Dopamine stimulation of cardiac β-adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393

1. Mechanisms underlying β-adrenoceptor stimulation by dopamine were examined on guinea-pig Langendorff-perfused hearts and isolated cells from the right atrium, by using the chronotropic effects and the enhancement of L-type Ca²⁺ current (I_Ca,L) in the presence of prazosin as indicators of β-adrenoceptor stimulation. Dopamine-induced overflow of noradrenaline (NA) concentrations was measured by high-performance liquid chromatography.
2. Dopamine caused positive chronotropic effects with an EC₅₀ of 2.5 μM and induced NA overflow with a similar EC₅₀ (1.3 μM). The chronotropic effect of dopamine was abolished by bisoprolol (1 μM).
3. The effects of dopamine were maintained during prolonged application, whereas the effects of tyramine faded with time. Dopamine (3 μM) restored the chronotropic effects and the NA release suppressed by pretreatment with tyramine, suggesting a de novo synthesis of NA during the exposure to dopamine.
4. Dopamine (3 μM)-induced NA release was not affected by tetrodotoxin, ω-conotoxin, rauwolscine, ICI118551 or sulpiride, but was inhibited by desipramine, a NA uptake inhibitor (IC₅₀ ∼1 μM). It was also not affected by GBR12909 and bupropion, dopamine uptake inhibitors in the central nervous system.
5. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393, a D₁ receptor partial agonist, potently inhibited the 3 μM dopamine-induced release of NA (IC₅₀ ∼0.1 μM). D₁ receptors are not involved in the DA-induced release of NA, since {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (3 μM), a potent D₁ antagonist, inhibited the NA release only slightly, and dihydrexidine (1 μM) and chloro-APB (1 μM), full D₁ agonists, caused no significant NA release.
6. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 inhibited tyramine-induced overflow of NA, and potentiated the field stimulation-induced NA release. {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 and desipramine retarded the decay of the stimulation-induced tachycardia in a similar manner. These results indicate that {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 is a potent monoamine transport inhibitor and a useful tool for the functional evaluation of indirectly-acting sympathomimetic agonists in the heart. In the presence of {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393 (10 μM), dopamine at 1 μM showed no chronotropic effect.
7. Voltage clamp experiments with isolated atrial cells revealed that dopamine is a weak partial agonist. The EC₅₀ for I_Ca,L stimulation by dopamine was high (13 μM). As a result, dopamine at 1 μM did not affect I_Ca,L. Bisoprolol abolished the stimulation of I_Ca,L by dopamine (30 μM), and dihydrexidine (1 μM) did not affect I_Ca,L.
8. It was concluded that the cardiac effects of dopamine at clinically relevant concentrations (<1 μM) result almost exclusively from the indirect effect of β adrenoceptor stimulation, involving the release of NA from sympathetic nerve terminals. The roles of the direct stimulation of β adrenoceptors by dopamine at these concentrations and the stimulation of postjunctional D₁ receptors seem negligible. The desipramine- and {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393-sensitive monoamine transporter mediates the release of NA.

     

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (I_Ca(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on I_Ca(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison.
2. In human atrial cells, 100 μM pimobendan increased I_Ca(L) (evoked by depolarization to +10 mV from a holding potential of −40 mV) by 250.4±45.0% (n=15), with the concentration for half-maximal stimulation (EC₅₀) being 1.13 μM. I_Ca(L) was increased by 100 μM UD-CG 212 by 174.5±30.2% (n=10) with an EC₅₀ value of 1.78 μM in human atrial cells. These two agents inhibit PDE III selectively.
3. A selective PDE IV inhibitor, rolipram (1–100 μM), did not itself affect I_Ca(L) in human atrial cells. However, 100 μM rolipram significantly enhanced the effect of 100 μM UD-CG 212 on I_Ca(L) (increase with UD-CG 212 alone, 167.9±33.9, n=5; increase with the two agents together, 270.0±52.2%; n=5, P<0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated I_Ca(L) by 52.9±9.3% (n=5) in human atrial cells.
4. In rabbit atrial cells, I_Ca(L) at +10 mV was increased by 22.1±9.0% by UD-CG 212 (n=10) and by 67.4±12.0% (n=10) by pimobendan (each at 100 μM). These values were significantly lower than those obtained in human atrial cells (P<0.0001). Rolipram (1–100 μM) did not itself affect I_Ca(L) in rabbit atrial cells. However, I_Ca(L) was increased by 215.7±65.2% (n=10) by the combination of 100 μM UD-CG 212 and 100 μM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 μM UD-CG 212 alone.
5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating I_Ca(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under β-adrenoceptor-mediated stimulation.

