In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties.

BACKGROUND AND PURPOSE: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H4 receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine). EXPERIMENTAL APPROACH: We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis. KEY RESULTS: A-940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice. CONCLUSIONS AND IMPLICATIONS: These data suggest that A-940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.

Use of the H3 receptor antagonist radioligand [3H]-A-349821 to reveal in vivo receptor occupancy of cognition enhancing H3 receptor antagonists.

BACKGROUND AND PURPOSE: The histamine H3 receptor antagonist radioligand [3H]-A-349821 was characterized as a radiotracer for assessing in vivo receptor occupancy by H3 receptor antagonists that affect behaviour. This model was established as an alternative to ex vivo binding methods, for relating antagonist H3 receptor occupancy to blood levels and efficacy in preclinical models. EXPERIMENTAL APPROACH: In vivo cerebral cortical H3 receptor occupancy by [3H]-A-349821 was determined in rats from differences in [3H]-A-349821 levels in the isolated cortex and cerebellum, a brain region with low levels of H3 receptors. Comparisons were made to relate antagonist H3 receptor occupancy to blood levels and efficacy in a preclinical model of cognition, the five-trial inhibitory avoidance response in rat pups. KEY RESULTS: In adult rats, [3H]-A-349821, 1.5 microg x kg(-1), penetrated into the brain and cleared more rapidly from cerebellum than cortex; optimally, [3H]-A-349821 levels were twofold higher in the latter. With increasing [3H]-A-349821 doses, cortical H3 receptor occupancy was saturable with a binding capacity consistent with in vitro binding in cortex membranes. In studies using tracer [3H]-A-349821 doses, ABT-239 and other H3 receptor antagonists inhibited H3 receptor occupancy by [3H]-A-349821 in a dose-dependent manner. Blood levels of the antagonists corresponding to H3 receptor occupancy were consistent with blood levels associated with efficacy in the five-trial inhibitory avoidance response. CONCLUSIONS AND IMPLICATIONS: When employed as an occupancy radiotracer, [3H]-A-349821 provided valid measurements of in vivo H3 receptor occupancy, which may be helpful in guiding and interpreting clinical studies of H3 receptor antagonists.

The histamine H3 receptor: an attractive target for the treatment of cognitive disorders.

The histamine H3 receptor, first described in 1983 as a histamine autoreceptor and later shown to also function as a heteroreceptor that regulates the release of other neurotransmitters, has been the focus of research by numerous laboratories as it represents an attractive drug target for a number of indications including cognition. The purpose of this review is to acquaint the reader with the current understanding of H3 receptor localization and function as a modulator of neurotransmitter release and its effects on cognitive processes, as well as to provide an update on selected H3 antagonists in various states of preclinical and clinical advancement. Blockade of centrally localized H3 receptors by selective H3 receptor antagonists has been shown to enhance the release of neurotransmitters such as histamine, ACh, dopamine and norepinephrine, among others, which play important roles in cognitive processes. The cognitive-enhancing effects of H3 antagonists across multiple cognitive domains in a wide number of preclinical cognition models also bolster confidence in this therapeutic approach for the treatment of attention deficit hyperactivity disorder, Alzheimer's disease and schizophrenia. However, although a number of clinical studies examining the efficacy of H3 receptor antagonists for a variety of cognitive disorders are currently underway, no clinical proof of concept for an H3 receptor antagonist has been reported to date. The discovery of effective H3 antagonists as therapeutic agents for the novel treatment of cognitive disorders will only be accomplished through continued research efforts that further our insights into the functions of the H3 receptor.

Characterization of a novel ATP-sensitive K+ channel opener, A-251179, on urinary bladder relaxation and cystometric parameters.

