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.

Use of an inverse agonist radioligand [3H]A-317920 reveals distinct pharmacological profiles of the rat histamine H3 receptor.

Selective radioligands for histamine H(3) receptors have been used to characterize H(3) receptor pharmacology by radioligand binding assays and to determine H(3) receptor distribution by tissue autoradiography. Here we report the synthesis and receptor binding characterization of [(3)H]A-317920 (furan-2-carboxylic acid(2-[4-[3-([3,5-(3)H]4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-1-methyl-2-oxo-ethyl)-amide), a high affinity inverse agonist radioligand for the rat H(3) receptor. The binding of [(3)H]A-317920 to rat cortical and cloned H(3) receptors revealed fast on- and slower off-rate kinetics with calculated K(d) values in agreement with those determined in saturation binding assays (0.2 nM for both receptors). Further, we compared [(3)H]A-317920 with the agonist [(3)H](N)-alpha-methylhistamine ([(3)H]NalphaMH) as radioligand tools to study receptor pharmacology. Agonists and antagonists displaced [(3)H]NalphaMH with one-site binding characteristics and Hill slopes approached unity. In contrast, although antagonists exhibited one-site binding, [(3)H]A-317920 displacement by agonists was best fit by two-site binding models, and the potencies of the high affinity, GDP-sensitive sites correlated with the potencies defined in [(3)H]NalphaMH binding. Unlike [(125)I]iodoproxyfan, [(3)H]A-317920 exhibits potent and selective binding to rat H(3) receptors with low binding to non-H(3) sites, including cytochrome P450. These findings show that [(3)H]A-317920 is a potent rat H(3) receptor antagonist radioligand and has utility for studying H(3) receptor pharmacology.

Molecular modeling and pharmacological analysis of species-related histamine H(3) receptor heterogeneity.

Presynaptic histamine H(3) receptors (H(3)R) regulate neurotransmitter release in the central nervous system, suggesting an important role for H(3) ligands in human diseases such as cognitive disorders, sleep disturbances, epilepsy, or obesity. Drug development for many of these human diseases relies upon rodent-based models. Although there is significant sequence homology between the human and rat H(3)Rs, some compounds show distinct affinity profiles. To identify the amino acids responsible for these species disparities, various mutant receptors were generated and their pharmacology studied. The N-terminal portion was shown to determine the species differences in ligand binding since a chimeric H(3)R containing N-terminal human and C-terminal rat receptor sequences exhibited similar pharmacology to the human receptor. Sequence analysis and molecular modeling studies suggested key amino acids at positions 119 and 122 in transmembrane region 3 play important roles in ligand recognition. Mutant receptors changing amino acids 119 or 122 of the human receptor to those in the rat improved ligand binding affinities and functional potencies of antagonist ligands, confirming the significant role that these amino acids play in species-related pharmacological differences. A model has been developed to elucidate the ligand receptor interactions for H(3)Rs, and pharmacological aspects of this model are described.

In vivo and in vitro peroxisome proliferation properties of selected clofibrate analogues in the rat. Structure-activity relationships.