     

Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones

1. The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique.
2. Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 μM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (afterdepolarizing) tail current (I_ADP) (measured under ‘hybrid'' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 μM).
3. L-Glutamate (0.25–1 mM, in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), 1S,3S-ACPD (50 μM), ibotenate (Ibo; 25 μM, in the presence of 100 μM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 μM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 μM), or quisqualate (10 μM, in the presence of 20 μM CNQX), but not the mGluR II agonist 2S,1′S,2′S-2-(2′-carboxycyclopropyl)-glycine (L-CCGI, 1 μM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus I_ADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)-glycine (DCG-IV, 10 μM, in the presence of 100 μM DL-APV) was also active.
4. The excitatory effects induced by 10 μM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (+)-α-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5–1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-α-methyl-L-AP4 (MAP4, 1 mM).
5. The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 μM), and by the selective mGluR II agonists (mGluR I antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 μM).
6. The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM).
7. It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

1. The effects of endothelin-1 (ET-1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole-cell clamp techniques.
2. ET-1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization.
3. In voltage-clamp experiments, ET-1 decreased the basal L-type Ca²⁺ current (I_Ca(L)) dose-dependently with a half-maximal inhibitory concentration (EC₅₀) of 0.42 nM and maximal inhibitory response (E_max) of 49.5%. The delayed rectifying K⁺ current (I_K) was also reduced by 33.2±11.1% at 1 nM. In addition, an inwardly rectifying K⁺ current was activated by ET-1 at higher concentrations (EC₅₀=4.8 nM). These ET-1-induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ET_A receptor antagonist.
4. When I_Ca(L) was inhibited by ET-1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in I_Ca(L), suggesting the involvement of cyclic AMP in the effects of ET-1 on I_Ca(L). In contrast, 1 nM ET-1 further decreased I_Ca(L) in the presence of 10 μM ACh, suggesting that ET-1 activates some additional mechanism(s) which inhibit I_Ca(L). The ET-1-induced I_Ca(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H-89 (5 μM). However, the I_Ca(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET-1 under basal conditions.
5. ET-1 failed to inhibit I_Ca(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml⁻¹, >6 h) only reduced the ET-1-induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on I_Ca(L).
6. Intracellular perfusion of 8-bromo cyclicAMP (8-Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET-1 on I_Ca(L). This 8-Br cyclicAMP-resistant component (17.5±14.4%, n=20) was not affected by combined application of 8-Br cyclicAMP with 8-bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol-12-myristate-13-acetate (TPA; 50 nM).
7. In summary, ET-1 exerts negative chronotropic effects on the SA node via ET_A-receptors. ET-1 inhibits both I_Ca(L) and I_K, and increases background K⁺ current. The inhibition of I_Ca(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative.

     

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT; 1 nM–100 μM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC₅₀; 68–84 nM) was more sensitive than the longitudinal muscle (EC₅₀; 1.3–1.44 μM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations.
2. Pretreatment with tetrodotoxin (1 μM), propranolol (1 μM), atropine (1 μM), guanethidine (10 μM) or L-NAME (100 μM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells.
3. 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, α-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10–100 μM.
4. Inhibition by 5-HT was not decreased by either pindolol (1 μM), ketanserin (1 μM), tropisetron (10 μM), MDL72222 (1 μM) or {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (10 μM), but was antagonized by the following compounds in a competitive manner (with pA₂ values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86).
5. Ro 20-1724 (20 μM) and rolipram (10 μM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 μM) nor dipyridamole (10 μM) altered the response of 5-HT.
6. 5-HT (1 nM–1 μM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle.
7. From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT₇ receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT₇ receptors.

     

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder

1. Cumulative concentration-response curves (CRC) to prostaglandin E₁ (PGE₁), PGE₂, PGD₂ and PGF_2α (0.01–30 μM) and to the thromboxane A₂ (TXA₂) receptor agonist U-46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation.
2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF_2α>U-46619>PGE₂ whereas weak contractile responses were obtained with PGD₂ and PGE₁. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM.
3. The selective TXA₂ antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK_B value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF_2α and it was a non-surmountable antagonist of PGE₂ (apparent pK_B of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U-46619 (apparent pK_B value of 5.88±0.04; n=4).
4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U-46619 (0.01–3 μM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK_B of 8.54±0.14 (n=4 for each antagonist concentration). PGF_2α in the range 0.01–10 μM (n=7), but not PGE₂ and PGE₁ (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5).
5. Cumulative additions of U-46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U-46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2).
6. We concluded that TXA₂ can induce direct contraction of human isolated urinary bladder through the classical TXA₂ receptor. Prostanoid receptors, fully activated by PGE₂ and PGF_2α are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE₂ seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA₂ and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

     

Pharmacological characterization of endothelin-induced rat pulmonary arterial dilatation

