Characterization of opioid receptors modulating the function of capsaicin-sensitive neurons in guinea-pig atria

Transmural nerve stimulation of isolated atria, obtained from reserpine-pretreated guinea-pigs, in the presence of atropine and the β₁-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. [D-Ala², N-Me-Phe⁴, Gly⁵-ol] enkephalin (DAGO), [D-Ala², D-Leu⁵]enkephalin (DADLE), morphine and dynorphin dose dependently reduced the cardiac response to transmural nerve stimulation. The δ receptor selective agonist [D-Pen², D-Pen⁵]enkephalin (DPDPE), and the κ receptor agonist, U50488, were unable to modify the response. The inhibitory effect of all the active opioid agonists was antagonized by naloxone but not by the selective δ and κ opioid receptor antagonists, ICI 174.864 and MR 2266. These results suggest the presence on sensory nerve terminals of inhibitory opioid receptors belonging to the μ, but not to the δ and κ subtypes.     

Inhibition of P-type ATPases by [(dihydroindenyl)oxy]acetic acid (DIOA), a K+ -Cl- cotransporter inhibitor

[(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K⁺–Cl⁻ cotransporter (IC₅₀ = 10 μM). Here we found that DIOA inhibited activities of P-type ATPases such as dog kidney Na⁺,K⁺-ATPase (IC₅₀ = 53 μM), hog gastric H⁺,K⁺-ATPase (IC₅₀ = 97 μM) and rabbit muscle Ca²⁺-ATPase (IC₅₀ = 127 μM). In the membrane preparation of the LLC-PK1 cells stably expressing rabbit gastric H⁺,K⁺-ATPase, DIOA inhibited activities of the endogenous Na⁺,K⁺-ATPase (IC₅₀ = 95 μM) and the exogenous H⁺,K⁺-ATPase (IC₅₀ = 75 μM). 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl⁻ channel blocker, had no effects on the DIOA-elicited inhibition of the P-type ATPases. These findings suggest that lower concentration of DIOA (< 20–30 μM) should be used for evaluation of the activity of K⁺–Cl⁻ cotransporter without affecting the activities of coexisting Na⁺,K⁺-ATPase and/or H⁺,K⁺-ATPase in cells.     

Effects of adenosine A2 receptor agonists on the excitation of capsaicin-sensitive afferent sensory nerves in airway tissues

We examined the effects of adenosine analogues on the asthmatic reactions induced by the stimulation of capsaicin-sensitive afferent sensory nerves. Intravenous (i.v.) injection of adenosine A₂ receptor agonists, 5′-(N-ethylcarboxamido)-adenosine (NECA) and 2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamido-adenosine (CGS 21680), dose dependently inhibited capsaicin-induced guinea-pig bronchoconstriction (1–1000 nmol kg⁻¹), whereas i.v. administration of the adenosine A₁ receptor agonist, N⁶-cyclo-hexyladenosine (CHA), did not affect it (1000 nmol kg⁻¹). Intratracheal injection of NECA (0.05–5 nmol site⁻¹) and CGA 21 680 (0.05−5 nmol site⁻¹) also reduced capsaicin-induced constriction in a dose-dependent manner. However, NECA (1000 nmol kg⁻¹) failed to inhibit substance P-induced guinea-pig bronchoconstriction. NECA (1–1000 nmol kg⁻¹) dose-dependently inhibited cigarette smoke-induced rat tracheal plasma extravasation, but not substance P-induced reaction. NECA (0.1–10 μM) and CGS 21 680 (10 μM) significantly blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea-pig lung, whereas CHA (10 μM) had no effect. This evidence suggests that adenosine A₂ receptors modulate negatively the excitation of capsaicin-sensitive afferent sensory nerves and substance P release from their endings in airway tissues.     

Effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of catalpalactone on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalpalactone at 5–30 μM decreased intracellular dopamine content with the IC₅₀ value of 22.1 μM. Catalpalactone at 5–20 μM, but not 30 μM, did not alter cell viability. Catalpalactone at 20 μM inhibited tyrosine hydroxylase (TH) and aromatic-l-amino acid decarboxylase (AADC) activities. Catalpalactone also decreased cyclic AMP levels and inhibited TH phosphorylation. In addition, catalpalactone at 20 μM reduced the increases in dopamine levels induced by L-DOPA (20–50 μM). Catalpalactone (5–30 μM) associated with L-DOPA (50–100 μM) enhanced L-DOPA-induced cytotoxicity at 48 h, which was prevented by N-acetyl-l-cysteine. These results suggest that catalpalactone inhibited dopamine biosynthesis by reducing TH and AADC activities and enhanced L-DOPA-induced cytotoxiciy in PC12 cells.     