We have examined, relative to clofibric acid (CPIB), the effects of a chemical series of phenoxyacetic acids and of two asymmetric CPIB analogues, the R(+)- and S(-)-enantiomers of 2-(4-chlorophenoxy)propionic acid (4-CPPA) and 2-(4-chlorophenoxy)butyric acid (4-CPBA), on hepatic peroxisome proliferation both in vivo and in vitro utilizing cholesterol-fed rats and primary cultured rat hepatocytes respectively. Peroxisome proliferation was assessed by measuring changes in peroxisomal fatty acyl-CoA oxidase (FACO) and microsomal laurate hydroxylase (LH) activities as well as by electron microscopic examination of 3,3'-diaminobenzidine-stained liver slices. CPIB and enantiomers of 4-CPPA and 4-CPBA (0.6 mmol/kg/day for 7 days) produced hepatomegaly, lowered serum cholesterol levels, and caused 4.7- to 12.9-fold and 2.9- to 6.1-fold increases in hepatic FACO and LH activities, respectively, in cholesterol-fed rats. Electron micrographs of liver cells showed an increased number of peroxisomes from cholesterol-fed rats given S(-)-4-CPBA and CPIB. Likewise, these compounds (0.03 to 1.0 mM) induced FACO and LH in primary rat hepatocyte cultures after 72 hr. R(+)- and S(-)-Enantiomers of 4-CPPA produced similar concentration-dependent and maximal increases in both FACO and LH activities, whereas enantiomeric selectivity [S(-) greater than R(+)] for the induction of these two enzymes was observed with the isomers of 4-CPBA. The increases in the activities of FACO and LH caused by S(-)-4-CPBA were similar to those elicited by 1.0 mM CPIB (58.6- and 9.8-fold respectively). These results show that the enantiomers of 4-CPPA and 4-CPBA induce the peroxisome proliferation-associated enzymes FACO and LH in vivo and in vitro, and that the S(-)-isomer of 4-CPBA causes a greater induction of FACO and LH in vitro than its corresponding R(+)-isomer, indicating that these two enzymes are induced in an enantioselective manner. Optimal induction of the peroxisome proliferation-associated enzymes FACO and LH in rat hepatocyte cultures was produced by phenoxyacetic acids possessing (1) a chlorine atom at the 4-position of the phenyl ring, (2) a dimethyl or mono-ethyl substitution at the alpha-carbon atom of the carboxylic acid side chain; and (3) an S(-)-orientation for chiral analogues possessing a mono-ethyl group at the alpha-carbon atom of the carboxylic acid side chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Subtypes of alpha 1-adrenoceptors in DDT1 MF-2 and BC3H-1 clonal cell lines.

We examined which subtype(s) of alpha 1-adrenoceptors are expressed in the widely used DDT1 MF-2 and BC3H-1 cell lines. Pretreatment with chloroethylclonidine (CEC) inactivated 76-85% of the specific [125I]BE 2254 binding sites in membrane preparations from both cell lines. Competition with subtype-selective competitive antagonists showed primarily the alpha 1B subtype in both cell lines. However, in BC3H-1 cells 5-methyl-urapidil showed complex behavior suggesting that about half of the binding sites had a lower affinity. Chloroethylclonidine pretreatment eliminated [3H]inositol phosphate responses to norepinephrine in both cell lines. Measurement of intracellular Ca2+ with fura-2 in DDT1 MF-2 cells showed that norepinephrine induced a complex response involving both transient and sustained components. Chloroethylclonidine pretreatment blocked both responses, while chelation of extracellular Ca2+ left the transient response intact but eliminated the sustained component. These results support previous work that these cell lines contain alpha 1B-adrenoceptors linked to inositol phosphate formation and mobilization of intracellular Ca2+. However, these results show that alpha 1B-adrenoceptors can be linked to Ca2+ influx as well as intracellular mobilization, and support the existence of pharmacologically distinct alpha 1B variants.

Inducible expression of alpha 1B-adrenoceptors in DDT1 MF-2 cells: comparison of receptor density and response.

Hamster DDT1 MF-2 smooth muscle cells natively express alpha 1B-adrenoceptors which are linked to phosphatidylinositol hydrolysis. We studied the relationship between alpha 1B-adrenoceptor density and response in this cell line by stable transfection with an isopropyl-beta-D-thiogalactoside (IPTG)-inducible vector (pOP alpha 1B) containing the hamster alpha 1B-adrenoceptor cDNA. Transfected cells showed a 2-fold increase in receptor density compared to untransfected cells due to constitutive activity of the uninduced vector. Induction of vector expression caused a time-dependent increase in receptor density, reaching a maximum 8-fold increase after 48 h. Exposure to different concentrations of inducing agent for 16 h caused a graded increase in both receptor density and norepinephrine-stimulated [3H]inositol phosphate (InsP) formation. A linear correlation between receptor density and maximum InsP response was observed. Induction of receptor expression did not alter the potency of norepinephrine in stimulating [3H]InsP formation, suggesting that there was no receptor reserve, even at very high expression levels. This inducible expression system should be useful for relating receptor density and responsiveness, and comparing the coupling efficiency of closely related subtypes in activating different signal transduction mechanisms.