1. The aim of study was to characterize endothelin (ET)-induced vasodilatation in isolated extrapulmonary rat arteries (EPA) and in intrapulmonary arteries (IPA) preconstricted with 1 μM phenylephrine.
2. The ET-3 (1 nM–100 nM)- and ET-1 (10 nM–100 nM)-induced transient vasodilatations in EPA were more potent than those in IPA. The vasodilatation induced by ET-3 (100 nM) was larger than that induced by ET-1 (100 nM).
3. Both the ET_B antagonist, BQ788 (3 μM) and or endothelium denudation, but not the ET_A antagonist, BQ123 (3 μM), abolished the vasodilatation induced by ET-1 or ET-3 (100 nM each) in EPA and in IPA. The ATP-sensitive K+channel blocker, glibenclamide (20 μM) and the nitric oxide synthase inhibitor, N^G-monomethyl-L-arginine (L-NMMA, 1 mM) suppressed the ET-induced vasodilatation in EPA and in IPA.
4. We conclude that the vasodilatation induced by endothelins is markedly reduced in rat isolated IPA, and suggest that the endothelial ET_B-mediated vasodilatation varies depending on rat pulmonary arterial regions. Furthermore, ET_B-mediated vasodilatation involves activation of ATP-sensitive K⁺ channels and of nitric oxide synthase in rat isolated EPA and IPA.

     

Pharmacological modulation of GABAA receptor-mediated postsynaptic potentials in the CA1 region of the rat hippocampus

1. It is unclear whether GABA_A receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP_As and DPSP_As, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA_A receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA_A receptor ligands, on each response.
2. The GABA uptake inhibitor NNC 05-711 (10 μM) enhanced whereas bicuculline (10 μM) inhibited both IPSP_As and DPSP_As.
3. (−)-Baclofen (5 μM), [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAGO; 0.5 μM), and carbachol (10 μM) caused substantial depressions (up to 99%) of DPSP_As that were reversed by CGP 55845A (1 μM), naloxone (10 μM) and atropine (5 μM), respectively. In contrast, 2-chloroadenosine (CADO; 10 μM) only slightly depressed DPSP_As. Quantitatively, the effect of each agonist was similar to that reported for IPSP_As.
4. The neurosteroid ORG 21465 (1–10 μM), the anaesthetic propofol (50–500 μM), the barbiturate pentobarbitone (100–300 μM) and zinc (50 μM) all enhanced DPSP_As and IPSP_As.
5. The benzodiazepine (BZ) agonist flunitrazepam (10–50 μM) and inverse agonist DMCM (1 μM) caused a respective enhancement and inhibition of both IPSP_As and DPSP_As. The BZω₁ site agonist zolpidem (10–30 μM) produced similar effects to flunitrazepam.
6. The anticonvulsant loreclezole (1–100 μM) did not affect either response.
7. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP_As and DPSP_As by activating GABA_A receptors that are subject to similar allosteric modulation.

     

A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (I_Ca), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the β-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of I_Ca and on the muscarinic activated potassium current I_K,ACh, in frog atrial and ventricular myocytes.
2. ODQ (10 μM) and LY 83583 (30 μM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 μM) on the isoprenaline (Iso)-stimulated I_Ca which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 μM) had no effect under similar conditions.
3. In the absence of SNAP, LY 83583 (30 μM) potentiated the stimulations of I_Ca by either Iso (20 nM), forskolin (0.2 μM) or intracellular cyclic AMP (5–10 μM). ODQ (10 μM) had no effect under these conditions, while MBlue (30 μM) inhibited the Iso-stimulated I_Ca.
4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 μM acetylcholine (ACh) on I_Ca.
5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I_K,ACh by ACh in the presence of intracellular GTP, and this inhibition was weakened when I_K,ACh was activated by intracellular GTPγS.
6. The potentiating effect of LY 83583 on Iso-stimulated I_Ca was absent in the presence of either DL-dithiothreitol (DTT, 100 μM) or a combination of superoxide dismutase (150 u ml⁻¹) and catalase (100 u ml⁻¹).
7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of β-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of MBlue.

     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Diazoxide- and leptin-activated KATP currents exhibit differential sensitivity to englitazone and ciclazindol in the rat CRI-G1 insulin-secreting cell line

