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.

     

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

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

     

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.

     

Role of the purinergic and noradrenergic components in the potentiation by endothelin-1 of the sympathetic contraction of the rabbit central ear artery during cooling

1. To examine the role of the purinergic and noradrenergic components in the potentiation of endothelin-1 on the vascular response to sympathetic nerve stimulation, we recorded the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1–8 Hz) under different conditions, at 37°C and during cooling (30°C).
2. Electrical field stimulation produced frequency-dependent contraction, which was reduced during cooling (about 60% for 8 Hz). Both at 37°C and 30°C, phentolamine (1 μM) or blockade of α₁-adrenoceptors with prazosin (1 μM) reduced, whereas blockade of α₂-adrenoceptors with yohimbine (1 μM) increased, the contraction to electrical field stimulation. This contraction was increased after desensitization of P2-receptors with α,β-methylene adenosine 5′-triphosphate (α,β-meATP, 3 μM) at 37°C but not at 30°C, and was not modified by blockade of P2-receptors with pyridoxalphosphate-6-azophenyl-2,4′-disulphonic acid (PPADS, 30 μM) at either temperature.
3. Endothelin-1 (1, 3 and 10 nM) at 37°C did not affect, but at 30°C it potentiated in a concentration-dependent manner the contraction to electrical field stimulation (from 28±6 to 134±22%, for 8 Hz). At 37°C, endothelin-1 in the presence of phentolamine or prazosin, but not in that of yohimbine, α,β-meATP or PPADS, potentiated the contraction to electrical stimulation. At 30°C, phentolamine or yohimbine reduced, prazosin or PPADS did not modify and α,β-meATP slightly increased the potentiation by endothelin-1 of the response to electrical stimulation.
4. The arterial contraction to ATP (2 mM) and the α₂-adrenoceptor agonist BHT-920 (10 μM), but not that to (−)-noradrenaline (1 μM), was potentiated by endothelin-1 at both 37°C and 30°C.
5. These results in the rabbit central ear artery suggest that the sympathetic response: (a) at 37°C, could be mediated mainly by activation of α₁-adrenoceptors, with low participation of P2-receptors, (b) is diminished during cooling, probably by a reduction in the participation of α₁-adrenoceptors, and in this condition the response could be mediated in part by P2-receptors, and (c) is potentiated by endothelin-1 during cooling, probably by increasing the response of both postjunctional α₂-adrenoceptors and P2-receptors.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

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

     

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.

     

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

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

     

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.

     

α2-Adrenoceptor-mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP-dependent and a cyclic AMP-independent mechanism

1. The aim of this study was to determine the conditions under which the α₂-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery.
2. UK14304 (0.3 μM) produced a small contraction of porcine isolated ear arteries which was 7.8±3.3% of the response to 60 mM KC1. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 μM U46619 (8.2±1.8% and 10.2±2.6% of 60 mM KC1 response, respectively). However, an enhanced α₂-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1–2 μM forskolin before the addition of UK14304 (46.9±9.6% of 60 mM KC1 response).
3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the α₁-adrenoceptor antagonist prazosin (0.1 μM), but were inhibited by the α₂-adrenoceptor antagonist rauwolscine (1 μM), indicating that the enhanced responses were mediated via postjunctional α₂-adrenoceptors.
4. In the presence of 0.1 μM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 μM forskolin produced an increase in [³H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 μM UK14304 prevented this increase.
5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9±9.6% of the 60 mM KC1 response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8±3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2±1.7%, which was not different from the control response in the same tissues (12.2±5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2±1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production.
6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide.
7. In conclusion, enhanced contractions to the α₂-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.

     

The mechanisms of enhancement and inhibition of field stimulation responses of guinea-pig vas deferens by prostacyclin analogues