BACKGROUND AND PURPOSE: ATP-sensitive K(+) channels (K(ATP)) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4-methyl-N-(2,2,2-trichloro-1-(3-pyridin-3-ylthioureido)ethyl)benzamide (A-251179) as a K(ATP) channel opener. EXPERIMENTAL APPROACH: Glyburide-sensitive membrane potential, patch clamp and tension assays were employed to study the effect of A-251179 in vitro. The in vivo efficacy of A-251179 was characterized by suppression of spontaneous contractions in obstructed rat bladder and by measuring urodynamic function of urethane-anesthetized rat models. KEY RESULTS: A-251179 was about 4-fold more selective in activating SUR2B-Kir6.2 derived K(ATP) channels compared to those derived from SUR2A-Kir6.2. In pig bladder smooth muscle strips, A-251179 suppressed spontaneous contractions, about 27- and 71-fold more potently compared to suppression of contractions evoked by low-frequency electrical stimulation and carbachol, respectively. In vivo, A-251179 suppressed spontaneous non-voiding bladder contractions from partial outlet-obstructed rats. Interestingly, in the neurogenic model where isovolumetric contractions were measured by continuous transvesical cystometry, A-251179 at a dose of 0.3 micromol kg(-1), but not higher, was found to increase bladder capacity without affecting either the voiding efficiency or changes in mean arterial blood pressure. CONCLUSIONS AND IMPLICATIONS: The thioureabenzamide analog, A-251179 is a potent novel K(ATP) channel opener with selectivity for SUR2B/Kir6.2 containing K(ATP) channels relative to pinacidil. The pharmacological profile of A-251179 is to increase bladder capacity and to prolong the time between voids without affecting voiding efficiency and represents an interesting characteristic to be explored for further investigations of K(ATP) channel openers for the treatment of overactive bladder.

Blockade of mGluR1 receptor results in analgesia and disruption of motor and cognitive performances: effects of A-841720, a novel non-competitive mGluR1 receptor antagonist.

BACKGROUND AND PURPOSE: To further assess the clinical potential of the blockade of metabotropic glutamate receptors (mGluR1) for the treatment of pain. EXPERIMENTAL APPROACH: We characterized the effects of A-841720, a novel, potent and non-competitive mGluR1 antagonist in models of pain and of motor and cognitive function. KEY RESULTS: At recombinant human and native rat mGluR1 receptors, A-841720 inhibited agonist-induced calcium mobilization, with IC50 values of 10.7+/-3.9 and 1.0 +/- 0.2 nM, respectively, while showing selectivity over other mGluR receptors, in addition to other neurotransmitter receptors, ion channels, and transporters. Intraperitoneal injection of A-841720 potently reduced complete Freund's adjuvant-induced inflammatory pain (ED50 = 23 micromol kg(-1)) and monoiodoacetate-induced joint pain (ED50 = 43 micromol kg(-1)). A-841720 also decreased mechanical allodynia observed in both the sciatic nerve chronic constriction injury and L5-L6 spinal nerve ligation (SNL) models of neuropathic pain (ED50 = 28 and 27 micromol kg(-1), respectively). Electrophysiological studies demonstrated that systemic administration of A-841720 in SNL animals significantly reduced evoked firing in spinal wide dynamic range neurons. Significant motor side effects were observed at analgesic doses and A-841720 also impaired cognitive function in the Y-maze and the Water Maze tests. CONCLUSIONS AND IMPLICATIONS: The analgesic effects of a selective mGluR1 receptor antagonist are associated with motor and cognitive side effects. The lack of separation between efficacy and side effects in pre-clinical models indicates that mGluR1 antagonism may not provide an adequate therapeutic window for the development of such antagonists as novel analgesic agents in humans.

In vivo evaluation of the potency and bladder-vascular selectivity of the ATP-sensitive potassium channel openers (-)-cromakalim, ZD6169 and WAY-133537 in rats.

