Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle

1. The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M₃ receptors in rabbit urinary bladder smooth muscle.
2. (+)-Cis-dioxolane induced concentration-dependent contractions of the iris sphincter muscle (pEC₅₀=6.41±0.10, E_max=181±17 mg, n=38) and urinary bladder smooth muscle (pEC₅₀=6.97±0.04, E_max=4.28±0.25 g, n=54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK_B values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30±0.07 and 9.40±0.04), AQ-RA 741 (6.35±0.04 and 6.88±0.03), darifenacin (9.56±0.05 and 9.12±0.05), methoctramine (5.75±0.07 and 5.81+0.06), oxybutynin (8.10±0.09 and 8.59±0.06), pirenzepine (6.79±0.05 and 6.89±0.04), secoverine (7.54±0.05 and 7.66±0.05), p-F-HHSiD (7.55±0.09 and 7.50±0.05) and zamifenacin (8.69±0.10 and 8.36±0.06). A significant correlation between the pK_B values in the bladder and the pK_B values in the iris was obtained.
3. In both tissues, the pK_B values correlated most favorably with pK_i values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors.
4. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically-defined muscarinic M₃ receptor.

     

Roles of M2 and M3 muscarinic receptors in cholinergic nerve-induced contractions in mouse ileum studied with receptor knockout mice

BACKGROUND AND PURPOSE: The functional roles of M(2) and M(3) muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve-induced contractions in the ileum using mutant mice lacking M(2) or M(3) receptor subtypes. EXPERIMENTAL APPROACH: Contractile responses to transmural electrical (TE) stimulation were isometrically recorded in ileal segments from M(2)-knockout (KO), M(3)-KO, M(2)/M(3)-double KO, and wild-type mice. KEY RESULTS: TE stimulation at 2-50 Hz frequency-dependently evoked a fast, brief contraction followed by a slower, longer one in wild-type, M(2)-KO or M(3)-KO mouse preparations. Tetrodotoxin blocked both the initial and later contractions, while atropine only inhibited the initial contractions. The initial cholinergic contractions were significantly greater in wild-type than M(2)-KO or M(3)-KO mice; the respective mean amplitudes at 50 Hz were 91, 74 and 68 % of 70mM K(+)-induced contraction. Pretreatment with pertussis toxin blocked the cholinergic contractions in M(3)-KO but not in M(2)-KO mice. Cholinergic contractions also remained in wild-type preparations, but their sizes were reduced by 20-30 % at 10-50 Hz. In M(2)/M(3)-double KO mice, TE stimulation evoked only slow, noncholinergic contractions, which were significantly greater in sizes than in any of the other three mouse strains. CONCLUSION AND IMPLICATIONS: These results demonstrate that M(2) and M(3) receptors participate in mediating cholinergic contractions in mouse ileum with the latter receptors assuming a greater role. Our data also suggest that the lack of both M(2) and M(3) receptors causes upregulation of noncholinergic excitatory innervation of the gut smooth muscle.     

Pharmacological characterization of pre- and postsynaptic prostanoid receptors in pig gastric fundus

This study characterized the subtype of prostanoid receptors on the cholinergic neurones and smooth muscle cells in circularly oriented muscle strips of the pig gastric fundus. Tissues were electrically stimulated (40 V, 4 Hz, 0.25 ms, 2 min) to induce tritium outflow after incubation with [3H]-choline. Indomethacin increased the electrically induced tritium outflow, suggesting an inhibitory effect of endogenous prostanoids. In the presence of indomethacin, PGE2 > PGF2alpha >PGI2 inhibited tritium release while the TP-receptor agonist U-46619 and PGD2 had no effect. The EP2-receptor agonist butaprost had no effect while the EP1- and EP3-receptor agonist sulprostone mimicked the effect of PGE2. The effect of sulprostone was not affected by AH 6809, that antagonizes EP1- and EP2-receptors, suggesting the presence of presynaptic EP3-receptors on the cholinergic nerve endings. All prostanoid receptor agonists, except butaprost, contracted the tissues concentration-dependently; the rank order of potency (U-46619 > sulprostone > PGE2 > PGF2alpha > PGD2 = PGI2) suggests the presence of TP- and EP1- and EP3-receptors on the circular smooth muscle cells.     