Opioid receptor types and antinociceptive activity in chronic inflammation: bothe κ- and μ-opiate agonistic effects are enhanced in arthritic rats

The antinociceptive effects obtained in arthritic rats with morphine, the opioid μ-agonist DAGO 9D-Al²,MePhe⁴,Gly-ol⁵ol⁵]enkephalin, the δ-selective agonist DTLET [D-Thr²,Leu⁵]enkephalyl-Thr, and the κ-agonist U-50,488H were compared to their corresponding effects in normal animals and morphine-pretreated arthritic rats, respectively, using a paw pressure test. The effects of the μ- and κ-agonists were increased in arthritic rats. While morphine-treated rats were cross-tolerant to the μ- and κ-agonists, no tolerance to the δ-selective agonist was found. The possibility that the potent action of morphine on this model for chronic inflammatory pain is mediated partly through κ-mechanisms is discussed.     

Angiotensin II-induced responses in vascular smooth muscle cells: inhibition by non-peptide receptor antagonists

The present study investigates the effect of angiotensin II and LR-B/081 (-methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetra-zol-5-yl) [1,1′-biphenyl]-4-yl] methyl]-1(6H)-pyrimidinyl] methyl]-3-thiophenecarboxylate), a novel non-peptide angiotensin II receptor antagonist, on both early and late responses in rat vascular smooth muscle cells. Angiotensin II induced a rapid and transient elevation of inositol trisphosphate intracellular levels, triggered the release of both prostaglandin E₂ and prostaglandin I₂ (EC₅₀ = 21 ± 3 and 16 ± 2 nM, respectively), and, in long-term studies, increased leucine and thymidine incorporation. All angiotensin II effects were antagonized by LR-B/081 and losartan, the reference non-peptide angiotensin AT₁-selective receptor antagonist, whereas they were unaffected by PD123177 (1-(4-amino-3-methylphenyl)methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine carboxylic acid), a non-peptide angiotensin AT₂-selective receptor antagonist. LR-B/081 displayed a much higher potency than losartan in inhibiting angiotensin II-induced prostaglandin E₂ (IC₅₀ = 0.15 ± 0.02 and 39 ± 9 nM, respectively) and prostaglandin I₂ release (IC₅₀ = 0.18 ± 0.04 and 134 ± 40 nM, respectively) and was also more potent in blocking the increase in protein synthesis (IC₅₀ = 242 ± 119 nM and 1221 ± 687 nM, respectively). Moreover, LR-B/081 and losartan blocked the response to angiotensin III but failed to inhibit the prostaglandin release stimulated by vasopressin or the mitogenic effect of serum. LR-B/081 and losartan were devoid of intrinsinc agonistic properties in the experimental conditions employed. The present results describe LR-B/081 as a novel, highly specific and potent, non-peptide angiotensin AT₁-selective receptor antagonist, that is capable of blocking angiotensin II-proliferative responses, which may be of relevance for cardiovascular diseases.     

Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures

We have examined the fluctuation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100–250 μ ) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca²⁺]_i were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K⁺ efflux. Co-incubation with promethazine (1 μ ) or ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic 0215 acid (4 m ) attenuated both the APAP-associated [Ca²⁺]_i changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca²⁺ may be an important early event in APAP-induced hepatotoxicity.     

Response of the guinea-pig urinary bladder to purine and pyrimidine nucleotides

The ability of purines and pyrimidines to cause and inhibit contractile responses was examined in strips of guinea-pig urinary bladder. Adenosine 5′-triphosphate (ATP), β, γ-methylene ATP (APPCP), adenosine 5′-diphosphate (ADP), guanosine 5′-triphosphate (GTP) and cytidine 5′-triphosphate (CTP) caused concentration-dependent contractions of the bladder. The order of potency was APPCP > ATP > GTP=CTP > ADP. Adenosine 5′-monophosphatedenosine, guanosine 5′-diphosphate, guanosine, 5′-monophosphate, guanosine, cytidine 5′-disphosphate, cytidine 5′-monophosphate and cytidine had no apparent contractile activity up to 10⁻ M. Cumulative administrations of 5 × 10⁻⁴ M APPCP, ATP, ADP, GTP or CTP resulted in complete desensitization of the tissue to the contractile activity of the nucleotide. Tissues desensitized to GTP or CTP were non-responsive to ATP suggesting cross-tachyphylaxis. Nucleotides lacking contractile activity or nucleosides did not alter the response of the bladder to ATP; except AMP and adenosine which significantly reduced the contraction. These results suggest that the polyphosphate structure, and not the base, is the determining portion of the nucleotide for promoting contractile activity and the development of tachyphylaxis.     