OBJECTIVE: To compare in vivo the potency and bladder-vascular selectivity of ATP-sensitive potassium channel openers (KCOs) (-)-cromakalim, WAY-133537 and ZD6169 and a muscarinic antagonist, tolterodine in rats. MATERIALS AND METHODS: Bladder and arterial pressures were monitored simultaneously, before and after increasing intravenous doses of compounds, in each of two urethane-anaesthetized rat bladder hyperactivity models: spontaneous non-voiding myogenic contractions secondary to partial outlet obstruction and volume-induced neurogenic contractions. RESULTS: (-)-Cromakalim, WAY-133537 and ZD6169 caused a dose-dependent suppression of spontaneous contractions in the obstructed model, with a 50% inhibition of the contraction area under the curve at doses of 0.06, 0.14 and 2.4 micro mol/kg (intravenous), respectively. Corresponding decreases in mean arterial pressure at these effective doses were 24%, 15% and 15%, respectively. The KCO potency rank order was the same and their relative potency highly comparable in the neurogenic model. There was complete inhibition of spontaneous contractions in obstructed rats at doses corresponding to approximately 50% inhibition of the neurogenic contractions. While tolterodine caused a dose-dependent inhibition of contractions in the neurogenic model, it was ineffective at inhibiting non-voiding contractions in obstructed rats. CONCLUSIONS: All KCOs tested caused significant decreases in arterial pressure at doses effective on the bladder in the model of obstructive instability, suggesting a lack of bladder-vascular selectivity. Similar KCO potency in both assays suggests no appreciable changes in KATP channel function as a result of partial outlet obstruction.

Activation of soluble guanylate cyclase causes relaxation of corpus cavernosum tissue: synergism of nitric oxide and YC-1.

Nitric oxide (NO) activates corpus cavernosum smooth muscle soluble guanylate cyclase (sGC) and increases the synthesis of cGMP that results in smooth muscle relaxation and ultimately, penile erection. To characterize sGC and define the potential synergy between NO and the allosteric activator YC-1 in corpus cavernosum, rat sGC was activated by either sodium nitroprusside (SNP) or YC-1, and YC-1 potentiated the effects of SNP with a 200-fold activation of sGC. Both SNP and YC-1 decreased the Km and increased the Vmax. ODQ significantly inhibited sGC activated by SNP with IC50 of 0.5 nM, but did not affect the sGC activated by YC-1 as well as basal sGC activity. SNP and YC-1 synergistically increased intracellular cGMP levels in rabbit corpus cavernosum smooth muscle cell cultures. YC-1 significantly relaxed rabbit cavernosum tissue strips in organ baths with an EC50 of 8.4 microM. In the presence of L-nitroarginine methyl ester to block endogenous NO production, co-administration of SNP shifted the dose response of YC-1 to the left, showing the synergism of SNP and YC-1 in tissue strips. In view of the clinical efficacy of phosphodiesterase-5 inhibitors, activation of sGC may provide an alternative means for enhancing the activity of neurally derived NO during sexual stimulation in the corpus cavernosum, representing a novel approach for the treatment of erectile dysfunction.

Nitric oxide independent activation of guanylate cyclase by YC-1 causes erectile responses in the rat.

PURPOSE: Activation of soluble guanylate cyclase with a subsequent increase in intracellular levels of cyclic guanosine monophosphate is necessary for normal erection. In vascular tissue 3(5'-hydroxymethyl-2'-furyl-1-benzyl indazole (YC-1) (Abbott Laboratories, North Chicago, Illinois) has been shown to stimulate soluble guanylate cyclase independent of nitric oxide. We studied whether YC-1 modulates erectile responses in the rat. MATERIALS AND METHODS: The effects of YC-1 given intracavernously or intraperitoneally on intracavernous pressure were investigated in rats. Functional effects of YC-1 on neuronal and endothelial nitric oxide relaxations were studied in 3 x 10(-6) M. 1-noradrenaline contracted preparations of rat isolated corpus cavernosum. RESULTS: Intracavernous YC-1 (10 micromol. kg.-1) produced erectile responses with a mean intracavernous pressure plus or minus standard error of mean of 81 +/- 17 cm. water (p <0.001) and a mean duration of 7.1 +/- 3.3 minutes (p <0.001). YC-1 (10 micromol. kg.-1) given intraperitoneally also increased the amplitude and duration of erectile responses to cavernous nerve stimulation. Mean peak intracavernous pressure increased from 63 +/- 6 to 10(2) +/- 16 cm. water (p <0.05). Erections induced by a submaximal dose of 25 microg. kg.-1 apomorphine s.c. increased in number after 10 micromol. kg.-1 YC-1 intraperitoneally (p <0.05). In vitro nerve induced relaxant responses were enhanced by increasing concentrations of YC-1. Relaxations at 20 Hz. were increased from a mean of 9% +/- 5% to 52% +/- 5% at a YC-1 concentration of 10(-5) M. (p <0.001). At this concentration carbachol induced relaxations were enhanced from a mean of 19% +/- 3% to 40% +/- 9% (p <0.05). CONCLUSIONS: YC-1 can evoke erectile responses when given intracavernously and it enhances erections induced by cavernous nerve stimulation and apomorphine when given systemically. In vitro YC-1 enhances electrically evoked relaxations in rat corpus cavernosum. YC-1 represents an interesting pharmacological principle that may be useful for treating erectile dysfunction.

