₀ estimates of HL-PST with the therapeutic plasma concentrations of NSAIDs corrected for the plasma unbound fraction was consistent with the view that mefenamic acid and salicylic acid, when administered at therapeutic doses, should impair the hepatic sulfation of those compounds that are substrates of phenol sulfotransferase. Received: 7 June 1999 / Accepted in revised form: 13 January 2000     

Kinetics and state-dependent effects of verapamil on cardiac L-type calcium channels

The voltage dependence and the kinetics of block by verapamil of L-type calcium current (I_Ca) were investigated in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. I_Ca was elicited repetitively in response to depolarizing voltage pulses from –80 mV to 0 mV at different pulse intervals and durations. Verapamil reduced the magnitude of I_Ca in a frequency-dependent manner without tonic component. The time course of I_Ca remained unchanged suggesting that not open but inactivated channels were affected by the drug. The interaction of verapamil with inactivated channels was investigated by the application of twin pulses. In the presence of verapamil, the duration of the first pulse significantly determined the magnitude of I_Ca during the second pulse. Variation of the duration of the first pulse between 12 and 3000 ms, followed by a pulse interval of 100 ms, resulted into a gradual decrease of I_Ca during the second pulse (180 ms), described by concentration-dependent monoexponential decay curves (t = 1060 ± 138 ms at 0.3 μM (n = 3); t = 310 ± 24 ms at 1 μM (n = 6), and t = 125 ± 7 ms at 10 μM (n = 5); means ± SEM). Under control conditions, the changes in I_Ca were comparably negligible. The recovery of I_Ca from block was analyzed by the application of a twin pulse protocol in which two depolarising voltage pulses at fixed length (1. pulse at 3 s and 2. pulse at 180 ms) were interrupted by variable pulse intervals (6 ms–60 s). Under control conditions, recovery from inactivation was fast (t = 11 ± 0.7 ms; means ± SEM; n = 3). In the presence of verapamil, recovery from block was about 500times slower than under control conditions, independent of the drug concentration (t = 5.05 ± 0.44 s at 0.3 μM (n = 3), t = 6.7 ± 0.69 s at 1 μM (n = 4), and t = 6.02 ± 0.9 s at 10 μM (n = 5); means ± SEM). Since development of block was dependent on the concentration of verapamil, whereas recovery from block was independent from the drug concentration, it is assumed that the described time constants for block and unblock reflect voltage-dependent net binding (τ_on) and unbinding (τ_off), respectively, of verapamil at its receptor sites. A computer simulation, including the time constants of block development at 0 mV and of recovery from block at –80 mV, predicted reasonably well the observed frequency-dependent block of I_Ca by verapamil. The development of either measured or calculated block of I_Ca, using 180 ms depolarising voltage pulses from –80 mV to 0 mV, was fitted by identical monoexponential association curves (t = 7 s each at 0.2 Hz and t = 1.7 s each at 1 Hz). When Ba²⁺ was used as the charge carrier, which removes the calcium-dependent inactivation of the current, verapamil (3 μM) was less efficient: I_Ca was decreased by 57 ± 6 % (means ± SEM; n = 6), whereas I_Ba was decreased by 24 ± 4 % (means ± SEM; n = 5). It is proposed that verapamil binds to calcium channels in their inactivated state at more positive potentials and dissociates from the channels in the resting state at more negative potentials. In the proposed scheme of periodical drug binding and unbinding, dependent on the state of the channels, the development of frequency-dependent block of I_Ca by verapamil is adequately predicted by the construction of cumulative association/dissociation curves which include the experimentally determined time constants of development and recovery from block at 0 mV and –80 mV, respectively. Received: 17 May 1996 / Accepted: 23 September 1996     

Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man

Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator (Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method using substrate-specific enzyme reactor-columns. Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine was detected in the surface epithelium of human bronchi (33 pmol/g), mouth (female 0.7 and male 8 pmol/sample), small and large intestine (800 and 16 pmol/g, respectively), gall bladder (12 pmol/g), vagina (6 pmol/sample), skin 1000 (pmol/g) and in pulmonary pleura (5 pmol/sample). Somewhat higher amounts of acetylcholine were found in rat tracheal and intestinal epithelium and in rat skin. The synthesizing enzyme choline acetyltransferase (ChAT) was demonstrated in human surface epithelium by immunohistochemistry and by Western blot analysis. Enzymatic ChAT activity was demonstrated in isolated epithelial cells of human bronchi and small intestine (3.5 and 28 nmol/mg protein/h, respectively). Applied acetylcholine (in nM concentrations) increased, whereas inhibition of ChAT activity by bromoacetylcholine (10 μM) reduced the growth of cultured human bronchial epithelial cells. Inhibition of cell growth occurred also in the presence of atropine (1 μM) together with (±)-tubocurarine (30 μM). In conclusion, the present experiments demonstrate a widespread existence of non-neuronal acetylcholine in surface cells of man. Non-neuronal acetylcholine may act as a local signalling molecule. Received: 19 September 1996 / Accepted: 12 December 1996     