1. In the guinea-pig isolated vas deferens preparation bathed in Tyrode''s solution, the prostacyclin analogues, cicaprost, TEI-9063, iloprost, taprostene and benzodioxane-prostacyclin, enhanced twitch responses to submaximal electrical field stimulation (20%-EFS). The high potency of cicaprost (EC₁₅₀=1.3 nM) and the relative potencies of the analogues (equi-effective molar ratios=1.0, 0.85, 1.6, 17 and 82, respectively) suggest the involvement of a prostacyclin (IP-) receptor.
2. Maximum enhancement induced by cicaprost in 2.5 mM K⁺ Krebs-Henseleit solution was similar to that in Tyrode solution (2.7 mM K⁺), but was progressively reduced as the K⁺ concentration was increased to 3.9, 5.9 and 11.9 mM. There was also a greater tendency for the other prostacyclin analogues to inhibit EFS responses in 5.9 mM standard K⁺ Krebs-Henseleit solution; this may be attributed to their agonist actions on presynaptic EP₃-receptors resulting in inhibition of transmitter release.
3. The EFS enhancing action of cicaprost was not affected by the α₁-adrenoceptor antagonist prazosin (100 and 1000 nM). Cicaprost (20 and 200 nM) did not affect contractile responses of the vas deferens to either ATP (5 μM) or α,β-methylene ATP (1 μM) in the presence of tetrodotoxin (TTX, 100 nM). In addition, enhancement by cicaprost of responses to higher concentrations of ATP (30 and 300 μM) in the absence of TTX, as shown previously by others, was not seen. Prostaglandin E₂ (PGE₂, 10 nM) and another prostacyclin analogue TEI-3356 (20 nM) enhanced purinoceptor agonist responses. Unexpectedly, TTX (0.1 and 1 μM) partially inhibited contractions elicited by 10–1000 μM ATP; contractions elicited by 1–3 μM ATP were unaffected. Further studies are required to establish whether a pre- or post-synaptic mechanism is involved.
4. In a separate series of experiments, cicaprost (5–250 nM), TEI-9063 (3–300 nM), 4-aminopyridine (10–100 μM) and tetraethylammonium (100–1000 μM) enhanced both 20%-EFS responses and the accompanying overflow of noradrenaline to a similar extent. In further experiments with the EP₁-receptor antagonist AH 6809, TEI-3356 (1.0–100 nM) and the EP₃-receptor agonist, sulprostone (0.1–1.0 nM) inhibited both maximal EFS responses and noradrenaline overflow, thus confirming previous reports of the high activity of TEI-3356 at the EP₃-receptor. Cicaprost had no significant effect on noradrenaline overflow at 10 and 100 nM, but produced a modest inhibition at 640 nM.
5. In conclusion, our studies show that prostacyclin analogues (particularly TEI-3356) can inhibit EFS responses of the guinea-pig vas deferens by acting as agonists at presynaptic EP₃-receptors. Prostacyclin analogues (particularly cicaprost and TEI-9063) can also enhance EFS responses through activation of IP-receptors. The mechanism of the enhancement has not been rigorously established but from our results we favour a presynaptic action to increase transmitter release.

     

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.

     

Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells

1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml⁻¹) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM–1 mM), ADP (10 nM–10 mM) and UTP (1 μM–100 mM), but not uridine (100 μM–10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg²⁺ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia.
2. In non-proliferating microglial cells, the ATP (10 μM)-induced outward current was antagonized by suramin (300 μM) or reactive blue 2 (50 μM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 μM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 μM) and was not altered by reactive blue 2 (50 μM) or 8-SPT (100 μM).
3. The current response to UTP (1 mM) disappeared when K⁺ was replaced in the pipette solution by an equimolar concentration of Cs⁺ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl⁻ was replaced with an equimolar concentration of gluconate⁻ (145 mM). The application of 4-aminopyridine (1 mM) or Cs⁺ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca²⁺-free bath medium, or in the presence of charybdotoxin (0.1 μM); the inclusion of U-73122 (5 μM) or heparin (5 mg ml⁻¹) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 μM) persisted under these conditions.
4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs⁺ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near−75 mV. Omission of Ca²⁺ from the Cs⁺ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K⁺ by Cs⁺ (150 mM) in the pipette solution abolished the UTP current.
5. Replacement of GTP (200 μM) by GDP-β-S (200 μM) in the pipette solution abolished the current evoked by UTP (1 mM).
6. When the pipette solution contained Cs⁺ (150 mM) instead of K⁺ and in addition inositol 1,4,5,-trisphosphate (InsP₃; 10 μM), an inward current absolutely dependent on extracellular Ca²⁺ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps.
7. A rise of the internal free Ca²⁺ concentration from 0.01 to 0.5 μM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K⁺ solution. The reversal potential was shifted to negative values, when the extracellular K⁺ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl⁻ concentration from 164 to 38 mM did not change the reversal potential.
8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP₃. The depletion of the intracellular Ca²⁺ pool appears to initiate a capacitative entry of Ca⁺ from the extracellular space. This Ca²⁺ then activates a Ca²⁺-dependent K⁺ current.