Activators of soluble guanylate cyclase for the treatment of male erectile dysfunction.

Soluble guanylate cyclase (sGC) is an important enzyme in corpus cavernosum smooth muscle cells as it is one of the regulators of the synthesis of cGMP. The efficacy of sildenafil (Viagra) in the treatment of male erectile dysfunction indicates the importance of the cGMP system in the erectile response as the increased levels of cGMP induce relaxation of the corpus cavernosum. sGC is physiologically activated by nitric oxide (NO) during sexual stimulation, and its activity can be pharmacologically enhanced by several NO-donors. Agents like YC-1 can also activate sGC after binding to a novel allosteric site in the enzyme, a site different from the NO binding site. YC-1 can relax rabbit cavernosal tissue and it facilitates penile erection in vivo. This review summarizes the enzymology, biochemistry and pharmacology of this novel allosteric site and its relevance for the regulation of penile function. This type of sGC activators represent a new class of compounds with a different pharmacological profile in comparison to the classical NO-donors and they could be beneficial for the treatment of male erectile dysfunction.

Comparison of alpha 1-adrenoceptor agonists in canine urethral pressure profilometry and abdominal leak point pressure models.

PURPOSE: We describe and compare the usefulness of 3 minimally invasive dog urethral function models to demonstrate the efficacy potential of alpha 1 agonists for stress urinary incontinence. From this overall composite dataset the efficacy profiles of the alpha 1A selective agonist A-61603 and the active metabolite of midodrine ST-1059 were specifically compared. MATERIALS AND METHODS: Isoflurane anesthetized multiparous female beagle dogs were used in all studies. Intraurethral pressure was measured using a 7Fr balloon catheter. Profilometry was performed using an 8Fr Millar transducer catheter. Bladder pressure required to produce leakage in response to external abdominal ballottements of increasing intensity was measured using a 5Fr transurethral catheter. Agonist responses were measured before and after increasing cumulative intravenous doses of each compound in each test. RESULTS: Agonist induced increases in maximal urethral closure and leak point pressure strongly correlated in linear fashion (R(2) = 0.94), as did measurements of agonist induced increases in proximal intraurethral pressure using the microtransducer or balloon catheter technique (R(2) = 0.87). A dose of 0.01 to 1 nmol./kg. A-61603 and 10 to 1,000 nmol./kg. ST-1059 intravenously each caused dose dependent increases in maximum urethral closure, leak point and intraurethral pressure. CONCLUSIONS: While the dose range for which alpha 1 agonists affect urethral pressure was adequately predicted by any of the 3 methods used, the leak point pressure assay described has the advantage of being a dynamic test that directly evaluates efficacy to protect against leakage caused by increases in abdominal pressure. This leak point pressure test appears be useful for the preclinical evaluation of compounds used to treat stress urinary incontinence.

Dual mechanism of action of nicorandil on rabbit corpus cavernosal smooth muscle tone.

The potential of ATP-sensitive potassium channel openers (KCOs) for the treatment of male erectile dysfunction has recently been suggested based on positive clinical outcomes following intra-cavernosal administration of pinacidil. Agents that increase the levels of cGMP via elevation of nitric oxide (NO) nitroglycerin, for example, are also effective in improving erectile function preclinically and clinically. The aim of the present study was to determine the effects and mechanism of the action of nicorandil on rabbit corpus cavernosum. The in vitro regulation of smooth muscle tone was assessed in isolated cavernosal tissues pre-contracted with phenylephrine. Nicorandil, but not its major metabolite, relaxed phenylephrine-precontracted cavernosum smooth muscle with an EC(50) of 15 microM. The effects of nicorandil were only partially reversed by the K(ATP) channel blocker glyburide (10 microM) or by a soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (ODQ, 3 microM). However, a combination of ODQ and glyburide completely blocked the relaxant effects of nicorandil. The results of the present study indicate that nicorandil can relax rabbit cavernosal tissue in vitro via a mechanism that involves activation of K(ATP) channels and stimulation of soluble guanylate cyclase.

