Rank Order Of Agonist Potency Research Articles

Pre- and postjunctional actions of endothelin in the rat iris sphincter preparation

Effects of endothelins (ETs) were studied in the rat iris sphincter preparation. Three peptides (ET-1, ET-2 and ET-3) caused contractile responses, and the rank order of agonist potency was: ET-1 = ET-2 > ET-3. The concentration-response curve to ET-1 was shifted to the right by the ETA receptor antagonist cyclo [D-Asp-L-Pro-D-Val-L-Leu-D-Trp] (BQ-123: 10(-7) M), the pA2 value of which was 7.41 +/- 0.09 (n = 4). ET-1 and ET-3, at the concentration of 10(-9) M, potentiated cholinergic contractions evoked by electrical field stimulation (5 and 20 Hz) without affecting the postjunctional sensitivity to carbachol. This potentiating effect was not influenced by BQ-123 (10(-6) M). The ET-evoked percentage increase in the stimulation-induced contraction observed at 5 Hz was significantly greater than that at 20 Hz. A release of immunoreactive ET was detected when the preparation was stimulated at 20 Hz (1.81 +/- 0.36 pg/sphincter n = 6). ET release evoked by 20 Hz stimulation was completely abolished by tetrodotoxin (10(-7) M). In conclusion, ET interacts with two different receptor types, ETA and non-ETA receptors (probably ETB) which exist post- and presynaptically at cholinergic neuroeffector junctions of the rat iris preparation. Stimulation of ETA receptor results in a direct muscle contraction and non-ETA receptor activation facilitates the acetylcholine output from cholinergic nerve endings. It is suggested that ET released from a tetrodotoxin-sensitive site is involved in the modulation of acetylcholine release in the rat iris sphincter preparation.

Comparison between the pharmacology of dopamine receptors mediating the inhibition of cell firing in rat brain slices through the substantia nigra pars compacta and ventral tegmental area

1. Electrophysiological recordings were made from presumed dopaminergic neurons in the substantia nigra pars compacta and ventral tegmental area of rat brain slices. The ability of selective dopamine receptor agonists to hyperpolarize neurones and inhibit cell firing, as well as the ability of dopamine receptor antagonists to block responses to quinpirole were compared. 2. Six dopamine receptor agonists were examined for their ability to hyperpolarize neurones within the substantia nigra pars compacta. Of these, the most potent ligand tested was naxagolide with an EC50 value of 20 nM and estimated maximum of 10 mV. The rank order of agonist potency was naxagolide > quinpirole > apomorphine > dopamine. 3. Quinpirole was more potent at inhibiting cell firing in the substantia nigra pars compacta (pIC50 = 7.65 +/ 0.06, n = 35) than in the ventral tegmental area (pIC50 = 7.24 +/- 0.06, n = 32; P < 0.01, Student's t test). 7-Hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), a putative D3 selective agonist, had a comparable potency to quinpirole in both the ventral tegmental area (pIC50 = 7.39 +/- 0.26, n = 4), and substantia nigra pars compacta (pIC50 = 7.71 +/- 0.20; n = 4). 4. The inhibition of cell firing by quinpirole was antagonized by haloperidol, S(-)-sulpiride, clozapine, and ritanserin. S(-)-sulpiride and haloperidol had the highest estimated affinities in the substantia nigra, with pA2 values of 8.97 (slope = 0.85) and 8.20 (slope = 2.09) respectively. The pA2 values for S(-)-sulpiride and haloperidol in the ventral tegmental area were 8.07 (slope = 0.87) and 8.11 (slope = 1.48)respectively. Clozapine had a lower functional affinity than S(-)-sulpiride and haloperidol in both the substantia nigra (pA2 = 6.47, slope = 1.19) and ventral tegmental area (pA2 = 6.53, slope 0.87). Ritanserin,a 5-HT2 receptor antagonist that also binds to D2.u. dopamine receptors, caused a slight but significant shift in the concentration-effect curve to quinpirole with an estimated pKA of 6.97 +/- 0.13(n =4) in the substantia nigra and pKA of 7.12 +/- 0.22 (n =4) in the ventral tegmental area.5. Comparison of these data with the binding affinity for cloned dopamine receptors demonstrates that the responses to quinpirole on dopaminergic neurones in both the A9 (substantia nigra) and A10(ventral tegmental area) brain areas are consistent with the activation of predominantly D2, and not D3 or D4 dopamine receptors. Furthermore, the similarity in functional affinity of antagonists for these receptors suggest that the mesolimbic selectivity of atypical neuroleptics, like clozapine, may be a consequence of their actions on other receptors or their effects elsewhere in the brain.

