A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

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.

     

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.

     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM).
2. In the prostatic half of the vas deferens the A₁ selective adenosine receptor agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A₁/A₂ adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC₅₀±s.e.mean values 6.15±0.24, 5.99±0.26 and 5.51±0.24, respectively). The responses to CPA were blocked by the A₁ adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM).
3. In the epididymal half of the vas deferens NECA potentiated (at ⩽100 nM) and inhibited (at ⩾1 μM) electrically-evoked contractions. In the presence of the non-selective α-adrenoceptor antagonist phentolamine (3 μM), the α₁-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC₅₀ values 6.05±0.25, 5.97±0.29 and 5.71±0.27, respectively). CPA (at 10 μM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC₅₀ 6.14±0.67); this effect was antagonized by DPCPX (30 nM, apparent pK_B 8.26±0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 μM), CPA (up to 1 μM) potentiated electrically-evoked contractions.
4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC₅₀ values 7.49±0.62, 7.65±0.74 and 5.84±0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK_B value of 7.64±0.64.
5. The α₁-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 μM) potentiated responses to phenylephrine (⩽1 μM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 μM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 μM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC₅₀ values 7.57±0.54 and 8.08±0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis.
6. These studies are consistent with the action of stable adenosine analogues at prejunctional A₁ and postjunctional A₁-like adenosine receptors. The prejunctional A₁ adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of α₁-adrenoceptor-, but not of ATP-induced contractility.

     

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.

     

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.

     

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[³H]-arginine to L-[³H]-citrulline.
2. Elevation of extracellular K⁺ to 20, 35 or 60 mM concentration-dependently augmented L-[³H]-citrulline production. The response to 35 mM KCl was abolished by N^G-nitro-L-arginine (100 μM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl₂ concentration up to 10 mM also prevented the K⁺ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity.
3. However, the effect of high K⁺ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine.
4. The 35 mM K⁺ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca²⁺ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K⁺-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω-conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX.
5. It was concluded that elevation of extracellular [K⁺] causes NO synthase activation by external Ca⁺ entering cells mainly through channels of the P/Q-type. Other Ca²⁺ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca²⁺ ([Ca²⁺]_i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca²⁺ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca²⁺ and an influx of extracellular Ca²⁺, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA₂ activation.
2. In RAW 264.7 cells UTP (100 μM) and thapsigargin (1  μM) caused 2 and 1.2 fold increases, respectively, in [³H]-AA release. The release of [³H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca²⁺-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA₂ inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml⁻¹) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA₂) had no effect on UTP and thapsigargin responses.
3. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 μM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca²⁺]_i rise.
4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM–3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release.
5. Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca²⁺]_i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA.
6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC.
7. Following treatment of cells with SK&F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca²⁺ influx, and the accompanying AA release, were down-regulated.
8. Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin.
9. We conclude that UTP-induced cPLA₂ activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca²⁺, which is essential for the activation of cPLA₂ by UTP and thapsigargin. The [Ca²⁺]_i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca²⁺]_i was also shown to be involved in AA release.

     

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.

     

Dependence of P2-nucleotide receptor agonist-mediated endothelium-independent relaxation on ectonucleotidase activity and A2A-receptors in rat portal vein

1. The mechanism of action of P2 nucleotide receptor agonists that produce endothelium-independent relaxation and the influence of ecto-ATPase activity on this relaxing effect have been investigated in rat portal vein smooth muscle.
2. At 25°C, ATP, 2-methylthioATP (2-MeSATP) and 2-chloroATP (2-ClATP), dose-dependently inhibited spontaneous contractile activity of endothelium-denuded muscular strips from rat portal vein. The rank order of agonist potency defined from the half-inhibitory concentrations was 2-ClATP (2.7±0.5 μM, n=7)>ATP (12.9±1.1 μM, n=9)⩾2-MeSATP (21.9±4.8 μM, n=4). In the presence of αβ-methylene ATP (αβ-MeATP, 200 μM) which itself produced a transient contractile effect, the relaxing action of ATP and 2-MeSATP was completely abolished and that of 2-ClATP strongly inhibited.
3. The non-selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 100 μM) did not affect the relaxation induced by ATP, 2-MeSATP, and 2-ClATP.
4. The A_2A-adenosine receptor antagonist ZM 241385 inhibited the ATP-induced relaxation in a concentration-dependent manner (1–100 nM). In the presence of 100 nM ZM 241385, the relaxing effects of 2-MeSATP and 2-ClATP were also inhibited.
5. ADP, AMP and adenosine also produced concentration-dependent inhibition of spontaneous contractions. The relaxing effects of AMP and adenosine were insensitive to αβ-MeATP (200 μM) but were inhibited by ZM 241385 (100 nM).
6. Simultaneous measurements of contraction and ecto-ATPase activity estimated by the degradation of [γ-³²P]-ATP showed that muscular strips rapidly (10–60 s) hydrolyzed ATP. This ecto-ATPase activity was abolished in the presence of EDTA and was inhibited by 57±11% (n=3) by 200 μM αβ-MeATP.
7. These results suggest that ATP and other P2-receptor agonists are relaxant in rat portal vein smooth muscle, because ectonucleotidase activity leads to the formation of adenosine which activates A_2A-receptors.