Functional implication of spare ATP-sensitive K(+) channels in bladder smooth muscle cells.

ATP-sensitive K(+) (K(ATP)) channels play important roles in the regulation of excitability in urinary bladder smooth muscle cells. Patch-clamp studies revealed that the current density was about 9-fold higher in the pig bladder smooth muscle cells, compared with guinea pig, although the rank order of potencies for suppression of electrical field-stimulated contraction of bladder strips by K(ATP) channel openers (KCOs) showed a nearly 1:1 correlation between pig and guinea pig. To investigate the existence of spare K(ATP) channels, P1075-evoked current and membrane potential responses were studied in bladder smooth muscle cells. During a 10-min exposure to P1075 (10 microM), K(ATP) currents ran down by approximately 30.5%, whereas membrane hyperpolarization remained constant. P1075 evoked membrane hyperpolarization with an EC(50) value of 0.20 +/- 0.02 microM, comparable to that required for smooth muscle relaxation (EC(50) = 0.11 +/- 0.01 microM). However, these potencies are 6-fold higher than those required for current activation (EC(50) = 0.73 +/- 0.4 microM). These findings demonstrate that the reduction in membrane excitability by KCOs is associated with membrane hyperpolarization, and that a low amount of K(ATP) channel opening is sufficient to suppress bladder smooth muscle contraction.

A-315456: a selective alpha(1D)-adrenoceptor antagonist with minimal dopamine D(2) and 5-HT(1A) receptor affinity.

In functional assays, A-315456, N-[3-(cyclohexylidene-(1H-imidazol-4-ylmethyl))phenyl]ethanesulfonamide, behaved as an alpha(1D)-adrenoceptor subtype selective antagonist (pA(2)=8.34) in the rat aorta. It was 83-fold less potent at the alpha(1B)-adrenoceptor subtype expressed in the rat spleen, and was inactive at the alpha(1A)-adrenoceptor subtype expressed in the rat vas deferens. Radioligand binding assays also revealed high affinity (pK(i)=8.71) for the alpha(1D)-adrenoceptor subtype and weaker affinities at the alpha(1A)-adrenoceptor (pK(i)=6.23) and alpha(1B)-adrenoceptor (pK(i)=7.86). In comparison to its potent affinity at the alpha(1D)-adrenoceptor subtype, A-315456 was 3020-, 794- and 38-fold weaker at the dopamine D(2)-, 5-HT(1A)-, and alpha(2a)-adrenoceptors, respectively. These studies indicate that A-315456 is a potent and selective alpha(1D)-antagonist that may serve as a useful pharmacological ligand to probe the physiological role of the alpha(1D)-adrenoceptor subtype in normal and disease states.

Pharmacological properties of A-204176, a novel and selective alpha1A adrenergic agonist, in in vitro and in vivo models of urethral function.

A-204176 (N-[5-(1H-imidazol-4-y1)-5,6,7,8-tetrahydro-1-naphthalenyl]methanesulfonamide) is a potent and selective alpha1A adrenoceptor agonist that binds with 17-fold and 9-fold greater affinity to the alpha1A (Ki=176 nM) than the alpha1b and alpha1d subtypes, respectively. In functional studies A-204176 is potent (pD2=6.4) and efficacious (83% of maximum control phenylephrine response) at rabbit urethra alpha1A receptors, with weaker potency and greatly reduced efficacy at rat spleen alpha1B (pD2=5.3, 11%) and rat aorta alpha1D (pD2=4.4, 10%) subtypes. In anesthetized female dogs, A-204176 is more potent than the non-selective alpha1 adrenoceptor agonist phenylpropanolamine (PPA) to increase measures of urethral tone and is more efficacious to increase pressure in the proximal region of the urethra. Significant increases on parameters of the urethral pressure profilometry were induced at 100 and 300 nmol/kg, i.v., by A-204176 and PPA, respectively. A-204176 was more potent than PPA to increase the abdominal pressure required to produce leakage. In the simultaneous measurement of intraurethral pressure and mean arterial blood pressure, A-204176 displays enhanced urethral selectivity relative to PPA. However, despite its selectivity for alpha1A versus alpha1B and alpha1D adrenoceptors in vitro, A-204176 did not display the degree of urethral selectivity in vivo that would have been expected. The observed effect of A-204176 on blood pressure may be due to the presence of extra-synaptic alpha1A adrenoceptors in the vasculature or to activation of spinal and supraspinal alpha1A adrenoceptors. These data indicate that A-204176 may represent a useful pharmacological tool to investigate the functional role of the alpha1A adrenoceptor in the urethra and to elucidate the lack of uroselectivity observed in vivo.