High-affinity inhibition of glutamate release from corticostriatal synapses by ω-agatoxin TK

To know which Ca²⁺ channel type is the most important for neurotransmitter release at corticostriatal synapses of the rat, we tested Ca²⁺ channel antagonists on the paired pulse ratio. ω-Agatoxin TK was the most effective Ca²⁺ channel antagonist (IC₅₀=127 nM; maximal EFFECT=211% (with >1 μM) and Hill COEFFICIENT=1.2), suggesting a single site of action and a Q-type channel profile. Corresponding parameters for Cd²⁺ were 13 μM, 178% and 1.2. The block of L-type Ca²⁺ channels had little impact on transmission, but we also tested facilitation of L-type Ca²⁺ channels. The L-type Ca²⁺ channel agonist, s-(−)-1,4 dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644 (5 μM)), produced a 45% reduction of the paired pulse ratio, suggesting that even if L-type channels do not participate in the release process, they may participate in its modulation.     

Centrally administered ß;-endorphin produces prolonged changes in δ-opioid ligand activity in vivo

Spontaneous reflex bladder contractions were recorded isometrically in urethane anesthetized rats. Bladder contractions were depressed by intracerebroventricular injections of the μ-opioid receptor agonist [D-A'la²,MePhe⁴,Gly(ol)⁵]enkephalin (DAGO) and the δ-agonist [2D-penicillamine,5D-penicillamine]enkephalin (DPDPE) respectively. The effect of DPDPE was selectively antagonized by ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib = -aminoisobutyric acid). However following the administration of β-endorphin the antagonistic action of ICI 174,864 could no longer be observed. In addition ICI 174,864 exhibited agonistic activity following β-endorphin and the effects of DPDPE were prolonged in a dose related manner by β-endorphin. These observations suggest that β-endorphin may produce complex changes in central δ-opioid receptor activity.     

Ca dependence of the response of three adenosine type receptors in rat hepatocytes

The effect of three different receptor-specific adenosine agonists on the rate of ureagenesis by isolated rat hepatocytes and the dependence on the external free Ca²⁺ concentration ([Ca²⁺]_e) were investigated. In the presence of high [Ca²⁺]_e all adenosine receptor agonists increased ureagenesis to similar levels. However, with low [Ca²⁺]_e the effects of each agonist varied as follows: (i) the adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyl-adenosine, increased ureagenesis depending partially on [Ca²⁺]_e, (ii) the adenosine receptor A₂ agonist, 2-p-(-2-carboxy-ethyl) phenethylamino-5′-N-ethylcarboxyamido adenosine hydrochloride, increased ureagenesis independently of [Ca²⁺]_e and (iii) in contrast, the adenosine receptor A₃ agonist N⁶-2-(-4-aminophenyl) ethyladenosine, increased ureagenesis only in the presence of high [Ca²⁺]_e. The adenosine receptor A₁ antagonist, 1-allyl-3,7-dimethyl-8-phenyl xanthine, inhibited the effect of the adenosine receptor A₁ agonist on ureagenesis, but not the effect of the adenosine A₂ or A₃ receptor agonists. The adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine, inhibited only the effect of the adenosine A₂ receptor agonist. Thus, in addition to A₁ and A₂ type adenosine receptors, rat hepatocytes possess an A₃-like adenosine receptor which responds to the addition of an adenosine A₃ agonist by accelerating ureagenesis a [Ca²⁺]_e dependent manner. Moreover, it was observed that in the presence of extracellular Ca²⁺ each agonist increased [Ca²⁺]_i and this effect was inhibited by the appropriate specific antagonist.     

Delta(9)-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Δ⁹-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca²⁺]_i spiking by Win55,212-2. In control experiments, 0.1 μM Win55,212-2 inhibited [Ca²⁺]_i spiking by 93 ± 5%. Win55,212-2 produced significantly less inhibition of [Ca²⁺]_i spiking following 18–24 h treatment with 1 μM THC (48 ± 5%) or treatment with 1 μM Win55,212-2 (29 ± 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 μM, remained stable between 1 and 7 days of preincubation and shifted the EC₅₀ of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.     

Salmeterol, a long-acting beta 2-adrenoceptor agonist mediating cyclic AMP accumulation in a neuronal cell line.

