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.

     

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation.
2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC₅₀ values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ET_A receptors. Little or no response was obtained to the ET_B-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ET_A receptors in this preparation.
3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ET_A receptor in the media of umbilical vein. High density of binding was obtained with the ET_A selective [¹²⁵I]-PD151242, with much lower levels detected with the ET_B selective [¹²⁵I]-BQ3020.
4. Big ET-1 (EC₅₀=42.7 nM) and big ET-2_(1-38) (EC₅₀=99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2_(1-38) was more potent than its isoform big ET-2_(1-37) with concentration–response curves to big ET-2_(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1_(22-38) and big ET-2_(22-38) were inactive.
5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10⁻⁵ M) or by the dual NEP/ECE inhibitor phosphoramidon (10⁻⁵ M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10⁻⁵ M and 10⁻⁴ M).
6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ET_A receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3.
7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2_(1-37), big ET-2_(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.

     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists

1. Bradykinin (BK) and Lys-BK are peptides which are released at high nanomolar concentrations into the tear-film of ocular allergic patients. We hypothesized that these peptides may activate specific receptors on the ocular surface, especially the corneal epithelium (CE) and thus the CE cells may represent a potential target tissue for these kinins.
2. The purpose of the present studies, therefore, was to determine the presence of and the pharmacological characteristics of bradykinin receptors on normal cultured primary and SV40 virus-transformed human corneal epithelial (CEPI) cells by use of the accumulation of [³H]-inositol phosphates ([³H]-IPs) as a bioassay.
3. Bradykinin (BK) induced a maximal 1.95±0.24 fold (n=17) and 2.51±0.29 fold (n=26) stimulation of [³H]-IPs accumulation in normal, primary (P-CEPI) and SV40-immortalized (CEPI-17-CL4) cells, respectively. This contrasted with a maximal 3.2–4.5 fold and 2.0–2.9 fold stimulation by histamine (100 μM) and platelet activating factor (100 nM) in both cell-types, respectively.
4. The molar potencies of BK and some of its analogues in the CEPI-17-CL4 cells were as follows: BK (EC₅₀=3.26±0.61 nM, n=18), Lys-BK (EC₅₀=0.95±0.16 nM, n=5), Met-Lys-BK (EC₅₀=2.3± 0.42 nM, n=5), Ile-Ser-BK (EC₅₀=5.19±1.23 nM, n=6), Ala³-Lys-BK (EC₅₀=12.7±2.08 nM, n=3), Tyr⁸-BK (EC₅₀=19.3±0.77 nM, n=3), Tyr⁵-BK (EC₅₀=467±53 nM, n=4) and des-Arg⁹-BK (EC₅₀=14.1±2.7 μM, n=4). The potencies of BK-related peptides in normal, P-CEPI cells were similar to those found in transformed cells, thus: BK, EC₅₀=2.02±0.69 nM (n=7), Tyr⁸-BK, EC₅₀=14.6±2.7 nM (n=3), Tyr⁵=BK, EC₅₀=310±70 nM (n=4) and des-Arg⁹-BK, EC₅₀=12.3± 3.8 μM (n=3).
5. The bradykinin-induced responses were competitively antagonized by the B₂-receptor selective BK antagonists, Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]BK; Icatibant; molar antagonist potency=2.9 nM; pA₂=8.54±0.06, n=4; and slope=1.04±0.08) and D-Arg⁰[Hyp³,Thi^5,8, DPhe⁷]-BK (K_B=371 nM; pK_B=6.43±0.08, n=4) in CEPI-17-CL4 cells. The antagonist potency of Hoe-140 against BK in normal, P-CEPI cells was 8.4±1.8 nM (pK_i=8.11±0.12, n=4), this being similar to the potency observed in the immortalized cells.
6. This rank order of potency of agonist BK-related peptides, coupled with the antagonism of the BK-induced [³H]-IPs by the specific B₂-receptor antagonists, strongly suggests that a B₂-receptor subtype is involved in mediating functional phosphoinositide (PI) responses in the CEPI-17-CL4 and P-CEPI cells.
7. In conclusion, these data indicate that the P-CEPI and CEPI-17-CL4 cells express BK receptors of the B₂-subtype coupled to the PI turnover signal transduction pathway. The CEPI-17-CL4 cells represent a good in vitro model of the human corneal epithelium in which to study further the role of BK receptors in its physiology and pathology, such as in allergic/inflammatory conditions, potential wound healing and other functions of the cornea.