Analysis of agonist and antagonist activities of phenylglycine derivatives for different cloned metabotropic glutamate receptor subtypes

The metabotropic glutamate receptors (mGluRs) consist of at least seven different subtypes and are coupled to intracellular signal transduction via G proteins. However, the lack of specific antagonists for the mGluRs limited the precise characterization of the role of the individual mGluRs. In this study, we investigated the agonist and antagonist activities of a series of phenylglycine derivatives for the mGluRs by examining their effects on the signal transduction of representative mGluR1, mGluR2, and mGluR4 subtypes expressed individually in Chinese hamster ovary cells. The phenylglycine derivatives examined included (S)- and (R)-forms of 3-hydroxyphenylglycine (3HPG), 4-carboxy-phenylglycine (4CPG), 4-carboxy-3-hydroxyphenylglycine (4C3HPG), 3-carboxy-4-hydroxyphenylglycine (3C4HPG), and (+)- and (-)-alpha-methyl-4-carboxyphenylglycine (alpha M4CPG). Among these 10 compounds, (S)-3HPG acted as an agonist for mGluR1, while (S)-4C3HPG, (S)-3C4HPG, and (S)-4CPG served as effective agonists for mGluR2. The rank order of agonist potencies for mGluR2 was L-glutamate > (S)-4C3HPG > (S)-3C4HPG > (S)-4CPG. No other phenylglycine derivatives showed any definite agonist activity on either mGluR1 or mGluR2. Among the phenylglycine derivatives with no mGluR1 agonist activity, (S)-4C3HPG, (S)-3C4HPG, (S)-4CPG, and (+)-alpha M4CPG effectively antagonized the action of L-glutamate on mGluR1. The rank order of antagonist potencies was (S)-4C3HPG > or = (S)-4CPG > or = (+)-alpha M4CPG > (S)-3C4HPG. The Schild plot analysis indicated that (RS)-4C3HPG, (S)-4CPG, and (+)-alpha M4CPG all act as competitive antagonists for mGluR1 with pA2 values of 4.38, 4.46, and 4.38, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of P2-purinoceptor subtypes on the smooth muscle and endothelium of rabbit coronary artery.

We studied purinoceptor subtypes mediating constriction and dilatation in isolated rabbit coronary arteries with and without endothelium by comparing the effects of adenosine, ATP and the ATP analogues, alpha, beta-methylene ATP and 2-methylthio ATP. For contraction, the rank order of agonist potency was alpha, beta-methylene ATP > 2-methylthio ATP >> ATP; adenosine did not produce contraction. Removal of the endothelium did not affect these responses, but they were abolished by previous desensitisation with alpha, beta-methylene ATP. For relaxation, adenosine, ATP, and 2-methylthio ATP were equipotent, but alpha, beta-methylene ATP had no vasodilator effect and caused further contraction of the preparations. A transient contraction often preceded the relaxations to ATP at higher concentrations. The dilator responses to adenosine, ATP, and 2-methylthio ATP were significantly reduced but not abolished in preparations denuded of endothelium; previous desensitisation with alpha, beta-methylene ATP had no effect on the relaxant responses but abolished the initial transient contractions to ATP and 2-methylthio ATP. These results indicate the presence of vasodilator P1- and P2Y-purinoceptors on smooth muscle and, to a lesser extent, on endothelium and P2X-purinoceptors, mediating transient vasoconstriction on smooth muscle alone. The presence of smooth muscle P2Y-purinoceptors is unusual and has functional implications regarding the nature of the response of the coronary artery to ATP.