Species-related pharmacological heterogeneity of histamine H(3) receptors.

We compared radioligand binding and functional data for histamine H(3) receptor ligands across different tissues or species to evaluate the basis for pharmacological evidence of receptor heterogeneity previously reported. Agonist binding affinities showed correlation coefficients near unity in comparing human, dog, rat, and guinea pig cerebral cortical histamine H(3) receptors. Antagonist binding affinities revealed lower correlations for human compared to dog, rat, or guinea pig, suggesting species-based pharmacological differences. The functional potencies of histamine H(3) receptor antagonists in field-stimulated guinea pig ileum were highly correlated to binding affinities for guinea pig, dog, and, to a lesser extent, rat cerebral cortex. However, antagonist binding affinity at human cerebral cortex did not correlate well with guinea pig ileum functional potency. These results suggest significant interspecies histamine H(3) receptor heterogeneity, consistent with recent receptor gene sequence data. Therefore, genetic heterogeneity, rather than peripheral and central histamine H(3) receptor diversity, is responsible for the pharmacological differences observed.

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.

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.

Alpha 1-adrenergic receptor subtypes.

Investigators have not yet reached a consensus on the number and signaling mechanisms of alpha 1-adrenergic receptor (AR) subtypes. Two native subtypes (alpha 1A and alpha 1B) can be distinguished pharmacologically, and three subtypes (alpha 1B, alpha 1C, and alpha 1D) have been cloned. One of the cloned subtypes (alpha 1D) was originally thought to encode the pharmacologically defined alpha 1A subtype. However, recent data suggest otherwise, and many investigators now agree that the alpha 1A subtype has probably not yet been cloned. The relationship between the cloned receptors and the native subtypes must be understood, and any additional cDNA clones obtained, before the drug specificities and second messenger pathways of alpha 1-AR subtypes can be clearly defined. Little is yet known about the cellular and tissue distribution of these subtypes, their developmental profiles, or their functional importance. Molecular cloning of complementary DNA sequences for the remaining subtypes will help to clarify the number and properties of these subtypes. Identification of drugs that can selectively target particular subtypes is an important goal that may result in therapeutic advances in numerous disease states, including benign prostatic hyperplasia. The newly recognized complexity of the adrenergic receptors presents us with both important challenges and new therapeutic targets. The potential impact of this field on medical therapeutics remains to be clearly defined.

Increased voltage-dependent calcium influx produced by alpha 1B-adrenergic receptor activation in rat medullary thyroid carcinoma 6-23 cells.

We characterized norepinephrine (NE)-activated Ca2+ influx in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line using fura-2. NE caused a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i), which was completely reversed by addition of nifedipine or removal of extracellular Ca2+. Bay K8644, KCl-induced depolarization, and ATP also increased [Ca2+]i in rMTC 6-23 cells, effects that were also reversed by nifedipine. Release of intracellular Ca2+ by thapsigargin was not blocked by nifedipine, and NE caused nifedipine-sensitive increases in [Ca2+]i even in the presence of thapsigargin. NE-stimulated increases in [Ca2+]i were mimicked by the alpha 1-adrenergic receptor (AR) agonist phenylephrine but not by the beta-AR agonist isoproterenol. The response to NE was blocked by the alpha-AR antagonist phentolamine and by pretreatment with the alpha 1B-selective alkylating agent chloroethylclonidine (CEC) but was not blocked by alpha 1A-selective concentrations of the subtype-selective antagonist 5-methylurapidil. alpha 1-AR binding sites labeled by 125I-BE 2254 in membranes from this cell line were highly sensitive to inactivation by CEC (> 80%), and competition with subtype-selective antagonists suggested the presence of a homogeneous population of alpha 1B-ARs. NE, epinephrine, and phenylephrine, but not KCl, ATP, or isoproterenol, caused large increases in [3H]inositol phosphate (InsP) formation in these cells. This [3H]InsP response was greatly reduced by CEC pretreatment, and competitive antagonists blocked this response with an alpha 1B-like pharmacology. Northern blots of poly(A)+ RNA from rMTC 6-23 cells showed single transcripts hybridizing to the hamster alpha 1B-AR (2.2-kilo-base) and less prominently to the rat alpha 1D-AR (4.0-kilobase) cDNAs but no detectable hybridization to the bovine alpha 1C-AR cDNA. The phospholipase C inhibitor U-73122 reduced the [3H] InsP response to NE in a concentration-dependent manner but had little or no effect on the NE-induced increases in [Ca2+]i. Phorbol myristate acetate also increased [Ca2+]i in rMTC 6-23 cells, although this response was not blocked by nifedipine. We conclude that activation of alpha 1B-like ARs (including possibly both alpha 1B- and alpha 1D-ARs) increases voltage-dependent Ca2+ influx in rat rMTC 6-23 cells. This effect appears to be independent of release of intracellular Ca2+, activation of phospholipase C, and/or activation of protein kinase C. This cell line should be very useful in defining the mechanisms underlying the known effects of alpha 1-ARs on voltage-gated Ca2+ influx, which plays an important functional role in vascular smooth muscle.

