Himanshu Shah

Human ADAM33: protein maturation and localization

ADAM33 (a disintegrin and metalloprotease) was recently found to be a novel asthma susceptibility gene. Domain-specific antibodies were used to study its expression and processing. When the pro-domain and catalytic domain were expressed by a stable-transfected cell line, the pro-domain was removed by cleavage within a putative furin cleavage site. The catalytic domain was active in an alpha(2)-macroglobulin complex formation assay and mutation of the catalytic site glutamic acid (E346A) eliminated activity. In transient transfections using the full-length protein, a pro-form and mature form were detectable and alternate glycosylation was demonstrated at sites within the catalytic domain. ADAM33 was detected on the cell surface, with the majority of protein detected intracellularly. The E346A mutation had no significant effect on protein processing. Endogenous ADAM33 was detected in bronchus tissue, bronchial smooth muscle cells, and MRC-5 fibroblasts, consistent with a role in the pathophysiology of asthma.

Receptor reserve analysis of the human CCR3 receptor in eosinophils and CCR3-transfected cells.

A novel pharmacological study of CCR3 receptor reserve in a CCR3‐transfected cell (CREM3) and human eosinophils was done; functional responses measured were increases in intracellular calcium and chemotaxis. Eotaxin, eotaxin‐2, monocyte chemoattractant protein‐4 (MCP‐4), RANTES, and MCP‐3 induced similar maximal eosinophil chemotaxis, whereas MCP‐3 and RANTES induced submaximal calcium responses in eosinophils compared to eotaxin, MCP‐4, and eotaxin‐2. This suggested a receptor reserve in the chemotaxis response. Receptor reserve was quantitated for eotaxin. Occupancy of all CCR3 receptors was required for a maximal calcium response in both CREM3 and eosinophils (reserve = 1.0 or 0.17, respectively); the stimulus‐calcium response relationship was linear, indicating no receptor reserve. In contrast, in eosinophils a large receptor reserve (6.5) was found for chemotaxis, where occupancy of 15% receptors drove half‐maximal responses. These studies indicate that CCR3 interacts with G‐proteins that are poorly coupled to the calcium response, whereas coupling efficiency and/or amplification to the chemotaxis apparatus in human eosinophils is significantly greater. J. Leukoc. Biol. 67: 441–447; 2000.

Receptor reserve analysis of the human α2C-adrenoceptor using [35S]GTPγS and cAMP functional assays

Abstract Here we determine for norepinephrine, (5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline) (UK14,304), 5,6,7,8-tetrahydro-6-(2-propenyl)-4 H -thiazolo[4,5- d ]azepin-2-amine dihydrochloride (BHT-920), (2-[3-hydroxy-2,6-dimethyl-4- t -butylbenzyl]-2-imidazoline) (oxymetazoline), and (( R )-3-Hydroxy-α-[(methylamino)methyl]-benzenemethanol hydrochloride) (phenylephrine), affinities using a radiolabeled agonist and antagonist, and potency and efficacy values in membrane [ 35 S]guanosine-5′- O -(3-thiotriphosphate) ([ 35 S]GTPγS) binding and cAMP cellular inhibition assays, in Chinese hamster ovary cells (CHO-K1) expressing the human α 2c -adrenoceptor. These cells express a high ratio of receptor to G-protein because each agonist, but not several antagonists, displaced [ 3 H]UK14,304 with higher affinity than [ 3 H]rauwolscine. The rank order of potency of high affinity K i and EC 50 in both functional assays was norepinephrine ≥UK14,304>BHT-920>oxymetazoline>phenylephrine. The receptor reserve of G-protein activation and cAMP responses was measured with the irreversible antagonist, benextramine; K A values of norepinephrine or UK14,304 were similar (289, 271 or 150, 163 nM, respectively). A 20-fold greater receptor occupancy was required for agonist-induced half-maximal [ 35 S]GTPγS binding compared to cAMP inhibition, indicating significant signal amplification in cells. Therefore, the G-protein activation assay is better at distinguishing full and partial agonists.

Postjunctional α2C-adrenoceptor contractility in human saphenous vein

Abstract The postjunctional α 2 -adrenoceptor-mediated contractility was characterized in human saphenous vein derived from coronary artery bypass graft surgery. Human saphenous vein contracted to α 2 -adrenoceptor selective agonists BHT-920 (5,6,7,8-Tetrahydro-6-(2-propenyl)-4 H -thiazolo[4,5-d]azepin-2-amine dihydrochloride; p D 2 =6.7±0.1) and UK 14,304 (5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline; p D 2 =7.2±0.1). BHT-920-induced contractions were inhibited by the α 2 -adrenoceptor antagonist yohimbine (17-Hydroxy-yohimban-16-carboxylic acid methyl ester hydrochloride; p A 2 =8.7±0.5), but not by the α 1 -adrenoceptor antagonist prazosin (1-[4-Amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]-piperazine hydrochloride; 300 nM). In contrast, prazosin (p K b =7.9±0.2) potently antagonized contractions elicited by the α 1 -adrenoceptor agonist phenylephrine (( R )-3-Hydroxy-α-[(methylamino)methyl] benzenemethanol hydrochloride; p D 2 =4.9±0.1), indicating that both α 2 - and α 1 -adrenoceptor evoke human saphenous vein contractions. Functional antagonist activity estimates (p A 2 or p K b ) obtained for the α-adrenoceptor antagonists ARC 239 (2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl-1,3-(2 H ,4 H )-isoquinolindione dihydrochloride), WB 4101 (2-(2,6-Dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and HV 723 (α-ethyl-3,4,5-trimethoxy-α-(3-((2-(2-methoxyphenoxy) ethyl)amino)propyl)benzeneacetonitrile) against BHT-920-induced human saphenous vein contractions were 7.0±0.6, 8.3±0.6 and 7.7±0.3, respectively. The α 2 -adrenoceptor subtype affinities (p K i ) obtained in recombinant human α 2A -, α 2B - and α 2C -adrenoceptor competition binding assays were 8.6, 8.3 and 8.6 for yohimbine; 6.3, 8.4 and 7.0 for ARC 239; 8.4, 7.5 and 8.4 for WB 4101 and 7.5, 7.4 and 7.9 for HV 723, respectively. Taken together, the binding and functional antagonist activity estimates obtained in these investigations indicate that α 2C -adrenoceptor is the predominant postjunctional α 2 -adrenoceptor subtype in human saphenous vein.

Pharmacological Characterization of a Novel α2C-Adrenoceptor Agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N′-methylurea (Compound A)

We define the pharmacological and pharmacokinetic profiles of a novel α2C-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N′-methylurea]. This compound has high affinity (Ki) for the human α2C-adrenoceptor (Ki = 12 nM), and 190- to 260-fold selectivity over the α2A- and α2B-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC50 = 166 nM) and efficacy (Emax = 64%) responses at the α2C-adrenoceptor, much lower potency and efficacy at the α2A-adrenoceptor (EC50 = 1525 nM; Emax = 8%) and α2B-adrenoceptor (EC50 = 5814 nM; Emax = 21%) subtypes, and low or no affinity and functional activity at the α1A-, α1B-, and α1D-adrenoceptor subtypes. In the human saphenous vein postjunctional α2C-adrenoceptor bioassay, compound A functions as a potent agonist (pD2 = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC50 = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α2C-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.