Substance P produces an inward current by suppressing voltage-dependent and -independent K + currents in bullfrog primary afferent neurons

A whole-cell patch-clamp study was carried out to examine the effect of substance P (SP) on the excitability of neurons in bullfrog dorsal root ganglia (DRG). SP (3 nM to 1 μM) produced an inward current associated with decreased membrane conductance at voltage range between −10 and −130 mV. Neurokinin A (NKA) and neurokinin B (NKB) also produced the inward current in DRG cells; the rank order of agonist potency was NKA = SP ⪢ NKB. An antagonist for SP receptors, [D-Arg 1, D-Trp 7,9, Leu 11]SP, did not prevent the response to SP. SP (3 nM to 1 μM) suppressed the voltage-dependent non-inactivating K + current, the M-current ( I M) by reducing the maximum M-conductance. A voltage-independent background K + current, I K(B), could be recorded at a hyperpolarizing voltage (≤−60 mV) from DRG neurons. SP (3 nM to 1 μM) produced the inward current associated with decreased I K(B) at a holding potential more negative than −60 mV. The SP-induced inward current reversed its polarity at the equilibrium potential for K ions. Intracellular dialysis with Cs + blocked the SP-induced responses. Depletion of intracellular ATP reduced SP-induced inward current. These results suggest that the SP-induced inward current was due to suppression of both the I M and I K(B) that are regulated by intracellular activity of ATP in bullfrog DRG neurons.

Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated cyclic AMP responses to NECA and 2-chloroadenosine.

5-Hydroxytryptamine (5-HT) contracts the guinea-pig isolated iliac artery via 5-HT1-like and 5-HT2 receptors.

The characterization of 5-hydroxytryptamine (5-HT) receptors mediating contractions of the guinea-pig isolated iliac artery was studied when the basal tone was slightly increased by prostaglandin F2 alpha (PGF2 alpha). In the presence of ketanserin (1 mumol/l), 5-HT and several 5-HT receptor agonists induced contractile responses with the rank order of agonist potency: 5-HT = 5-carboxyamidotryptamine (5-CT) = lysergol > ergometrine = methylergometrine > RU 24969 approximately 5-methoxytryptamine (5-MeOT) > methysergide > sumatriptan > tryptamine. Concentration-effect curves to the ergot alkaloids, lysergol, ergometrine, methylergometrine and methylsergide, were biphasic. In the presence of ketanserin (1 mumol/l), contractile responses to 5-HT, 5-CT, RU 24969, 5-MeOT, sumatriptan and tryptamine were antagonized by methiothepin (30 nmol/l) and flesinoxan (3 mumol/l) with approximate pKB values of 8.5-9.0 and 6.0-6.3, respectively. The first phase of contraction produced by the ergot alkaloids, lysergol, ergometrine, methylergometrine and methysergide, were blocked by methiothepin (30 nmol/l) and flesinoxan (3 mumol/l), respectively, with approximate pKB values about 8.4-8.7 and 6.2-6.4, respectively. The mechanism underlying the second phase of contraction remains to be established. Maximum responses of the concentration-effect curves to 5-HT (1 nmol/l-1 mumol/l) were concentration-dependently depressed by ketanserin (1 nmol/l-1 mumol) and spiperone (30 nmol/l-0.3 mumol/l) and reached approximately 60% of the 5-HT maximum response in the presence of ketanserin (1 mumol/l) and spiperone (0.1 mumol/l), respectively. Agonist potency of 5-HT was not affected by the antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences between endothelin receptors mediating contraction of guinea-pig aorta and pig coronary artery