1. The accumulation of cyclic AMP stimulated by salmeterol, a long-acting beta 2-adrenoceptor agonist and by isoprenaline, a non-selective beta-adrenoceptor agonist have been compared in the B50 neuroblastoma cell line. 2. Salmeterol produced a concentration-dependent increase in the accumulation of total [3H]-cyclic AMP in B50 cells yielding an EC50 value of 37 nM which was lower than that obtained with isoprenaline (294 nM). The maximum response to salmeterol was only 46% of that obtained with isoprenaline. 3. The beta 2-adrenoceptor antagonist, ICI 118551, inhibited the responses to both salmeterol (apparent KD 2.2 nM) and isoprenaline (apparent KD 1.6 nM). However, the beta 1-adrenoceptor antagonist, atenolol, produced no significant effect at concentrations up to 100 microM. 4. Salmeterol (1 microM) changed the concentration-response curve of isoprenaline in the manner of a partial agonist interacting with a full agonist. The KD of salmeterol obtained from the interaction was 55.6 nM. 5. Whereas salmeterol has a slow onset of action in airway smooth muscle compared to other beta 2-adrenoceptor agonists, in B50 monolayers both salmeterol and isoprenaline produced a rapid increase in cyclic AMP accumulation (t1/2 1.1 min and 0.4 min respectively). 6. Despite the existence of cyclic AMP efflux mechanisms that exist in this cell line it was possible to investigate the duration of agonist action by measuring intracellular levels of the second messenger. Replacement of drug-containing medium with fresh buffer led to a rapid reduction in intracellular levels of cyclic AMP in isoprenaline-stimulated cells whereas cyclic AMP accumulation was sustained for much longer periods in salmeterol-stimulated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca transport proteins and causing dysregulation of Ca homeostasis

Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca²⁺ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca²⁺ within these cells in the 5–60 μM concentration range (EC₅₀, 21 μM). TBBPA also causes cell death (LC₅₀, 18 μM) partly via apoptosis, involving Ca²⁺-dependent mitochondrial depolarisation. Studies on intracellular Ca²⁺ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA) at low concentrations (IC₅₀, 0.4 to 1.2 μM) and also activate the Ryanodine receptor Ca²⁺ channel within the 0.4–4 μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca²⁺ signalling, by directly affecting Ca²⁺ transport proteins.     

Differences in functional cyclic AMP compartments mediating lepolysis by isoprenaline and BRL 37344 in four adipocyte types

Triglyceride mobilization and adenylyl cyclase activation in adipocytes from Wistar rats, lean Zucker (Fa/?) rats, obese Zucker (fa/fa) rats and humans were investigated in concentration-response studies with (−)-isoprenaline and the atypical β₃-adrenoceptor selective agonist BRL 37344. Maximum FFA production by both agonists was identical in Wistar rat and lean Zucker rat adipocytes, while obese Zucker rat adipocytes and human adipocytes produced significantly less FFA, especially with BRL 37344. Maximum adenylyl cyclase activation by (−)-isoprenaline was similar for all types of adipocyte ghosts, whereas BRL 37344 was a partial agonist in all cases with the lowest intrinsic activity in human adipocytes. For (−)-isoprenaline the relationship between cAMP and lipolysis was steepest with Wistar rat adipocytes, followed by human and lean Zucker rat adipocytes, while obese Zucker rat cells showed a shallow relationship. For BRL 37344, the relationship was very steep and similar for all four adipocyte types, despite the marked differences in maximal lipolysis and cyclic AMP production. The results strongly argue in favour of cyclic AMP compartmentalization, the activity ratio between the functional and the non-functional compartment being least favourable in obese Zucker rat adipocytes. The atypical β₃-adrenoceptor agonist BRL 37344 very efficiently directs the generated cyclic AMP into the functional compartment in all four adipocytes types investigated.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes.

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.

Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).

In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.

Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.     

Modulation of the hyperpolarisation-activated current, Ih, in rat facial motoneurones in vitro by ZD-7288

ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs⁺ have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1–10 μM) inhibited a current that reversed at −43.7±3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K⁺ (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I_h. Depolarisation-activated transient (I_K(A)) and sustained outward currents were unaffected by ZD-7288. The IC₅₀ for block of I_h by ZD-7288 was around 0.2 μM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba²⁺ and Rb⁺ blocked an inwardly rectifying potassium (K⁺) current, confirming both the presence of I_K(IR) and its insensitivity to ZD-7288. Cs⁺ rapidly and reversibly blocked both I_h and I_K(IR). Inhibition of I_h by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I_h, and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I_h that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.