     

Peptidergic modulation of the sympathetic contraction in the rabbit ear artery: effects of temperature

1. The effects of neuropeptide Y, endothelin-1, arginine-vasopressin and angiotensin II on the vascular contraction to sympathetic nerve stimulation were studied in isolated segments, 2 mm long, from the rabbit central ear artery, a cutaneous vessel, during changes in temperature (24°–41°C).
2. Transmural electrical stimulation (1–8 Hz, at supramaximal voltage) produced frequency-dependent contraction, and this response, partially blocked by tetrodotoxin (1 μM) and phentolamine (1 μM), was reduced by cooling (30°C–24°C) and was not modified by warming (41°C), as compared to that recorded at 37°C.
3. Pretreatment with neuropeptide Y (10, 30 and 100 nM) increased in a concentration-dependent manner the vascular contraction to sympathetic stimulation at every temperature studied, but this potentiation was greater during cooling (34°C–24°C) than at 37°C or warming (41°C).
4. Pretreatment with endothelin-1 (3 and 10 nM) or vasopressin (0.1, 0.3 and 1 nM) increased in a concentration-dependent manner the vascular contraction to sympathetic stimulation during cooling (34°C–24°C), but not at 37°C or warming (41°C).
5. Pretreatment with angiotensin II (0.1, 0.3 and 1 μM) did not modify the contraction to sympathetic stimulation at any temperature studied.
6. These results suggest that neuropeptide Y, endothelin-1 and vasopressin, but not angiotensin II, modulate the cutaneous vasoconstriction to sympathetic nerve stimulation by potentiating this vasoconstriction during cooling.

     

Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells

1. We have used whole-cell patch clamping methods to study and characterize the cytolytic P2X₇ (P2_Z) receptor in the NTW8 mouse microglial cell line.
2. At room temperature, in an extracellular solution containing 2 mM Ca²⁺ and 1 mM Mg²⁺, 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (Bz-ATP; 300 μM), or ATP (3 mM), evoked peak whole cell inward currents, at a holding potential of −90 mV, of 549±191 and 644±198 pA, respectively. Current-voltage relationships generated with 3 mM ATP reversed at 4.6 mV and did not display strong rectification.
3. In an extracellular solution containing zero Mg²⁺ and 500 μM Ca²⁺ (low divalent solution), brief (0.5 s) application of these agonists elicited larger maximal currents (909±138 and 1818±218 pA, Bz-ATP and ATP, respectively). Longer application of ATP (1 mM for 30 s) produced larger, slowly developing, currents which reached a plateau after approximately 15–20 s and were reversible on washing. Under these conditions, in the presence of ATP, ethidium bromide uptake could be demonstrated. Further applictions of 1 mM ATP produced rapid currents of the same magnitude as those observed during the 30 s application. Subsequent determination of concentration-effect curves to Bz-ATP, ATP and 2-methylthio-ATP yielded EC₅₀ values of 58.3, 298 and 505 μM, respectively. These affects of ATP were antagonized by pyridoxal-phosphate-6-azophenyl- 2′, 4′-disulphonic acid (PPADS; 30 μM) but not suramin (100 μM).
4. In low divalent solution, repeated application of 1 mM ATP for 1 s produced successively larger currents which reached a plateau, after 8 applications, of 466% of the first application current. PPADS (30 μM) prevented this augmentation, while 5-(N,N-hexamethylene)-amiloride (HMA) (100 μM) accelerated it such that maximal augmentation was observed after only one application of ATP in the presence of HMA. At a bath temperature of 32°C, current augmentation also occurred in normal divalent cation containing solution.
5. These data demonstrate that mouse microglial NTW8 cells possess a purinoceptor with pharmacological characteristics resembling the P2X₇ receptor. We suggest that the current augmentation phenomenon observed reflects formation of the large cytolytic pore characteristic of this receptor. We have demonstrated that pore formation can occur under normal physiological conditions and can be modulated pharmacologically, both positively and negatively.

     

KATP-channel activation: effects on myocardial recovery from ischaemia and role in the cardioprotective response to adenosine A1-receptor stimulation