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

BACKGROUND AND PURPOSE

Despite the abundant expression of the UDP-sensitive P2Y₆ receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood.

EXPERIMENTAL APPROACH

We compared the effects of UDP and of the selective P2Y₆ receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [³H]-ACh overflow experiments.

KEY RESULTS

Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y₆ receptor antagonist, MRS2578. Effects mediated by urothelial P2Y₆ receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y₁ receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [³H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y₁ receptor antagonist, MRS2179. The P2Y₆ receptor agonist increased threefold ATP levels in the voided fluid.

CONCLUSIONS AND IMPLICATIONS

Activation of P2Y₆ receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y₁ receptors.     

Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle

1. The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), α,β-methylene-ATP (α,β-MeATP) and 2-methylthio-ATP (2-MeSATP) on longitudinally orientated smooth muscle strips from marmoset urinary bladder were investigated by use of standard organ bath techniques.
2. After being mounted in superfusion organ baths, 66.7% (n=249) of marmoset detrusor smooth muscle strips developed spontaneous tone, 48.2% of all strips examined developed tone equivalent to greater than 0.1 g mg⁻¹ of tissue and were subsequently utilized in the present investigation.
3. On exposure to ATP, muscle strips exhibited a biphasic response, a rapid and transient contraction followed by a more prolonged relaxation. Both responses were found to be concentration-dependent. ADP and 2-MeSATP elicited a similar response (contraction followed by relaxation), whereas application of α,β-MeATP only produced a contraction. The potency order for each effect was α,β-MeATP>>2-MeSATP⩾ATP>ADP (contractile response) and ATP=2-MeSATP⩾ADP>>α,β-MeATP (relaxational response).
4. Desensitization with α,β-MeATP (10 μM) abolished the contractile phase of the response to ATP, but had no effect on the level of relaxation evoked by this agonist. On the other hand, the G-protein inactivator, GDPβS (100 μM) abolished only the relaxation response to ATP. Suramin (general P2 antagonist, 100 μM) shifted both the contractile and relaxation ATP concentration-response curves to the right, whereas cibacron blue (P2Y antagonist, 10 μM) only antagonized the relaxation response to ATP. In contrast, the adenosine receptor antagonist, 8-phenyltheophylline (10 μM), had no effect on the relaxation response curve to ATP.
5. Incubation with tetrodotoxin (TTX, 3 μM) or depolarization of the muscle strip with 40 mM K⁺ Krebs failed to abolish the relaxation to ATP. In addition, neither N_ω-nitro-L-arginine (L-NOARG, 10 μM) nor methylene blue (10 μM) had any effect on the relaxation response curve. However, tos-phe-chloromethylketone (TPCK, 3 μM), an inhibitor of cyclicAMP-dependent protein kinase A (PKA), significantly (P<0.01) shifted the curve for the ATP-induced relaxation to the right.
6. It is proposed that marmoset detrusor smooth muscle contains two receptors for ATP, a classical P2X-type receptor mediating smooth muscle contraction, and a P2Y (G-protein linked) receptor mediating smooth muscle relaxation. The results also indicate that the ATP-evoked relaxation may occur through the activation of cyclicAMP-dependent PKA.

     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

Torcetrapib-induced blood pressure elevation is independent of CETP inhibition and is accompanied by increased circulating levels of aldosterone