Endothelin receptors mediating contraction were characterized and compared in rings from guinea-pig thoracic aorta and pig left circumflex coronary artery. In guinea-pig aorta, the following rank order of agonist potencies was found (mean EC 50 value, nM): endothelin-1 (5.0) = endothelin-2 (5.5) > vasoactive intestinal contractor (VIC; 11.0) > sarafotoxin S6b (39.8) >[Ala 3,11]endothelin-1 (121) > sarafotoxin S6a (> 150)> endothelin-3 (> 500). [Ala 1,3,11,15]Endothelin-1, endothelin-(16–21), sarafotoxin S6c and sarafotoxin S6d were neither agonists nor antagonists at concentrations up to 1, 10, 3 and 1 μM, respectively. Cyclo-( D-Trp- D-Asp-Pro- D-Val-Leu) (BQ-123; 0.1–1 μM) behaved as a competitive antagonist of endothelin-1 (pA 2 7.4±0.1, slope factor 0.91±0.17, n=4). In pig coronary artery, all endothelins and sarafotoxins were agonists, except for endothelin-(16–21). Sarafotoxin S6c, [Lys 4]sarafotoxin S6c, [Nle 6]sarafotoxin S6c and [Ala 1,3,11,15]endothelin-1 acted as partial agonists ( E max about 40% of that of endothelin-1). The rank order of agonist potencies was: sarafotoxin S6c (1.5) = [Lys 4]sarafotoxin S6c (1.5) > [Nle 6]sarafotoxin S6c (6.7) ≥ sarafotoxin S6a (7.5) ≥ endothelin-1 (12.6) ≥ sarafotoxin S6b (14.8) ≥ VIC (18.3) = endothelin-2(19.3) ≥ [Ala 1,3,11,15]endothelin-1 (41.7) ≥ [Ala 3,11]endothelin-1 (55.2) > endothelin-3 (96.8) > sarafotoxin S6d (> 200). Endothelin-(16–21) was neither agonist nor antagonist at 10 μM. The concentration-response curves of endothelin-3 and sarafotoxin S6a were biphasic, consisting of a higher sensitivity (40–45% of the total effect) and a lower sensitivity component. BQ-123 (0.1–1 μM) did not alter the concentration-response curve of endothelin-1. At 10 μM, BQ-123 shifted the curves of endothelin-1 and sarafotoxin S6c to the right, with mean pK B values of 5.21 and 5.04, respectively. Sarafotoxin S6c (0.3 μM), used as an antagonist, abolished the effects of [Ala 1,3,11,15]endothelin-1 and increased the threshold concentrations of endothelin-3, sarafotoxin S6a and, in the presence of 10 μM BQ-123, of endothelin-1. The results show that endothelin receptors mediating contraction of guinea-pig aorta and pig coronary artery are different. In guinea-pig aorta, these receptors conformed to the ET A type. In pig coronary artery, receptors of the ET B type, and probably other receptors that are poorly sensitive to BQ-123, participated in the contraction.

Effects of β‐adrenoceptor agonists in human bronchial smooth muscle

1. We have investigated the potency and duration of action of isoprenaline and a range of beta-adrenoceptor agonists as relaxants of inherent tone in human superfused, isolated bronchial smooth muscle, a tissue reported to contain a homogeneous population of beta 2-adrenoceptors. 2. All of the beta-adrenoceptor agonists caused concentration-related inhibition of inherent tone, with isoprenaline having an EC50 of 27 nM. The rank order of agonist potency was: formoterol > or = -salmeterol > or = clenbuterol > fenoterol = isoprenaline > terbutaline > or = salbutamol > quinprenaline. 3. Relaxant responses to salmeterol were fully reversed by the selective beta 2-adrenoceptor blocking drug, ICI 118551, demonstrating the involvement of beta 2-adrenoceptors. 4. Rt50, i.e. the time taken for 50% recovery from the effects of an EC50 concentration of agonist, differed considerably between the different beta 2-adrenoceptor agonists. Most agonists were short-acting, having Rt50 values less than 13 min. Quinprenaline was of moderate duration, with an Rt50 value of > or = 20 min. In contrast, salmeterol was extremely long-acting, with no sign of recovery within 4 h. 5. Estimates of relative potency and duration of action were similar to those previously determined for these agonists in the guinea-pig isolated trachea. These results suggest, therefore, that guinea-pig trachea is a suitable alternative to human bronchus for the evaluation of the actions of beta-adrenoceptor agonists on airways smooth muscle.

Characterization of the adenosine receptor mediating contraction in rat colonic muscularis mucosae

1. The objective of this study was to characterize the adenosine receptor mediating contraction in rat isolated colonic muscularis mucosae (RCMM). 2. Sequential additions of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; 0.01-10 microM) elicited reproducible, concentration-related contractions in RCMM. The effects of NECA were mimicked by the adenosine A1 receptor-selective agonists cyclopentyladenosine (CPA), R-phenylisopropyladenosine (R-PIA) and N-[1S, trans)2-hydroxycyclopentyl] adenosine (GR79236) and by S-PIA (the stereoisomer of R-PIA). The adenosine A2 agonists N-[(2-methylphenyl)methyl] adenosine (metrifudil) and 2-[p-(2-carboxyethyl)phenethylamine]-5'-N-ethylcarboxamidoadenosine (CGS21680) also produced contractions in RCMM but were 54 and 165 times less potent respectively than NECA. The rank order of agonist potency for contraction of RCMM was CPA > or = GR79236 = R-PIA > or = NECA > > S-PIA = metrifudil > CGS21680, which is identical to that reported for the inhibition of spontaneous rate in rat isolated right atria and inhibition of lipolysis in rat isolated adipocytes by these same agonists. 3. R-PIA, S-PIA and metrifudil behaved as partial agonists in RCMM. 4. The adenosine A1 receptor-selective antagonist 8-cyclopentyl-1,3- dipropylxanthine (DPCPX) inhibited the contractions produced by all the adenosine agonists tested, with pKB values between 9.2 and 9.5. The non-selective adenosine antagonist 8-phenyltheophylline (8-PT) antagonized the effects of NECA but also markedly potentiated (by 93.0 +/- 10.2% at 3 microM) the maximum contractile response to NECA in RCMM. Neither 8-PT (3 microM) nor DPCPX (0.1 microM) had any effect on the contractions produced by carbachol. 5. The contractile responses to NECA in RCMM were not affected by atropine (1 microM), tetrodotoxin(0.3 microM) or the P2 antagonist, suramin (100 microM).6. The present study confirms that contractions to adenosine agonists in the RCMM are mediated via adenosine Al receptors.