1. Optimization of myocardial energy substrate metabolism improves the recovery of mechanical function of the post-ischaemic heart. This study investigated the role of K_ATP-channels in the regulation of the metabolic and mechanical function of the aerobic and post-ischaemic heart by measuring the effects of the selective K_ATP-channel activator, cromakalim, and the effects of the K_ATP-channel antagonist, glibenclamide, in rat fatty acid perfused, working hearts in vitro. The role of K_ATP channels in the cardioprotective actions of the adenosine A₁-receptor agonist, N⁶-cyclohexyladenosine (CHA) was also investigated.
2. Myocardial glucose metabolism, mechanical function and efficiency were measured simultaneously in hearts perfused with modified Krebs-Henseleit solution containing 2.5 mM Ca²⁺, 11 mM glucose, 1.2 mM palmitate and 100 mu l⁻¹ insulin, and paced at 300 beats min⁻¹. Rates of glycolysis and glucose oxidation were measured from the quantitative production of ³H₂O and ¹⁴CO₂, respectively, from [5-³H/U-¹⁴C]-glucose.
3. In hearts perfused under aerobic conditions, cromakalim (10 μM), CHA (0.5 μM) or glibenclamide (30 μM) had no effect on mechanical function. Cromakalim did not affect glycolysis or glucose oxidation, whereas glibenclamide significantly increased rates of glycolysis and proton production. CHA significantly reduced rates of glycolysis and proton production but had no effect on glucose oxidation. Glibenclamide did not alter CHA-induced inhibition of glycolysis and proton production.
4. In hearts reperfused for 30 min following 30 min of ischaemia, left ventricular minute work (LV work) recovered to 24% of aerobic baseline values. Cromakalim (10 μM), administered 5 min before ischaemia, had no significant effect on mechanical recovery or glucose metabolism. CHA (0.5 μM) significantly increased the recovery of LV work to 67% of aerobic baseline values and also significantly inhibited rates of glycolysis and proton production. Glibenclamide (30 μM) significantly depressed the recovery of mechanical function to <1% of aerobic baseline values and stimulated glycolysis and proton production.
5. Despite the deleterious actions of glibenclamide per se in post-ischaemic hearts, the beneficial effects of CHA (0.5 μM) on the recovery of mechanical function and proton production were not affected by glibenclamide.
6. The data indicate that the cardioprotective mechanism of adenosine A₁-receptor stimulation does not involve the activation of K_ATP-channels. Furthermore, in rat fatty acid perfused, working hearts, stimulation of K_ATP-channels is not cardioprotective and has no significant effects on myocardial glucose metabolism.

     

Outward current produced by somatostatin (SRIF) in rat anterior cingulate pyramidal cells in vitro

1. A high density of receptors for somatostatin (SRIF) exists in the anterior cingulate cortex but their function is unknown. Whole-cell patch clamp recordings were made from visualized deep layer pyramidal cells of the rat anterior cingulate cortex contained in isolated brain slices to investigate the putative effects of SRIF and to identify the receptor subtype(s) involved.
2. SRIF (1–1000 nM) produced a concentration-dependent outward current which was associated with an increased membrane conductance, was sensitive to Ba²⁺ (300 μM–1 mM), and was absent in the presence of a maximal concentration of the GABA_B receptor agonist, baclofen (100 μM). These observations suggest the outward current was carried by K⁺ ions.
3. SRIF analogues also elicited outward currents with a rank potency order of (EC₅₀, nM): octreotide (1.8)>BIM-23027 (3.7)>SRIF (20)=L-362,855 (20). BIM-23056 was without agonist or antagonist activity. Responses to L-362,855 were unlike those to the other agonists since they were sustained for the duration of the application.
4. The sst₂ receptor antagonist, L-Tyr⁸Cyanamid 154806 (1 μM), had no effect alone but partially reversed responses to submaximal concentrations of SRIF (100 nM, 44±6% reversal) and L-362,855 (100 nM, 70±6% reversal) and fully reversed the response to BIM-23027 (10 nM). In contrast, L-Tyr⁸Cyanamid 154806 did not antagonize the response to baclofen (10 μM).
5. We conclude that SRIF activates a K⁺ conductance in anterior cingulate pyramidal neurones via an action predominantly at sst₂ receptors.

     