BACKGROUND AND PURPOSE: Inhibition of cholesteryl ester transfer protein (CETP) with torcetrapib in humans increases plasma high density lipoprotein (HDL) cholesterol levels but is associated with increased blood pressure. In a phase 3 clinical study, evaluating the effects of torcetrapib in atherosclerosis, there was an excess of deaths and adverse cardiovascular events in patients taking torcetrapib. The studies reported herein sought to evaluate off-target effects of torcetrapib. EXPERIMENTAL APPROACH: Cardiovascular effects of the CETP inhibitors torcetrapib and anacetrapib were evaluated in animal models. KEY RESULTS: Torcetrapib evoked an acute increase in blood pressure in all species evaluated whereas no increase was observed with anacetrapib. The pressor effect of torcetrapib was not diminished in the presence of adrenoceptor, angiotensin II or endothelin receptor antagonists. Torcetrapib did not have a contractile effect on vascular smooth muscle suggesting its effects in vivo are via the release of a secondary mediator. Treatment with torcetrapib was associated with an increase in plasma levels of aldosterone and corticosterone and, in vitro, was shown to release aldosterone from adrenocortical cells. Increased adrenal steroid levels were not observed with anacetrapib. Inhibition of adrenal steroid synthesis did not inhibit the pressor response to torcetrapib whereas adrenalectomy prevented the ability of torcetrapib to increase blood pressure in rats. CONCLUSIONS AND IMPLICATIONS: Torcetrapib evoked an acute increase in blood pressure and an acute increase in plasma adrenal steroids. The acute pressor response to torcetrapib was not mediated by adrenal steroids but was dependent on intact adrenal glands.     

Subclassification of muscarinic receptors in the heart,urinary bladder and sympathetic ganglia in the pithed rat

Summary In pithed normotensive rats muscarinic receptors were characterized heart, urinary bladder and sympathetic ganglia; the selectivity of some classical muscarinic agents for these subtypes was investigated. The potencies in decreasing heart rate, increasing bladder pressure and increasing diastolic blood pressure were measured for the following, intraarterially administered cholinergic agonists: McN-A-343 ([4-m-chlorophenylcarbamoyloxy]-2-butynyltrimethylammonium), pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine. The selective M₁-antagonist pirenzepine, the mixed M₁/M₂-antagonist dexetimide and the cardioselective M₂-antagonist gallamine were used as tools for identification of the receptors. All data were obtained after intravenous pretreatment with a high dose of atenolol to eliminate tachycardia induced by stimulating sympathetic ganglionic muscarinic receptors.Dexctimide strongly antagonized the bradycardia as well as the increase in bladder pressure induced by pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine, whereas pirenzepine was much less effective. Gallamine antagonized the bradycardia, whereas no influence was found on the bladder contraction. Pilocarpine acted as a partial agonist in reducing heart rate as well as in increasing bladder pressure, whereas McN-A-343 was almost ineffective in doses up to 1 mg/kg.The hypertensive response to pilocarpine and carbachol was less pronounced than that produced by McN-A-343. Pirenzepine and dexetimide significantly antagonized the hypertensive response to McN-A-343 and pilocarpine, whereas gallamine was much less effective. The hypertensive response induced by carbachol was totally blocked by hexamethonium. The other agonists used in this study did not produce a significant increase in diastolic blood pressure in doses that produced a maximal effect on heart rate and urinary bladder pressure.Simultaneously, intraarterially infused acetylcholine dose-dependently and reversibly decreased the pressor response to intravenously administered McN-A-343.These data suggest that muscarinic receptors in rat sympathetic ganglia belong to the M₁-subtype, whereas the muscarinic receptors in rat heart and urinary bladder represent heterogenous populations of M₂-receptors. The agonists used in this study, though, could not discriminate between these heterogenous M₂-receptors.Like McN-A-343, pilocarpine appears to be a rather selective M₁-agonist. In this study the M₁/M₂ selectivity of muscarinic agents with pronounced M₂-agonist activity could not be evaluated since M₂-receptor stimulation interferes with the hypertensive response to M₁-receptor stimulation.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca concentrations in human airway smooth muscle cells