Coexisting beta 1- and atypical beta-adrenergic receptors cause redundant increases in cyclic AMP in human neuroblastoma cells.

In SK-N-MC human neuroblastoma cells, the cAMP response to 10 nM isoproterenol (ISO) is mediated primarily by beta 1-adrenergic receptors. However, responses to higher concentrations of ISO (100-1000 nM) were only weakly blocked by beta 1- and beta 2-selective antagonists. When beta 1 receptors were blocked with 10 microM CGP 20712A, catecholamines still maximally activated cAMP accumulation, with only small decreases in potency. In the presence of CGP 20712A, beta blockers inhibited the response to ISO stereoselectively but with relatively low potencies. Pindolol derivatives were partial agonists with low potencies, and the atypical agonist BRL 37344 was a partial agonist with an intermediate potency. All binding sites in these cells labeled by 125I-cyanopindolol were of the beta 1 subtype. Nuclease protection assays indicated that SK-N-MC cells contain mRNA for both the human beta 1- and beta 3-adrenergic receptors, with the beta 3 subtype mRNA being expressed 25-50% more abundantly than that for the beta 1 subtype. Northern blot hybridizations showed the presence of two beta 3 mRNA transcripts of 3.1 and 2.4 kilobases. These results suggest that beta 1- and atypical beta-adrenergic receptors coexist in these cells and cause redundant increases in cAMP formation. Although molecular approaches suggest that the atypical subtype is the beta 3, the observed drug specificity differs from that reported for the expressed recombinant human beta 3 receptor.

Effects of the stereochemical orientation of phenethylamines and imidazolines on alpha-adrenergic receptor-mediated DNA synthesis in primary cultured rat hepatocytes.

The hepatic alpha 1-adrenergic receptor mediates a variety of hepatic functions including respiration, glycogenolysis, gluconeogenesis, and growth. We have utilized a rat primary hepatocyte culture system to show that the alpha 1-adrenergic receptor can be activated in a stereoselective manner by a series of phenethylamines and catecholimidazolines resulting in the stimulation of DNA synthesis as determined by [3H]thymidine incorporation. The phenethylamines adhered to the Easson-Stedman hypothesis with a rank order of potency of (-)-(R)-norepinephrine (NE) greater than (+)-(S)-NE greater than the desoxy analog dopamine (DA) for the stimulation of DNA synthesis. However, the 2-substituted catecholimidazolines did not follow this trend and demonstrated an order of potency of the desoxy analog 3,4-dihydroxybenzyl imidazoline (DHT) greater than or equal to (-)-(R)-2-(3,4,alpha-trihydroxybenzyl)imidazoline (TBI) greater than (+)-(S)-TBI. 4-Substituted catecholimidazolines were less potent as inducers of DNA synthesis than the corresponding 2-substituted analogs with an order of potency of (+)-(R)-4-(3,4-dihydroxybenzyl)imidazoline (DBI) greater than (+,-)-(R,S)-DBI greater than (-)-(S)-DBI. When the beta-hydroxyl moiety of NE is replaced with an amino group as in 3,4-dihydroxyphenylethylenediamine, the isomers are less active than the beta-hydroxylated analogs and also demonstrate no stereoselectivity for the stimulation of DNA synthesis. These results demonstrate that the hepatic alpha 1-adrenergic receptor can recognize various isomeric forms of these compounds and that hepatocellular growth can be modulated in a stereoselective manner by phenethylamines and imidazolines.