A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) by either carbachol (pEC₅₀ [negative logarithm of the EC₅₀]=6.19±0.01, R_max [maximum response]=93.2±0.4%; n=14) or calcium ionophore A23187 (pEC₅₀=6.46±0.02, R_max=83.6±3.6%; n=8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC₅₀=6.31±0.06, R_max=94.7±4.6%; n=10; with L-NAME: pEC₅₀=6.33±0.04, R_max=93.4±6.0%; n=4).
2. The selective cannabinoid receptor antagonist, SR 141716A (1 μM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 μM) greatly reduced the R_max values for EDHF-mediated relaxations to carbachol (control, 93.2±0.4%; SR 141716A, 10.7±2.5%; n=5; P<0.001) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control, 84.8±2.1%; SR 141716A, 3.5±2.3%; n=6; P<0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting R_max.
3. Precontraction with 60 mM KCl significantly reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 68.8±5.6% versus 17.8±7.1%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 71.4±6.1% versus 3.9±0.45%) and anandamide (control 71.1±7.0% versus 5.2±3.6%). Similar effects were seen in the presence of 25 mM K⁺. Incubation of vessels with pertussis toxin (PTX; 400 ng ml⁻¹, 2 h) also reduced (P<0.01; n=4 for all) relaxations to 1 μM carbachol (control 63.5±7.5% versus 9.0±3.2%), {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (control 77.0±5.8% versus 16.2±7.1%) and anandamide (control 89.8±2.2% versus 17.6±8.7%).
4. Incubation of vessels with the protease inhibitor phenylmethylsulphonyl fluoride (PMSF; 200 μM) significantly potentiated (P<0.01), to a similar extent (∼2 fold), relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.45±0.04; PMSF, 6.74±0.10; n=4) and anandamide (pEC₅₀: control, 6.31±0.02; PMSF, 6.61±0.08; n=8). PMSF also potentiated carbachol responses both in the presence (pEC₅₀: control, 6.25±0.01; PMSF, 7.00±0.01; n=4; P<0.01) and absence (pEC₅₀: control, 6.41±0.04; PMSF, 6.88±0.04; n=4; P<0.001) of L-NAME. Responses to the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) were also potentiated by PMSF (pEC₅₀: control, 7.51±0.06; PMSF, 8.00±0.05, n=4, P<0.001).
5. EDHF-mediated relaxation to carbachol was significantly attenuated by the K⁺ channel blocker tetraethylammonium (TEA; 1 mM) (pEC₅₀: control, 6.19±0.01; TEA, 5.61±0.01; n=6; P<0.01). In contrast, TEA (1 mM) had no effect on EDHF-mediated relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (pEC₅₀: control, 6.47±0.04; TEA, 6.41±0.02, n=4) or on anandamide (pEC₅₀: control, 6.28±0.06; TEA, 6.09±0.02; n=5). TEA (10 mM) significantly (P<0.01) reduced the R_max for anandamide (control, 94.3±4.0%; 10 mM TEA, 60.7±4.4%; n=5) but had no effect on the R_max to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187.
6. BaCl₂ (100 μM), considered to be selective for blockade of inward rectifier K⁺ channels, had no significant effect on relaxations to carbachol or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, but caused a small shift in the anandamide concentration-response curve (pEC₅₀: control, 6.39±0.01; Ba²⁺, 6.20±0.01; n=4; P<0.01). BaCl₂ (1 mM; which causes non-selective block of K⁺ channels) significantly (P<0.01) attenuated relaxations to all three agents (pEC₅₀ values: carbachol, 5.65±0.02; {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, 5.84±0.04; anandamide, 5.95±0.02; n=4 for each).
7. Apamin (1 μM), a selective blocker of small conductance, Ca²⁺-activated, K⁺ channels (SK_Ca), 4-aminopyridine (1 mM), a blocker of delayed rectifier, voltage-dependent, K⁺ channels (K_v), and ciclazindol (10 μM), an inhibitor of K_v and adenosine 5′-triphosphate (ATP)-sensitive K⁺ channels (K_ATP), significantly reduced EDHF-mediated relaxations to carbachol, but had no significant effects on {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide responses.
8. Glibenclamide (10 μM), a K_ATP inhibitor and charybdotoxin (100 or 300 nM), a blocker of several K⁺ channel subtypes, had no significant effect on relaxations to any of the agents. Iberiotoxin (50 nM), an inhibitor of large conductance, Ca²⁺-activated, K⁺ channels (BK_Ca), had no significant effect on the relaxation responses, either alone or in combination with apamin (1 μM). Also, a combination of apamin (1 μM) with either glibenclamide (10 μM) or 4-aminopyridine (1 mM) did not inhibit relaxation to carbachol significantly more than apamin alone. Neither combination had any significant effect on relaxation to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or anandamide.
9. A combination of apamin (1 μM) with charybdotoxin (100 nM) abolished EDHF-mediated relaxation to carbachol, but had no significant effect on that to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Apamin (1 μM) and charybdotoxin (300 nM) together consistently inhibited the response to {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, while apamin (1 μM) and ciclazindol (10 μM) together inhibited relaxations to both carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. None of these toxin combinations had any significant effect on relaxation to anandamide.
10. It was concluded that the differential sensitivity to K⁺ channel blockers of EDHF-mediated responses to carbachol and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 might be due to actions on endothelial generation of EDHF, as well as its actions on the vascular smooth muscle, and suggests care must be taken in choosing the means of generating EDHF when making comparative studies. Also, the relaxations to EDHF and anandamide may involve activation of cannabinoid receptors, coupled via PTX-sensitive G-proteins to activation of K⁺ conductances. The results support the hypothesis that EDHF is an endocannabinoid but relaxations to EDHF and anandamide show differential sensitivity to K⁺ channel blockers, therefore it is likely that anandamide is not identical to EDHF in the small rat mesenteric artery.