Molecular characterization of human SUR2-containing K(ATP) channels.

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone. Quantification of RNA for SUR2 exon 17+ and SUR2 exon 17- splice variants using real-time Taqman PCR indicated differential levels of expression in brain, kidney, skeletal muscle, heart and small intestine. Interestingly, the SUR2 exon 17+ variant is the major species expressed in all tissues examined in this study. Each of the SUR2 splice variants transiently expressed with the inward rectifier Kir 6.2 formed functional K(ATP) channels in HEK 293 cells as assessed either by changes in DiBAC(4)(3) fluorescence responses or glyburide-sensitive whole cell currents. Collectively, our findings demonstrate that various SUR2 splice variants have distinct expression patterns and can form functional K(ATP) channels.

Pharmacological and molecular analysis of ATP-sensitive K(+) channels in the pig and human detrusor.

The pharmacological and molecular properties of ATP-sensitive K(+) channels present in pig detrusor smooth muscle were investigated. In isolated pig detrusor strips, ATP-sensitive K(+) channel openers inhibited contractions elicited by low frequency field-stimulation in a concentration-dependent manner. The inhibitory effects of P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine] were attenuated by glyburide with a pA(2) value of 7.38 (slope=1.08). The potency of the inhibitory effects of the K(+) channel openers on the field-stimulated contractions correlated well with those evoked by the muscarinic receptor agonist, carbachol (r=0.93) and furthermore, to relaxation of the pre-contracted (25 mM potassium chloride, KCl) human detrusor (r=0.95). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed the presence of mRNA for sulfonylurea receptors SUR1 and SUR2B in both pig and human detrusor. Considering the similarities in the molecular and pharmacological profile of ATP-sensitive K(+) channels between the pig and the human detrusor, it is concluded that the pig detrusor may serve as a suitable in vitro model for the evaluation of novel K(+) channel openers with potential use in urological disorders in humans.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.

Characterization and modulation of [125I]iberiotoxin-D19Y/Y36F binding in the guinea-pig urinary bladder.

The radioligand binding characteristics of the Ca(2+)-activated K(+) channel ligand [125I]iberiotoxin-D19Y/Y36F were examined in guinea-pig urinary bladder membranes. Saturation analysis revealed a single class of high affinity binding sites in the bladder with a K(D) value of 45.6 pM and a B(max) value of 112 fmol/mg protein. Specific binding was displaced by unlabeled iberiotoxin and penitrem A, but not by blockers of other classes of K(+) channels including alpha-dendrotoxin, margatoxin and apamin. The indole alkaloids, paxilline and verruculogen, significantly increased binding by 4.5- and 4.3-fold, respectively. Tetraacetic acid derivatives such as ethylenediamine tetraacetic acid and ethyleneglycoltetraacetic acid enhanced specific [125I]iberiotoxin-D19Y/Y36F binding about 2.5-fold, which was not attributable to calcium chelation. This increase was due to a significant change in ligand binding affinity (K(D)=6.3 pM), but not due to a change in the B(max), indicating that these compounds may enhance toxin binding via allosteric interactions. Collectively, these results demonstrate that the binding sites for [125I]iberiotoxin-D19Y/Y36F present in the urinary bladder shows a pharmacological profile typical of maxi-K(+) channels and can be modulated, not only by previously known indole alkaloids, but also by tetraacetic acid analogs.