Increased airway smooth muscle mass due to cell proliferation contributes to airway hyper-responsiveness and remodeling in patients with asthma. Prostaglandin E₂ (PGE₂) inhibits proliferation of airway smooth muscle cells, but the role of prostanoid EP receptor subtypes in mechanisms involved has not been fully elucidated yet. We investigated the effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in human airway smooth muscle cells. Cell numbers were assessed by mitochondria-dependent reduction of 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate to formazan (WST-1 assay). RT-PCR data showed that human airway smooth muscle cells express EP2, EP3, and EP4 but not EP1 receptor mRNA. PGE₂ (1 nM–1 μM) inhibited cell proliferation induced by 5% fetal bovine serum (FBS) in a concentration-dependent manner. (16S)-9-deoxy-9β-chloro-15-deoxy-16-hydroxy-17, 17-trimethylene-19, 20-didehydro PGE₂ sodium salt (ONO-AE1-259-01; EP2 receptor agonist) and 16-(3-methoxymethyl)phenyl-ω-tetranor-3,7-dithia PGE₂ (ONO-AE1-329; EP4 receptor agonist) inhibited the 5% FBS-induced cell proliferation. ONO-AE1-259-01 and ONO-AE1-329 also significantly increased the cytosolic cAMP levels. In contrast, 11,15-O-dimethyl PGE₂ (ONO-AE-248; EP3 receptor agonist) elicited an oscillatory increase in [Ca²⁺]_i but did not affect the cell growth or cAMP levels. [(17S)-2,5-ethano-6-oxo-17,20-dimethyl PGE₁] (ONO-DI-004; EP1 receptor agonist) did not affect cell growth, cAMP levels, or [Ca²⁺]_i. In conclusion, PGE₂ inhibits FBS-induced cell proliferation mostly via EP2 and EP4 receptor activation and subsequent cAMP elevation. The EP3 receptor agonist causes an increase in [Ca²⁺]_i without affecting cell growth. There is no functional expression of the EP1 receptor. Research on prostanoid EP receptors may lead to novel therapeutic strategies for treatment of asthma.     

Characterization of the recombinant human prostanoid DP receptor and identification of L-644,698, a novel selective DP agonist

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D₂ (PGD₂) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3′:5′-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway.
2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (K_i) of 0.9 nM. This K_i value was comparable to the K_i values obtained in this study for ligands that have previously shown high affinity for DP: PGD₂ (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM).
3. L-644,698 was found to be a full agonist with an EC₅₀ value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC₅₀): PGD₂ (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM).
4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP₁ (>25,400 fold), EP₂ (∼300 fold), EP_3-III (∼4100 fold), EP₄ (∼10000 fold), FP (>25,400 fold), IP (>25,400 fold) and TP (>25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described.
5. PGJ₂ and Δ¹²-PGJ₂, two endogenous metabolites of PGD₂, were also tested in this system and shown to be effective agonists with K_i and EC₅₀ values in the nanomolar range for both compounds. In particular, PGJ₂ was equipotent to known DP specific agonists with a K_i value of 0.9 nM and an EC₅₀ value of 1.2 nM.

     

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.     

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose

5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck.

Experimental approach

Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS).

Key results

After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (±)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > α-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT_1A and 5-HT_1A/1B receptor antagonists respectively. Inhibitors of 5-HT_1B/1D, 5-HT₂, 5-HT_2B/2C, 5-HT₃, 5-HT₄, 5-HT_5A and 5-HT₆ receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, α-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca²⁺-activated K⁺ and ATP-dependent K⁺ channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na⁺-, Ca²⁺-and voltage-gated K⁺ (K_v)-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol.

Conclusions and implications

5-HT relaxed the pig urinary bladder neck through muscle 5-HT₇ receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT_1A receptors and K_v channels modulated 5-HT-induced relaxations whereas postjunctional K⁺ channels were not involved in such responses. 5-HT₇ receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.     

Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines

Background and purpose:

Prostaglandin (PG) E₂ and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE₂ signals principally through prostanoid receptors of the EP₂ and/or EP₄ subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP₂ and EP₄ receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE₂-induced IL-8 production.

Experimental approach:

To identify the causative receptor, we knocked-down and over-expressed EP₂ and EP₄ receptor subtypes in colonic epithelial cells and studied the effect of several selective EP₂/EP₄ receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE₂ and Bmax values for the EP₂ and EP₄ receptor on colonic epithelial cells were determined by radioligand-binding assays with [³H]PGE₂.