Coupling efficiencies of human alpha 1-adrenergic receptor subtypes: titration of receptor density and responsiveness with inducible and repressible expression vectors.

We compared the efficiencies with which human alpha 1-adrenergic receptor (AR) subtypes activate inositol phosphate (InsP) formation and increase intracellular Ca2+ in transfected cell lines. Expression of human alpha 1a-, alpha 1b-, and alpha 1d-AR cDNAs under the repressible control of anhydrotetracycline in human embryonic kidney (HEK) 293 cells, which normally express no alpha 1-ARs, was used to compare responses to norepinephrine (NE) at different receptor densities. Maximal NE-stimulated InsP formation was found to increase with increasing density of each subtype, whereas basal levels and responses to sodium fluoride did not change. A comparison of multiple subclones over equivalent ranges of receptor expression showed that activation of each subtype resulted in different maximal responses (alpha 1a > alpha 1b > alpha 1d) in HEK 293 cells. Analogous studies were carried out in human SK-N-MC cells, which normally express low levels of all three alpha 1-AR subtypes, using an isopropyl-beta-D-thiogalactoside-inducible expression system. Induction with isopropyl-beta-D-thiogalactoside increased the density of individual alpha 1-AR subtypes by 4-6-fold over the level of endogenous expression. Increased expression of each of these subtypes in SK-N-MC cells did not alter the EC50 value for NE in stimulating InsP formation or releasing [Ca2+]i but did increase maximal responses to NE. Similar to our findings in HEK 293 cells, a comparison of responses at similar expression levels in SK-N-MC cells showed different maximal responses stimulated by each subtype, for both InsP (alpha 1a > alpha 1b > or = alpha 1d) and [Ca2+]i (alpha 1a > alpha 1b > alpha 1d) responses. These studies show that agonist-occupied human alpha 1-AR subtypes have different efficiencies in activating phospholipase C in human cell lines. In both HEK 293 and SK-N-MC cells, alpha 1a-ARs couple most efficiently, whereas alpha 1d-ARs couple very poorly.

Selectivity of agonists for cloned alpha 1-adrenergic receptor subtypes.

The potencies and intrinsic activities of agonists in activating cloned alpha 1-adrenergic receptor (AR) subtypes were compared. The hamster alpha 1B-, bovine alpha 1C-, or rat alpha 1A/D-ARs were expressed at high levels in human embryonic kidney 293 cells. Catecholamines and phenylethylamines, but not lower efficacy agonists, were more potent in inhibiting radioligand binding to the expressed alpha 1A/D subtype than to the alpha 1B or alpha 1C subtypes; this selectivity remained in the presence of different buffers, nucleotides, and cations. Activation of all three subtypes caused substantial increases in [3H]inositol phosphate formation in cells grown in 96-well plates. Pretreatment with phenoxybenzamine decreased maximal responses to norepinephrine (NE) with only small decreases in apparent potency, suggesting similar small receptor reserves for all three subtypes. The catecholamines NE, epinephrine, and 6-fluoro-NE were full agonists with similar potencies at the three subtypes; alpha-methyl-NE was also a full agonist but was about 20-fold less potent at alpha 1B-ARs than at alpha 1C- or alpha 1A/D-ARs. Phenylephrine had similar potencies at all three subtypes but gave a submaximal response at alpha 1B-ARs. Methoxamine was a full agonist at alpha 1C- and alpha 1A/D-ARs, with about 20-fold greater potency at the alpha 1C subtype, but showed lower intrinsic activity at alpha 1B-ARs. A number of imidazolines, amidephrine, and SKF 89748 had substantial intrinsic activity at alpha 1C-ARs but little or no intrinsic activity at the other two subtypes. We conclude that the potencies of many agonists in competing for radioligand binding sites are related to their potencies in activating functional responses but that this relationship is not the same for all subtypes. NE and epinephrine activate all three cloned alpha 1-AR subtypes with similar potencies and intrinsic activities, but many widely used agonists show significant selectivity for different alpha 1-AR subtypes.