1. The effects of the antidiabetic agent englitazone and the anorectic drug ciclazindol on ATP-sensitive K⁺ (K_ATP) channels activated by diazoxide and leptin were examined in the CRI-G1 insulin-secreting cell line using whole cell and single channel recording techniques.
2. In whole cell current clamp mode, the hyperglycaemic agent diazoxide (200 μM) and the ob gene product leptin (10 nM) hyperpolarised CRI-G1 cells by activation of K_ATP currents. K_ATP currents activated by either agent were inhibited by tolbutamide, with an IC₅₀ for leptin-activated currents of 9.0 μM.
3. Application of englitazone produced a concentration-dependent inhibition of K_ATP currents activated by diazoxide (200 μM) with an IC₅₀ value of 7.7 μM and a Hill coefficient of 0.87. In inside-out patches englitazone (30 μM) also inhibited K_ATP channel currents activated by diazoxide by 90.8±4.1%.
4. In contrast, englitazone (1–30 μM) failed to inhibit K_ATP channels activated by leptin, although higher concentrations (>30 μM) did inhibit leptin actions. The englitazone concentration inhibition curve in the presence of leptin resulted in an IC₅₀ value and Hill coefficient of 52 μM and 3.2, respectively. Similarly, in inside-out patches englitazone (30 μM) failed to inhibit the activity of K_ATP channels in the presence of leptin.
5. Ciclazindol also inhibited K_ATP currents activated by diazoxide (200 μM) in a concentration-dependent manner, with an IC₅₀ and Hill coefficient of 127 nM and 0.33, respectively. Furthermore, application of ciclazindol (1 μM) to the intracellular surface of inside-out patches inhibited K_ATP channel currents activated by diazoxide (200 μM) by 86.6±8.1%.
6. However, ciclazindol was much less effective at inhibiting K_ATP currents activated by leptin (10 nM). Ciclazindol (0.1–10 μM) had no effect on K_ATP currents activated by leptin, whereas higher concentrations (>10 μM) did cause inhibition with an IC₅₀ value of 40 μM and an associated Hill coefficient of 2.7. Similarly, ciclazindol (1 μM) had no significant effect on K_ATP channel activity following leptin addition in excised inside-out patches.
7. In conclusion, K_ATP currents activated by diazoxide and leptin show different sensitivity to englitazone and ciclazindol. This may be due to differences in the mechanism of activation of K_ATP channels by diazoxide and leptin.

     

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3′:5′-cyclic monophosphate induced by NO-donors in human whole platelets

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate.
2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC₅₀ being 13.7 μM and 15.8 μM, respectively.
3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC₅₀=32.7 μM, NaNP and 25.0 μM, SNAP), while acetylsalicylic acid (up to 1000 μM) and diclofenac (up to 100 μM) were ineffective.
4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.
5. Indomethacin (10 μM), indoprofen (30 μM), diclofenac (100 μM) and acetylsalicylic acid (1000 μM) showed a comparable efficacy in inhibiting platelet thromboxane B₂ (TXB₂) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase.
6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.
7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on γ-aminobutyric acid (GABA)-induced currents (I_GABA) was also examined.
2. DM inhibited 30 μM glycine-induced current (I_Gly), without affecting the current caused by 30 μM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 μM, and reversible. The half-maximum inhibitory concentration (IC₅₀) of DM was 3.3 μM, about 85 times higher than that of strychnine.
3. DM 3 μM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 μM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 μM shifted the curve for glycine in a nearly parallel fashion.
4. The effect of 10 μM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 μM, inhibited I_Gly equally at −50 mV and +50 mV. The effect of 3 μM DM on I_Gly showed no use-dependence. Blockade by strychnine 0.1 μM showed no voltage- or use-dependence.
5. The results indicate that DM inhibits I_Gly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl⁻ channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl⁻ channel of the complex.

     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.

     

Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A₁ receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area.
2. The concentration-response curve for the inhibitory effects of the selective adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC₅₀=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC₅₀=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope.
3. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non-selective mGluR agonist (which activates both group I and II mGluR), (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; 100 μM). L-CCG-I had no consistent effects and ACPD (100 μM) decreased by 19.4±1.8% (n=4) the fe.p.s.p. slope.
4. The concentration-response curve for the inhibitory effects of CPA (2–100 nM) on the fe.p.s.p. slope (EC₅₀=8.2 (6.9–9.6) nM) was displaced to the right by the group III mGluR selective agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 25 μM) (EC₅₀=17.7 (13.1–21.9) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by L-AP4 (25 μM) was blocked in the presence of the mGluR antagonist (selective for the group III mGluR), (R,S)-α-methyl-4-phosphonophenylglycine (MPPG; 200 μM).
5. Both the direct effect of DHPG on synaptic transmission and the attenuation of the inhibitory effect of CPA (10 nM) were prevented in the presence of the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C.
6. The attenuation by L-AP4 (25 μM) of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope was also prevented by the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C. But this effect seemed to be distinct from the direct effect of L-AP4 (25 μM) on synaptic transmission, which was not modified by the protein kinase C selective inhibitors.
7. We conclude that agonists of metabotropic glutamate receptors (Groups I and III) are able to attenuate the inhibitory effects of adenosine A₁ receptor activation in the hippocampus. This interaction may have pathophysiological relevance in hypoxia, in which there is marked release of both excitatory amino acids and the important endogenous neuroprotective substance, adenosine.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.