Key results:

PGE₂ had the highest affinity for the EP₄ receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP₄ receptor agonist, ONO-AE1-329, and abolished by silencing the EP₄ receptor gene by using siRNA techniques, a selective EP₄ receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.

Conclusions and implications:

These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE₂ acting via the EP₄ receptor subtype.     

Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations

BACKGROUND AND PURPOSE

The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP₃ receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated.

EXPERIMENTAL APPROACH

Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted.

KEY RESULTS

L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP₃ antagonists. For ligands of low to moderate lipophilicity, potency increments for EP₃ and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC₅₀ of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H₁ antagonism above a pA₂ limit of 8.0. In contrast, L-798106 (EP₃), L-826266 (EP₃, TP) and the lipophilic H₁ antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP₃) had a faster onset. Agonism and antagonism on the vas deferens EP₃ system were overall much faster, although trends were similar.

CONCLUSIONS AND IMPLICATIONS

High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP₃ antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP₃ antagonists is discussed.     

5-Hydroxytryptamine potentiates neurogenic contractions of rat isolated urinary bladder through both 5-HT(7) and 5-HT(2C) receptors

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC₅₀ value of 6.86 ± 0.24. SB-269970 (0.01, 0.1 and 1 μM), a selective 5-HT₇ receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA₂ value was 8.16 with a slope of 0.46 ± 0.08. Neither ketanserine nor SB-204741, 5-HT_2A and 5-HT_2B receptors antagonists, respectively, affected the concentration–response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT_2C receptor antagonist SB-242084 (0.1 and 1 μM). In the presence of 1 μM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT₇ receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA₂ value of 8.77 and a slope not significantly different from unity (0.91 ± 0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT_2C receptor agonist (0.01–10 μM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT_2A agonist) had no effect. SB-242084 (0.1 and 1 μM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT₇ and 5-HT_2c receptors.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.     

Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck

Background and purpose:

As adenosine 5′-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck.

Experimental approach:

Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O₂ and 5% CO₂ for isometric force recordings.

Key results:

EFS, in the presence of atropine, guanethidine and N^G-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5′-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5′-triphosphate, uridine 5′-diphosphate and α,β-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y₁ and P1 receptor blockers 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist''s potency was: 5′-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N⁶-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A_2A antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na⁺ channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X₁ and P2X₃ receptors.

Conclusions and implications:

ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A_2A receptors after breakdown to adenosine, and P2Y₁ receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency.     

Pharmacological characterization and expression of VIP and PACAP receptors in isolated cranial arteries of the rat

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) are potent vasodilators in animals and humans. PACAP infusion but not VIP infusion precipitates migraine attacks in migraine patients. The vascular effects of VIP and the two varieties of PACAP (PACAP-27 and PACAP-38) were investigated versus selective antagonists in segments of rat middle cerebral arteries (MCA), basilar arteries (BA) and middle meningeal arteries (MMA) using myographs. The luminal and abluminal effects of VIP were studied using perfusion myograph. mRNA expression of the relevant receptors (VPAC₁, VPAC₂ and PAC₁) was examined by in situ hybridization. There was no significant difference in relaxant potency of the peptides in the MCA. In BA the relaxant potency was VIP > PACAP-27 = PACAP-38. Relaxant responses were either absent or very weak in MMA. VIP was found to be somewhat more potent in BA than in the MCA. Maxadilan, a selective PAC₁-receptor agonist, showed no relaxant effect in either vessel. The VPAC₂-antagonist PG 99-465 alone proved ineffective in the MCA, while it had a weak effect on BA. The VPAC₁-antagonist PG 97-269 inhibited relaxation induced by both VIP and the PACAPs in cerebral vessels. In combination, the two antagonists demonstrated better effect than either alone. VIP applied luminally via perfusion myograph caused no dilatation, indicating lack of endothelial involvement. In situ hybridization demonstrated the presence of mRNA for all three receptors in the smooth muscle cells of the vessels. In conclusion, migraine-like headache induced by PACAP-38 infusion is unlikely to be caused by direct vasodilator action on intracranial vessels.