     

Involvement of ETA and ETB receptors in the activation of phospholipase D by endothelins in cultured rat cortical astrocytes

1. This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [³²P]phosphatidylbutanol in [³²P]orthophosphate prelabelled cells stimulated in the presence of 25 mM butanol.
2. The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal effects evoked by the ET_B-preferring agonists, ET-3, S6c and IRL 1620, were significantly lower than the maximal response to the non-selective agonist ET-1.
3. The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET_A receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2±3.5% of the ET-1 response) was defined at low (up to 1 μM) concentrations of BQ-123, yielding an estimated K_i value for BQ-123 of 21.3±2.5 nM. In addition, the presence of 1 μM BQ-123 significantly reduced the maximal response to ET-1 but did not change the pD₂ value.
4. Increasing concentrations of the ET_B selective antagonist BQ-788 inhibited the S6c response with a K_i of 17.8±0.8 nM. BQ-788 also inhibited the effect of ET-1, although, in this case, two components were defined, accounting for approximately 50% of the response, and showing K_i values of 20.9±5.1 nM and 439±110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 μM BQ-788, also revealing two components. Only one of them, corresponding to 69.8±4.4% of the response, was sensitive to BQ-788 which showed a K_i value of 28.8±8.9 nM.
5. Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123.
6. In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET_A and ET_B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET_A and ET_B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET_B receptors were not blocked, and 30–50% and 50–70%, respectively, when ET_B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative effects shown by endothelins on cultured and reactive astrocytes.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

Prostanoid receptors of the EP3 subtype mediate inhibition of evoked [3H]acetylcholine release from isolated human bronchi

1. The release of neuronal [³H]acetylcholine (ACh) from isolated human bronchi after labelling with [³H]choline was measured to investigate the effects of prostanoids.
2. A first period of electrical field stimulation (S₁) caused a [³H]ACh release of 320±70 and 200±40 Becquerel (Bq) g⁻¹ in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S_n, n=2, 3, and 4) released less [³H]ACh, i.e. decreasing S_n/S₁ values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively).
3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E₂, nocloprost, and sulprostone (EP₁ and EP₃ selective) inhibited evoked [³H]ACh release in a concentration dependent manner with IC₅₀ values between 4–14 nM and maximal inhibition of about 70%.
4. The inhibition of evoked [³H]ACh release by prostaglandin E₂, nocloprost and sulprostone was not affected by the DP-, EP₁- and EP₂-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA₂ values) at the respective receptors.
5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP₁-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [³H]ACh release.
6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [³H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [³H]ACh release.
7. In conclusion, applied prostanoids appear to inhibit [³H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP₃ subtype.

     

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype

1. We have demonstrated recently that exogenous prostaglandin E₂ (PGE₂) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE₂ and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [³H]-ACh release.
2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 μM), PGE₂ (0.1 nM–1 μM) inhibited EFS-evoked [³H]-ACh release in a concentration-dependent manner with an EC₅₀ and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF_2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [³H]-ACh release by 48%, 41% and 35%, respectively. PGD₂ (1 μM) had no significant effect on [³H]-ACh release.
3. The selective TP-receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE₂ and PGF_2α.
4. A number of EP-receptor agonists mimicked the ability of PGE₂ to inhibit [³H]-ACh release with a rank order of potency: GR63799X (EP₃-selective)>PGE₂>M&B 28,767 (EP₃ selective)>17-phenyl-ω-trinor PGE₂ (EP₁-selective). The EP₂-selective agonist, AH 13205 (1 μM), did not affect EFS-induced [³H]-ACh release.
5. AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA₂ at DP-, EP₁- and EP₂-receptors, failed to reverse the inhibitory effect of PGE₂ or 17-phenyl-ω-trinor PGE₂ on [³H]-ACh release.
6. These results suggest that PGE₂ inhibits [³H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP₃-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [³H]-ACh release when compared to receptors of the EP₃-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

     

The effects of recombinant rat μ-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase

1. The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca²⁺]_i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃), [Ca²⁺]_i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
2. Opioid receptor binding was assessed with [³H]-diprenorphine ([³H]-DPN) as a radiolabel. Ins(1,4,5)P₃ and cyclic AMP were measured by specific radioreceptor assays. [Ca²⁺]_i was measured fluorimetrically with Fura-2.
3. Scatchard analysis of [³H]-DPN binding revealed that the B_max varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [³H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pK_i values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na⁺ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pK_i value of 6.76±0.04.
4. Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC₅₀=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml⁻¹ for 24 h)-sensitive and naloxone-reversible manner (K_i=1.7 nM). Morphine (1 μM) and [D-Ala², MePhe⁴, gly(ol)⁵]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen², D-Pen⁵], enkephalin (DPDPE, 1 μM) did not.
5. Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P₃ formation, with a pEC₅₀ of 7.95±0.15 (n=5). PTX (100 ng ml⁻¹ for 24 h) abolished, whilst Ni²⁺ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P₃ response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P₃ formation. Fentanyl (1 μM) also caused an increase in [Ca²⁺]_i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca²⁺]_i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca²⁺]_i.
6. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca²⁺]_i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.