Comparative effects of the two endothelin ETA receptor antagonists, BQ-123 and FR139317, on endothelin-1-induced contraction in guinea-pig iliac artery

The effects of two recently introduced endothelin ETA receptor antagonists, BQ-123 and FR139317, were investigated and compared in guinea-pig isolated iliac artery. Endothelins and sarafotoxins induced contraction of guinea-pig iliac artery with a pharmacological profile characteristic of the ETA receptor. The rank order of agonist potency was (mean EC50 values, nM): endothelin-1(11.7) ≥ endothelin-2(14.9) ≥ vasoactive intestinal contractor (19.5) > sarafotoxin S6b (49.8) ≥ [Ala3,11]endothelin-1 (55.0) > sarafotoxin S6a (>100) > endothelin-3 (≥1000). The C-terminal hexapeptide, endothelin-(16–21), sarafotoxin S6c and sarafotoxin S6d were neither agonists nor antagonists at concentrations up to 10, 3 and 1 μM, respectively. Both FR139317 (1–10 μM) and BQ-123 (0.1–1 μM) surmountably antagonized the effects of endothelin-1. Schild analysis suggested competitive antagonism for FR139317 (Schild slope 1.32 ± 0.21, pA2 5.82 ± 0.16, n = 5), but not for BQ-123 (Schild slope 0.28 ± 0.08, n = 5), which was however more potent (apparent pKB 6.6–7.2) than FR139317. The potency of FR139317 was particularly low with respect to the reported affinity for ETA receptors, suggesting heterogeneity among ETA receptors. Thus, the endothelin receptor present in guinea-pig iliac artery has the following features: (1) rank order of agonist potencies of the ETA type; (2) low potency of FR139317 and (3) non-competitive antagonism by BQ-123.

5-HT 1 receptors mediating contraction in bovine cerebral arteries: a model for human cerebrovascular ‘5-HT 1Dβ‘ receptors

We report on the pharmacological profile of the 5-HT receptor which induces contraction of the bovine isolated cerebral arteries. Several 5-HT receptor agonists were tested for their ability to induce vasoconstriction in bovine pial arteries and their potencies were compared to that of 5-HT. The rank order of agonist potency can be summarized as 5-carboxamidotryptamine ( 5- CT) = RU 24969 ≧ 5- HT > sumatriptan > α- methyl-5- HT > methysergide > 2- methyl-5- HT > ((±)-2- dipropylamino-8- hydroxy-1,2,3,4- tetrahydronaphthalene (8-OH-DPAT). Only methysergide induced a contraction which was smaller than that elicited by 5-HT. Antagonists with selective affinity at 5-HT 1A/1B (propranolol), 5-HT 1C (mesulergine), 5-HT 2 (ketanserin, mianserin) and 5-HT 3 (MDL 72222) sites were inactive to block the 5-HT-induced contraction. In contrast, the 5-HT 1/5-HT 2 receptor antagonists methiothepin and metergoline inhibited the 5-HT-induced response with relatively high affinity (pA 2 = 8.16 ± 0.26 and 6.73 ± 0.05, respectively). Overall, this pharmacological profile indicated clearly that a 5-HT 1 receptor, must closely related to the 5-HT 1D subtype, is responsible for the 5-HT-induced contraction of bovine cerebral arteries. Correlation analysis of the potencies of a series of 5-HT receptor agonists and antagonists in bovine and human cerebrovascular preparations showed a highly significant positive correlation (r=0.94, P=0.0051). Analyses of the correlation between the agonist and antagonist potencies at bovine cerebrovascular receptors and their published affinities at the cloned human 5-HT 1Dα and 5-HT 1Dβ (or human 5-HT 1B) receptors showed a highly significant correlstion only with the 5-HT 1Dβ (r=0.82; P=0.006) subtype. We conclude that cerebral vasoconstriction in bovine cerebral arteries is mediated by a receptor homologous to the human cerebrovascular 5-HT 1D receptor and that bovine pial arteries appear to be the best available pharmacological model for the human cerebrovascular 5-HT 1D receptor. Further, the results suggest that bovine cerebrovascular 5-HT 1D receptors resemble the cloned human 5-HT 1Dβ (or human 5-HT 1B) receptor subtype.