Comparison of alpha 1-adrenergic receptor subtypes and signal transduction in SK-N-MC and NB41A3 neuronal cell lines.

We compared the alpha 1-adrenergic receptor subtypes in two neuronal cell lines, SK-N-MC (human neuroepithelioma) and NB41A3 (murine neuroblastoma). 125I-BE 2254 labeled alpha 1-adrenergic receptor binding sites in membranes from both cell lines. Pretreatment with the alpha 1B-selective alkylating agent chloroethylclonidine (CEC) completely eliminated these binding sites in NB41A3 cells but caused only a 50% loss in SK-N-MC cells. Displacement with subtype-selective antagonists suggested that NB41A3 cells express only the alpha 1B subtype, whereas SK-N-MC cells express a pharmacologically heterogeneous receptor population, including both alpha 1A and alpha 1B subtypes. Norepinephrine increased [3H] inositol phosphate formation in both cell lines, but with different sensitivities to pertussis toxin and the presence of extracellular Ca2+. CEC pretreatment eliminated this response in NB41A3 cells but caused a maximal 42% reduction in SK-N-MC cells. Use of subtype-selective antagonists showed that the [3H]inositol phosphate response involved only the alpha 1B subtype in NB41A3 cells but a combination of subtypes in SK-N-MC cells. Norepinephrine induced both transient and sustained increases in intracellular Ca2+ concentrations in both cell lines, as measured with fura-2. CEC pretreatment abolished the Ca2+ response in NB41A3 cells but had little effect in SK-N-MC cells. In SK-N-MC cells the Ca2+ response was potently blocked by alpha 1A-selective antagonists. Chelation of extracellular Ca2+ eliminated the sustained component of the Ca2+ signal in both cell lines. Poly(A)+ RNA from NB41A3, DDT1MF-2, BC3H1, and MDCK-D1 cell lines showed one or more prominent transcripts (2.2-4.2 kilobases) that strongly hybridized to the hamster alpha 1B cDNA probe but not to the bovine alpha 1C or rat alpha 1D cDNA probes. Poly(A)+ RNA from SK-N-MC cells showed multiple transcripts (1.3-5.6 kilobases) that hybridized to both hamster alpha 1B and rat alpha 1D but not bovine alpha 1C cDNA probes. We conclude that NB41A3 cells contain exclusively alpha 1B-adrenergic receptors linked to inositol phosphate formation and mobilization of intracellular Ca2+, whereas at least two alpha 1-adrenergic receptor forms, which resemble the alpha 1A and alpha 1B subtypes, coexist in SK-N-MC cells. The CEC-insensitive alpha 1A-like subtype in SK-N-MC cells is capable of increasing inositol phosphate formation and mobilizing intracellular Ca2+.

Inducible expression of beta 1- and beta 2-adrenergic receptors in rat C6 glioma cells: functional interactions between closely related subtypes.

We examined the role of beta 1- and beta 2-adrenergic receptor (AR) density and ratio in catecholamine-stimulated cAMP responses in rat C6 glioma cells. These cells, which normally express both subtypes, were stably transfected with an isopropylthio-beta-D-galactoside-inducible vector containing either beta 1AR or beta 2AR coding sequences, and receptor expression was controlled by the time and concentration of isopropylthio-beta-D-galactoside exposure. Induction of the dominant beta 1AR subtype increased the potencies of isoproterenol (ISO) and other agonists in stimulating cAMP accumulation by 20-40-fold without changing maximal response. Induction of beta 2AR expression caused 7-13-fold increases in the potency of ISO, epinephrine, and zinterol, but not of norepinephrine, and a 20-40% loss in maximal response to all agonists. Selective antagonists showed that both subtypes contributed in a nonadditive manner in the response to ISO under different conditions. After beta 2AR induction, the effects of ISO were not blocked by the beta 1-selective antagonist CGP 20712A but were shifted 100-fold to the right by the beta 2-selective antagonist ICI 118,551. However, in the presence of ICI 118,551, CGP 20712A caused an additional 100-fold decrease in ISO potency, and Schild analysis revealed complex interactions between the two subtypes. Each antagonist alone caused smaller shifts to the right in the dose-response curve to NE and, when present simultaneously, completely abolished the NE response. We conclude that beta 1ARs and beta 2ARs have different efficiencies in activating cAMP accumulation in C6 glioma cells. Activation of coexisting subtypes results in complex and sometimes synergistic interactions between the two subtypes, which vary with agonist concentration, selectivity, subtype density, and ratio.