     

Presynaptic imidazoline receptors and non-adrenoceptor[3H]-idazoxan binding sites in human cardiovascular tissues

1. In segments of human right atrial appendages and pulmonary arteries preincubated with [³H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [³H]-noradrenaline release was investigated.
2. In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [³H]-noradrenaline release. The inhibition was not affected by blockade of α₂-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA₂ against aganodine and DTG: 5.55 and 5.21, respectively).
3. In the presence of 1 μM rauwolscine, [³H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine.
4. In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [³H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta.
5. When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α₂-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [³H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [³H]-noradrenaline release.
6. In human atrial appendages, non-adrenoceptor [³H]-idazoxan binding sites were identified and characterized. The binding of [³H]-idazoxan was specific, reversible, saturable and of high affinity (K_D: 25.5 nM). The specific binding of [³H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model.
7. The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I₂-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue.
8. It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I₁ and I₂ imidazoline binding sites.

     

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.

     

Antagonist properties of (?)-pindolol and WAY 100635 at somatodendritic and postsynaptic 5-HT1A receptors in the rat brain

1. The aim of the present work was to characterize the 5-hydroxytryptamine_1A (5-HT_1A) antagonistic actions of (−)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT_1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices.
2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT_1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (−)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 μM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (−)-Pindolol also prevented the 5-HT_1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 μM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT₄ receptors persisted in the presence of 3 μM (−)-pindolol.
3. In the hippocampus, (−)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC₅₀=92 nM; apparent K_B=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC₅₀=522 nM; apparent K_B=115.1 nM). The block by (−)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action.
4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (−)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC₅₀=598 nM; apparent K_B=131.7 nM) or 100 nM ipsapirone (IC₅₀=132.5 nM; apparent K_B=124.9 nM). The effect of (−)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 μM (−)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT.
5. In vivo labelling of brain 5-HT_1A receptors by i.v. administration of [³H]-WAY 100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (−)-pindolol. (−)-Pindolol (15 mg kg⁻¹) injected i.p. either subchronically (2 day-treatment before i.v. injection of [³H]-WAY 100635) or acutely (20 min before i.v. injection of [³H]-WAY 100635) markedly reduced [³H]-WAY 100635 accumulation in all 5-HT_1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (−)-pindolol to prevent [³H]-WAY 100635 accumulation in the DRN and the CA1 and CA3 hippocampal areas.
6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC₅₀=4.9 nM, apparent K_B=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC₅₀=0.80 nM, apparent K_B=0.28 nM).

     

Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ET_A and ET_B receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ET_A receptor antagonist, BQ-123; the ET_B receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ET_B receptor selective antagonist, BQ-788; and the ET_A/ET_B antagonist, TAK-044.
2. In 3rd generation mesenteric arteries, ET-1 (10⁻¹³10⁻⁷ M) produced concentration-dependent contractions (pD₂ 9.86). SRTX S6c (10⁻¹²10⁻⁷ M) also induced concentration-dependent contractions in 53% of arteries studied, although the E_max was much less than that obtained with ET-1 (10.7±2.9% vs 101.9±2.6% of the 60 mM KCl-induced contraction).
3. Neither ET_B receptor desensitization, by a supra-maximal concentration of SRTX S6c (10⁻⁷ M), nor incubation with BQ-788 (3×10⁻⁸ M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1.
4. In the presence of BQ-123 (10⁻⁶ M), responses to low concentrations of ET-1 (up to 10⁻¹⁰ M) were unaffected but responses to concentrations of ET-1 above 10⁻¹⁰ M were significantly inhibited.
5. SRTX S6c desensitization followed by incubation with BQ-123 (10⁻⁶ M) or co-incubation with BQ-788 (3×10⁻⁸ M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10⁻⁸ M and 3×10⁻⁷ M).
6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ET_A and ET_B receptors. The relative role of ET_B receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ET_B receptor desensitization or BQ-788. The effect of ET_B receptor desensitization or blockade is only revealed in the presence of ET_A receptor blockade, suggesting the presence of a ‘crosstalk'' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

     

Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca²⁺ ([Ca²⁺]_i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca²⁺]_i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized.
2. Histamine and substance P stimulated [³H]-inositol monophosphate ([³H]-IP₁) accumulation in U373 MG cells. Concentration-response curves of [³H]-IP₁ accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li⁺ yielded best-fit EC₅₀ values of 19.1±1.5 μM for histamine and 5.7±1.3 nM for substance P.
3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 μM histamine resulted in a transient 597±50 nM increase in [Ca²⁺]_i. The best-fit EC₅₀ for histamine was 4.6±2.2 μM. The initial, transient, histamine response was often followed by further small transient increases in [Ca²⁺]_i.
4. Treatment of U373 MG cells with 5 μM thapsigargin, followed by the readdition of 1.8 mM Ca²⁺ to the perfusion buffer, resulted in a steady-state level of [Ca²⁺]_i 97±5 nM above pretreated levels (measured 400 s after readdition of Ca²⁺). Perfusion of histamine (100 μM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca²⁺]_i. This effect of histamine was normally reversible upon washout. The best-fit EC₅₀ for the histamine response was 0.8±0.2 μM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca²⁺]_i.
5. Neither 100 μM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn²⁺ in U373 MG cells pretreated with 5 μM thapsigargin, indicating that the depressant effect on steady-state raised [Ca²⁺]_i was probably not due to a block of Ca²⁺ entry.
6. The depressant effect of histamine on [Ca²⁺]_i was blocked by 1 μM mepyramine, and was partially reduced by pre-incubation with 1 μM staurosporine (61±7% reduction) and with Ro 31-8220 (24±10% and 50±6% reduction by 1 and 10 μM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine.
7. Neither 1 μM staurosporine nor 10 μM KN-62 inhibited the binding of [³H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17±1% and 55±2% by 1 and 10 μM Ro 31-8220, respectively. However, [³H]-IP₁ accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 μM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 μM, similarly potentiated the response to 100 μM histamine in 3 out of 4 experiments. KN-62 (10 μM) did not stimulate histamine-induced [³H]-IP₁ accumulation.
8. In HEPES buffer to which no Ca²⁺ had been added, histamine stimulated a transient 451±107 nM increase in [Ca²⁺]_i. Pretreatment with 1 μM and 10 μM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca²⁺]_i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca²⁺]_i. H-89 did not alter the histamine response.
9. The effect of histamine in stimulating Ca²⁺ extrusion was not confined to U373 MG cells, since 100 μM histamine also caused a rapid decrease in steady-state levels of [Ca²⁺]_i in thapsigargin-treated human HeLa cells.
10. The results indicate that agonists which increase [Ca²⁺]_i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca²⁺]_i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca²⁺-extrusion pump.

     

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist,in normal and diseased human tissues

1. We have synthesized a new low molecular weight, non-peptide radioligand, [¹²⁵I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ET_A receptor.
2. Analysis of saturation binding assays demonstrated that [¹²⁵I]-PD164333 bound with high affinity to a single population of receptors (n⩾3 individuals ±s.e.mean) in human aorta (K_D=0.26±0.08 nM; B_max=8.8±3.95 fmol mg^-1 protein), left ventricle from the heart (K_D=0.16±0.02 nM; B_max=34.2± 3.02 fmol mg^-1 protein) and kidney (K_D=1.24±0.16 nM; B_max=125.3±35.07 fmol mg^-1 protein). In each case Hill slopes were close to unity.
3. In kinetic experiments, the binding of [¹²⁵I]-PD164333 to ET_A receptors in sections of heart was time-dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K₁ of 2.66±0.213×10⁸ M^-1 min^-1, and a half-time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [¹²⁵I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min^-1, a half-time for dissociation of 216 min and a K_D calculated from these kinetic data of 0.01 nM.
4. Unlabelled PD164333 inhibited the binding of [¹²⁵I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a K_DET_A of 0.99±0.32 nM and K_DET_B of 2.41±0.22 μM, giving a selectivity of 2500 fold. ET_A-selective ligands competed monophasically for [¹²⁵I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, K_D=3.93 ±0.18 nM; {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 K_D=3.53±0.69 nM. In contrast, the ET_B selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding.
5. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA₂=8.84) and a slope of unity.
6. In the human brain, autoradiography revealed high levels of [¹²⁵I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [¹²⁵I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion.
7. These results show [¹²⁵I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ET_A receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

     

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
2. In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA₂ value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D₁ receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [³H]-N-methylscopolamine ([³H]-NMS) bound to striatal M₄ receptors with a monophasic inhibitory curve and a pK_i value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC₅₀ of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC₅₀=50 nM) and stimulating at higher concentrations (EC₅₀=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pK_i value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
5. As in rat striatum, in CHO cell membranes the displacement of [³H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
6. Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [³⁵S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [³⁵S]-GTPγS binding with a pA₂ value of 7.48.
7. These results show that at the striatal M₄ receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

     

Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.