Agonist pharmacology of the neuronal α7 nicotinic receptor expressed in Xenopus oocytes

The potencies and efficacies of seven agonists at chick α7 nicotinic receptors expressed in Xenopus oocytes were determined by whole cell recording. (+)-Anatoxin-a was the most potent agonist (EC 50 = 0.58 μM) and acetylcholine was the least potent (EC 50 = 320 μM). The rank order of agonist potencies was: (+)-anatoxin-a ⪢ cytisine > (−)-nicotine > (+)-nicotine > DMPP > 1-acetyl-4-methylpiperazine methiodide > acetylcholine. DMPP evoked only very small currents: comparison of maximally effective agonist concentrations showed that DMPP was only one-fifth as efficacious as other agonists. Previously published IC 50 values for rat brain [ 125I]α-bungarotoxin sites show a similar agonist profile, and the identity of homo-oligomeric α7 receptors with native α-bungarotoxin-sensitive neuronal nicotinic receptors is discussed.

Cellular signaling responses mediated by a novel nucleotide receptor in rabbit microvessel endothelium

The adenine nucleotide, ATP, elicits an elevation in intracellular ionized calcium concentration ([Ca2+]i) and phospholipase C-mediated phosphatidylinositol hydrolysis and stimulates the synthesis of the prostaglandins E2 and I2 in cultured endothelial cells derived from rabbit cardiac muscle. Use of various ATP analogues indicated that these events did not fit the classical definition of P1 or P2 purinergic receptors and, furthermore, indicated that the receptor(s) mediating these activities was not specific for purines. The rank order of agonist potency on prostaglandin release, elevations in [Ca2+]i, and inositol phosphate response was UTP > or = ATP > ADP > ADP[beta]S = 2-methylthio ATP > adenosine, suggesting that these three cellular responses are coupled to the same or similar receptors. However, the sensitivity of these three cellular responses to added nucleotides was somewhat different. The half-maximum effective concentration (EC50) for ATP stimulation of prostaglandin release was 100 microM, for inositol phosphate turnover it was 25 microM, and for elevations in [Ca2+]i it was < 1 microM. Similar discrepancies in EC50 UTP values for these three cellular responses were also noted. These observations indicate that purine and pyrimidine nucleotides elicit at least three cellular responses in rabbit cardiac muscle microvessel endothelial cells, all demonstrating similar rank orders of potency. However, the differences in EC50 suggest that if these responses are mediated by a single receptor type, it exhibits divergent coupling to various cellular signaling pathways.

Functional characterization of three adenosine receptor types

1. The purpose of the present study was to classify adenosine receptors into A1 and A2 subtypes in a wide range of isolated tissues and cell types (rat adipocytes and atria, guinea-pig ileum and atria (A1); guinea-pig aorta, dog coronary artery and human platelets and neutrophils (A2)) using the R- and S-diastereoisomers of N-phenylisopropyladenosine (PIA), N-cyclopentyladenosine (CPA), the novel compound, N-[(1S,trans)-2-hydroxycyclopentyl]adenosine (GR79236), N-[(2-methylphenyl)methyl]adenosine (metrifudil), 2-(phenylamino)adenosine (CV1808), and 2[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]-N- ethylcarboxamidoadenosine (CGS21680); N-ethylcarboxamidoadenosine (NECA) was used as a standard. 2. Results obtained in all tissue preparations previously reported to contain A1-receptors could be described by a single rank order of agonist potency: CPA > or = GR79236, R-PIA > or = NECA >> S-PAI > or = metrifudil > or = CV1808, CGS21680. 3. In contrast, two distinct rank orders of agonist potency were observed in preparations previously reported to contain A2-receptors. In dog coronary artery, human neutrophils and platelets the rank order of potency was: CV1808, CGS21680 > or = NECA > R-PIA > or = metrifudil > or = CPA > GR79236 S-PIA. However, in guinea-pig aorta the rank order was: NECA > metrifudil > R-PIA, CPA > CV1808, GR79236 > or = S-PIA, CGS21680. 4. The results of this study are consistent with the existence of three types of adenosine receptor: A1-and two subtypes of A2-receptor. The receptor present in dog coronary artery, human platelets and neutrophils, probably corresponds to the A2a subtype, whilst that present in the guinea-pig aorta may be of the A2b subtype.