Effect of age on cholinergic muscarinic responsiveness and receptors in the rat urinary bladder.

The effects of age on urinary bladder responsiveness to muscarinic agonists and on the Bmax and Kd of the binding of [3H]quinuclidinyl benzilate (QNB) to muscarinic receptors of the bladder were studied. Bladder bodies and bases were isolated from Fischer 344 rats, ages seven, 16 and 27 months. No age-dependent change in maximum KCl-elicited isotonic contractions was observed in either bladder region. The bladder base showed an age-dependent increase in the maximum contractions (Emax) elicited by muscarinic agonists. The Emax values for bladder bases from rats 27 months of age were 44 per cent, 58 per cent and 76 per cent greater than those from rats seven months of age for acetylcholine, bethanechol and oxotremorine, respectively. No such alteration in responsiveness was observed in the bladder body with age. There were no age-related changes in ED50 values for the three agonists in either bladder region. Analysis of [3H]QNB binding in the bladder base demonstrated a modest 18 per cent increase in the Bmax (fmol./mg. tissue) from seven to 16 months and a significant 39 per cent decrease from 16 to 27 months. In the bladder body, the Bmax progressively increased by 25 per cent from seven to 27 months. The Kd values of [3H]QNB did not change with age in either region. The data demonstrate that the age-related increase in the responsiveness of the bladder is regionally specific and cannot be explained by a change in the number or affinity of muscarinic receptors.

Two novel and potent 3-[(o-methoxyphenyl)piperazinylethyl]-5-phenylthien.

The synthesis and in vitro characterization of A-119637 and A-123189, two novel, selective and potent alpha1D antagonists, are described.

Cloning of the human alpha 1d-adrenergic receptor and inducible expression of three human subtypes in SK-N-MC cells.

We have cloned the human alpha 1d-adrenergic receptor (AR) and compared the pharmacological properties of the three recombinant human alpha 1-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mixture of alpha 1-AR subtypes, and the use of an inducible expression system allowed us to directly compare the recombinant and native subtypes without concern for cell-specific processing or microenvironment. The human alpha 1d-AR was expressed from a cDNA/gene fusion construct cloned from human SK-N-MC cell cDNA and human genomic libraries. This receptor is deduced to contain 572 amino acids with 98% identity to the rat alpha 1d-AR in the transmembrane domains and, when expressed in human embryonic kidney 293 cells, has alpha 1-AR binding properties similar to those of the rat alpha 1d-AR. Norepinephrine increased inositol phosphate formation and mobilized intracellular Ca2+ in transfected 293 cells. Reverse transcription-polymerase chain reaction analysis of the three cloned human subtypes (alpha 1a, alpha 1b, and alpha 1d) in mRNA from SK-N-MC cells, which natively express alpha 1A- and alpha 1B-like pharmacology, showed abundant alpha 1a and alpha 1d but fewer alpha 1b transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vectors. Upon induction, alpha 1-AR density was increased with the recombinant subtype comprising 67-80% of total alpha 1-ARs. Inhibition curves for (+)-niguldipine and 5-methylurapidil fit best to a two-site model in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of both compounds, causing most inhibition curves to fit best to a one-site model. (+)-Niguldipine was 100-fold more potent at the alpha 1a-AR than at alpha 1b- or alpha 1d-ARs, whereas 5-methylurapidil had similar potencies at alpha 1a- and alpha 1d-ARs and about 10-fold lower affinity at the alpha 1b-AR. We conclude that the complex alpha 1A- and alpha 1B-like pharmacology observed in native SK-N-MC cells is due to expression of all three subtypes in different proportions, independently of cell-specific processing or environmental factors, and that the alpha 1a-AR cDNA encodes the pharmacologically defined alpha 1A subtype.