     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

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.

     

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea

1. The aim of the present study was to investigate the effects of bradykinin and [des-Arg⁹]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg⁹]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B₂ receptor antagonist, D-Arg⁰-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg⁹]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg⁹]-bradykinin was concentration-dependently inhibited by the B₁ receptor antagonist, [des-Arg⁹,Leu⁸]-bradykinin (0.01–1 μM), whereas the B₂ receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin were inhibited concentration-dependently.
4. Two nitric oxide (NO) biosynthesis inhibitors N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) and N^G-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg⁹]-bradykinin.
5. Prostaglandin E₂ (PGE₂, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE₂.
6. The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
7. These data demonstrate that bradykinin and [des-Arg⁹]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B₂-receptors and B₁-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

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.

     

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.

     

Relationships between structure and vascular activity in a series of benzylisoquinolines

1. In the present work, the properties of 3-methyl isoquinoline, 3,4-dihydropapaverine, tetrahydropapaverine and tetrahydropapaveroline were compared with those of papaverine and laudanosine. The work includes functional studies on rat isolated aorta contracted with noradrenaline, caffeine or KC1, and a determination of the affinity of the compounds for α₁-adrenoceptors and calcium channel binding sites, with [^#H]-prazosin, [^#H]-nitrendipine and [^#H]-(+)-cis-diltiazem binding to rat cerebral cortical membranes. The effects of papaverine derivatives on the different molecular forms of cyclic nucleotide phosphodiesterases (PDE) isolated from bovine aorta were also determined.
2. The three papaverine derivatives show greater affinity than papaverine for the [^#H]-prazosin binding site. They are therefore more selective as inhibitors of [^#H]-prazosin binding as opposed to [^#H]-(+)-cis-diltiazem, while papaverine appears to have approximately equal affinity for both. [^#H]-nitrendipine binding was not affected by either papaverine or papaverine derivatives in concentrations up to 100 μM. 3-Methylisoquinoline had no effect on any of the binding sites assayed.
3. Contractions evoked by noradrenaline (1 μM) in rat aorta were inhibited in a concentration-dependent manner by 3,4-dihydropapaverine, tetrahydropapaverine and with a lower potency, by tetrahydropapaveroline. In Ca²⁺-free solution, tetrahydropapaverine and to a lesser extent, tetrahydropapaveroline, inhibited the noradrenaline (1 μM) evoked contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (10 mM). This suggests that these alkaloids do not act at the intracellular level, unlike papaverine which inhibits the contractile response to caffeine and noradrenaline.
4. Inositol phosphates formation induced by noradrenaline (1 μM) in rat aorta was inhibited by tetrahydropapaverine (100 μM) and tetrahydropapaveroline (300 μM), thus suggesting that α_1D-adrenoceptors are coupled to phosphoinositide metabolism in rat aorta.
5. Unlike papaverine, which has a significant effect on all the PDE isoforms, the three alkaloids assayed did not have an inhibitory effect on the different forms of PDE isolated from bovine aorta.
6. These results provide evidence that papaverine derivatives with a partially or totally reduced isoquinoline ring have a greater affinity for α₁-adrenoceptors and a lower affinity for benzothiazepine sites in the Ca²⁺-channel than papaverine. This structural feature also implies a loss of the inhibitory activity on PDE isoforms. The planarity of the isoquinoline ring (papaverine) impairs the interaction with the α₁-adrenoceptor site and facilitates it with the Ca²⁺-channels and PDEs, whereas the more flexible tetrahydroisoquinoline ring increases the binding to α₁-adrenoceptors.

     

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).