Adenosine A1‐receptor stimulated increases in intracellular calcium in the smooth muscle cell line, DDT1MF‐2

1. The effect of a range of adenosine receptor agonists on intracellular free calcium concentration ([Ca2+]i) has been studied in the hamster vas deferens smooth muscle cell line DDT1MF-2. 2. Adenosine receptor agonists elicited a rapid and maintained increase in [Ca2+]i in fura-2 loaded DDT1MF-2 cells. The initial rise could be maintained in the absence of extracellular calcium, whereas the maintained or plateau phase was dependent upon the presence of extracellular calcium and appeared to be associated with calcium influx. The rank order of agonist potencies was N6-cyclopentyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, KD 0.14 nM) and 8-phenyltheophylline (KD 112 nM). 4. Pretreatment with the 5-lipoxygenase inhibitor AA861 (20 microM) produced only a small (14 +/- 2%) inhibition of the [Ca2+]i response elicted by N6-cyclopentyladenosine (300 nM), in nominally Ca(2+)-free buffer containing 0.1 mM EGTA. The cyclo-oxygenase inhibitor, indomethacin (2 microM) was without effect. 5. The Ca(2+)-influx associated with the plateau phase required the continued presence of agonist on the receptor. The antagonist DPCPX (100 nM) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re-applied after the cells had been stimulated with N6-cyclopentyladenosine (CPA; 300 nM) in experiments initiated in nominally Ca(2+)-free buffer. 6. Pretreatment with pertussis toxin (200 ng ml-1 for 4 h) inhibited the CPA (100 nM) stimulated intracellular Ca2+ release and Ca2+ influx but was without effect on the response to histamine (100 microM). 7.These data suggest that adenosine A(1)-receptor activation in DDT(1)MF-2 cells stimulates release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+) through Ca(2+) entry pathways in the plasma membrane which required the continued presence of agonist on the receptor.

Inhibition of human monocyte TNF production by adenosine receptor agonists

Adenosine receptor agonists and agents enhancing pericellular concentrations of adenosine possess antiinflammatory properties. In the present study, we found that R-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamido adenosine (NECA), other agonists of adenosine receptors and dipyridamole, an adenosine uptake inhibitor, inhibited tumor necrosis factor (TNF) production by endotoxin-stimulated human monocytes in a concentration- dependent manner with no inhibition of interleukin-6. The rank order of agonist potency is characteristic of neither A1 nor A2 receptors and suggests the involvement of another receptor subtype. The effect of R-PIA on TNF was in part abolished by the antagonist 8-sulfophenyltheophylline. In endotoxin-treated rats, R-PIA pretreatment (2.5 mg/kg) reduced serum TNF levels by 98%, with no modification of serum IL6 levels. TNF inhibition could be an important mechanism by which adenosine analogs exert their antiinflammatory action.

Eefects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells

Rank order of agonist potency for activation of adenylate cyclase by the naturally occurring prostanoids PGE 2, PGF 2α, PGD 2, the stable PGI 2 analogue iloprost, and the TXA 2 mimetic U 46619, provides evidence for the existence of a distinct PGE-receptor on guinea-pig duodenal enterocytes. This PGE-receptor is likely to be of the EP 2-subtype since the specific EP 2-agonist 11-deoxy-PGE 1 stimulated adenylate cyclase activity with a 20-fold higher potency than the EP 1-agonist 17-phenyltrinor-PGE 2 and the EP 3-agonists MB 28767 and GR 63799. In addition, sulprostone (acting on both EP 1- and EP 3-receptors) was ineffective. Since the specific EP 1-antagonist SC 19220 did not inhibit PGE 2-stimulated adenylate cyclase activity, the involvement of EP 1-receptors could be further excluded. The synthetic prostaglandin E-analogues misoprostol and nocloprost stimulated adenylate cyclase almost identically, though they were about 10-fold less potent than the natural PGE 2.