The contribution of different types of calcium channels to electrically-evoked adenosine release from rat hippocampal slices

The role of L-, N- and P-type voltage-dependent calcium channels (VDCCs) in the release of adenosine from rat hippocampal slices was investigated by evaluating the effect of the L-channel activator 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644) and of three calcium channel antagonists: the L-channel antagonist nifedipine, the N-channel blocker ω-conotoxin GVIA (ω-CgTx) and the P-channel blocker ω-agatoxin IVA (ω-Aga-IVA). Adenosine and inosine release, evoked by 5 min electrical stimulation at 10 Hz of hippocampal slices, was assayed by HPLC with ultraviolet absorbance or fluorimetric detection. Nifedipine (100 nM) did not affect adenosine and inosine release evoked by electrical stimulation. Bay K 8644 (100 nM) brought about a statistically significant increase in adenosine evoked release (70%). At a higher concentration (1 μM) Bay K 8644 had no significant effect either on adenosine or inosine release evoked by electrical stimulation. The increase in adenosine release elicited by 100 nM Bay K 8644 was abolished by nifedipine (100 nM). Both ω-CgTx (10 μM) and ω-Aga-IVA (200 nM) caused a statistically significant reduction (77–78%) in evoked release of adenosine. When the previously demonstrated glutamate-dependent component of the release of adenosine was suppressed in the presence of the NMDA and non-NMDA receptor antagonists, D(–)-2-amino-7-phosphonoheptanoic acid (D-AP7, 100 μM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 μM), the remaining release of adenosine was again significantly reduced by ω-CgTx (10 μM) (60%) and ω-Aga-IVA (200 nM) (73%). These data suggest that, while L-type VDCCs are involved in the regulation of the evoked release of adenosine only when activated by Bay K 8644, both P- and N-channels play a direct role in the calcium entry involved in the coupling process between electrical stimulation and adenosine release. Received: 4 September 1996 / Accepted: 8 October 1996     

Potent mechanism-based inhibition of human CYP3A in vitro by amprenavir and ritonavir: comparison with ketoconazole

Objective: Biotransformation of triazolam to its α-hydroxy and 4-hydroxy metabolites by human liver microsomes in vitro was used as an index of human cytochrome P ₄₅₀ 3A (CYP3A) activity. Results: The reaction was strongly inhibited by co-incubation with the viral protease inhibitors ritonavir (IC₅₀=0.14 μM) and amprenavir (IC₅₀=2.5–2.9 μM), and by the azole derivative ketoconazole (IC₅₀ = 0.07 μM). Pre-incubation of microsomes with ritonavir or amprenavir increased inhibitory potency (IC₅₀ reduced to 0.07 μM and 1.4 μM, respectively). This was not the case with ketoconazole. Conclusions: Thus, ritonavir and amprenavir are highly potent mechanism-based inhibitors of human CYP3A isoforms. Received: 11 January 2000 / Accepted in revised form: 9 March 2000     

Effects of avitriptan, a new 5-HT1B/1D receptor agonist, in experimental models predictive of antimigraine activity and coronary side-effect potential

Several acutely acting antimigraine drugs, including ergotamine and sumatriptan, have the ability to constrict porcine arteriovenous anastomoses as well as the human isolated coronary artery. These two experimental models seem to serve as indicators, respectively, for the therapeutic and coronary side-effect potential of the compounds. Using these two models, we have now investigated the effects of avitriptan (BMS-180048; 3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-N-methyl-1H-indole-5-methanesulfonamide monofumarate), a new 5-HT_1B/1D receptor agonist. In anaesthetized pigs, avitriptan (10, 30, 100 and 300 μg·kg^–1) decreased the total carotid blood flow by exclusively decreasing arteriovenous anastomotic blood flow; capillary blood flow was increased. The mean ± SEM i.v. dose of avitriptan eliciting a 50% decrease (ED₅₀) in the porcine carotid arteriovenous anastomotic blood flow was calculated to be 76 ± 23 μg·kg^–1 (132 ± 40 nmol·kg^–1) and the highest dose (300 μg·kg^–1) produced a 72 ± 4% reduction. In recent comparative experiments (DeVries et al. 1996), the mean ± SEM ED₅₀ (i.v.) of sumatriptan in decreasing carotid arteriovenous anastomotic blood flow was 63 ± 17 μg·kg^–1 (158 ± 43 nmol·kg^–1), with a reduction of 76 ± 4% by 300 μg·kg^–1, i.v. Both avitriptan (pD₂: 7.39 ± 0.09; E_max: 13.0 ± 4.5% of the contraction to 100 mM K⁺) and sumatriptan (pD₂: 6.33 ± 0.09; E_max: 15.5 ± 2.3% of the contraction to 100 mM K⁺) contracted the human isolated coronary artery. The above results suggest that avitriptan should be able to abort migraine headaches in patients, but may exhibit sumatriptan-like effects on coronary arteries. Initial clinical studies have demonstrated the therapeutic action of the drug in acute migraine. Received: 23 August 1996 / Accepted: 19 October 1996