Adenosine A1‐receptor stimulation of inositol phospholipid hydrolysis and calcium mobilisation in DDT1 MF‐2 cells

1. The effect of adenosine receptor-stimulation on inositol phospholipid hydrolysis and calcium mobilization has been investigated in the hamster vas deferens smooth muscle cell line DDT1 MF-2. 2. Adenosine receptor stimulation increased the accumulation of total [3H]-inositol phosphates in DDT1 MF-2 cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was N6-cyclopentyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine greater than adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (KD 1.2 nM), PD 115,199 (KD 39 nM) and 8-phenyltheophylline (KD 31 nM). 4. The inositol phosphate response to 2-chloradenosine (10 microM) was not significantly altered when the extracellular Ca2+ ion concentration was reduced from 2.4 mM to 1.2 mM or 0.6 mM. Under calcium-free conditions, however, a reduced but still significant response to 2-chloroadenosine was evident (39 +/- 10% of the response in calcium-containing medium). 5. The 5-lipoxygenase inhibitor AA861 (10 and 100 microM) inhibited the inositol phosphate response to 2-chloroadenosine by 40 +/- 9% and 60 +/- 4% respectively. The cyclo-oxygenase inhibitor, indomethacin, however, was without significant effect at 1 microM. 6. 2-Chloroadenosine stimulated an increase in intracellular free Ca2+ ion concentration in fura-2 loaded DDT1 MF-2 cells in calcium-free medium containing 0.1 mM EGTA, which could be inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM). 7. These data suggest that adenosine A1-receptor stimulation results in inositol phospholipid hydrolysis and calcium mobilization from intracellular stores in DDT1 MF-2 cells.

Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells

Extracellular ATP and UTP caused increases in the concentration of cytoplasmic free calcium ([Ca 2+] i) and the intracellular level of inositol 1,4,5-trisphosphate (IP 3), a second messenger for calcium mobilization, prior to the release of prostacyclin (PGI 2) from cultured bovine pulmonary artery endothelial (BPAE) cells. The agonist specificity and dose-dependence were similar for nucleotide-mediated increases in IP 3 levels, [Ca 2+] i and PGI 2 release. An increase in [Ca 2+]; and PGI 2 release was observed after addition of ionomycin, a calcium ionophore, to BPAE cells incubated in a calcium-free medium. The addition of ATP to the ionomycin-treated cells caused no further increase in [Ca 2+] i or PGI 2 release. The inability of ATP to cause an increase in [Ca 2+] i or PGI 2 release in ionomycin-treated cells was apparently due to the ionomycin-dependent depletion of intracellular calcium stores since the subsequent addition of extracellular calcium caused a significant increase in both [Ca 2+] i and PGI 2 release. Introduction of BAPTA, a calcium buffer, into BPAE cells inhibited ATP-mediated increases in [Ca 2+] i and PGI 2 release, further evidence that PGI 2 release is dependent upon an increase in [Ca 2+] i. The increase in [Ca 2+] i elicited by ATP apparently caused the activation of a calmodulin-dependent phospholipase A 2 since trifluoperazine, an inhibitor of calmodulin, and quinacrine, an inhibitor of phospholipase A 2, prevented the stimulation of PGI 2 release by ATP. Furthermore, ATP caused the specific hydrolysis of [ 14C]arachidonyl-labeled phosphatidylcholine and the generation of free arachidonic acid, the rate-limiting substrate for PGI 2 synthesis, prior to the release of PGI 2 from BPAE cells. These findings suggest that the increase in PGI 2 release elicited by ATP and UTP is at least partially dependent upon a phospholipase C-mediated increase in [Ca 2+] i and the subsequent activation of a phosphatidylcholine-specific phospholipase A 2. ATP analogs modified in the adenine base or phosphate moiety caused PGI 2 release with a rank order of agonist potency of adenosine 5′- O-(2-thiodiphosphate) (ADPβS) > 2-methylthioATP (2-MeSATP) > ATP, whereas α, β methyleneATP and β, γ methyleneATP had no effect on PGI 2 release.