NS-49, a novel alpha 1a-adrenoceptor-selective agonist characterization using recombinant human alpha 1-adrenoceptors.

alpha 1-Adrenoreceptors comprise a heterogeneous family and subtype-selective ligands are valuable in studying the functional role of each receptor subtype. Using the Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a, alpha 1b, and alpha 1d)1, we have compared a newly synthesized phenethylamine class agonist (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride (NS-49) with imidazoline class agonist oxymetazoline in their binding affinities and intrinsic activities in causing transient increases of cytosolic Ca2+ concentrations ([Ca2+¿i response). Radioligand binding studies with 2-[beta-(4-hydroxyl-3-[125I]iodophenyl)ethylamino-methyl]tetralone ([125I]HEAT) showed NS-49 and oxymetazoline had higher affinities at alpha 1a-than at alpha 1b- and alpha 1d-subtypes (-log Ki values at alpha 1a-, alpha 1b-and alpha 1d-subtype: 6.18, 5.13, and 5.38 for NS-49: 8.19, 6.50, and 6.44 for oxymetazoline, respectively). In functional studies, both oxymetazoline and NS-49 worked as a selective and partial agonist at alpha 1a-subtype: however, NS-49 is more efficacious than oxymetazoline. NS-49 is the phenethylamine class of alpha 1-adrenoceptor partial agonist relatively selective and efficacious for the human alpha 1a-adrenoceptor subtype, NS-49 would be potentially useful for studying the physiological role of alpha 1-adrenoceptor subtype.

Selectivity of the imidazoline alpha-adrenoceptor agonists (oxymetazoline and cirazoline) for human cloned alpha 1-adrenoceptor subtypes.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

KMD-3213, a novel, potent, alpha 1a-adrenoceptor-selective antagonist: characterization using recombinant human alpha 1-adrenoceptors and native tissues.

alpha 1-Adrenoceptors (ARs) comprise a heterogeneous family, and subtype-selective ligands are valuable for studying the functional role of each receptor subtype. We characterized a newly synthesized, alpha 1-AR antagonist, KMD-3213, by using Chinese hamster ovary cells stably expressing the three cloned human alpha 1-ARs (alpha 1a, alpha 1b, and alpha 1d), as well as native rat and human tissues. KMD-3213 potently inhibited 2-[2-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-alpha-tetralone binding to the cloned human alpha 1a-AR, with a Ki value of 0.036 nM, but had 583- and 56-fold lower potency at the alpha 1b- and alpha 1d-ARs, respectively. KMD-3213 inhibited norepinephrine-induced increases in intracellular Ca2+ concentrations in alpha 1a-AR-expressing Chinese hamster ovary cells with an IC50 of 0.32 nM but had a much weaker inhibitory effect on the alpha 1b- and alpha 1d-ARs. Using pharmacologically well characterized native rat tissues [submaxillary gland (alpha 1A-AR-expressing tissue), liver (alpha 1B-AR-expressing tissue), and heart (mixed alpha 1A- and alpha 1B-AR-expressing tissue)], binding studies showed that inhibition curves for KMD-3213 in submaxillary gland and liver best fit a one-site model (with Ki values of 0.15 and 16 nM, respectively), whereas KMD-3213 had high and low affinity sites in heart membranes. Chloroethylclonidine treatment of rat heart membranes completely eliminated the low affinity sites for KMD-3213. Furthermore, in human liver and prostate KMD-3213 could identify high and low affinity sites, the Ki values of which corresponded well to those for the cloned human alpha 1a- and alpha 1b-ARs, respectively. Moreover, the affinity of KMD-3213 was found to be approximately 10-fold higher at the cloned human alpha 1a-AR than at the cloned rat alpha 1a-AR. KMD-3213 is a potent and highly selective antagonist for the human alpha 1a-AR and would be useful for studying the physiological roles of human alpha 1-AR subtypes.

Cloning, functional expression and tissue distribution of human alpha 1c-adrenoceptor splice variants.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Nitric oxide reductase cytochrome P-450 gene, CYP 55, of the fungus Fusarium oxysporum containing a potential binding-site for FNR, the transcription factor involved in the regulation of anaerobic growth of Escherichia coli.

A unique cytochrome P-450 (P-450) is involved in fungal denitrification, acting as a nitric oxide reductase. The phylogenetic classification of the P-450 (P-450nor) into the group of bacterial P-450s along with its involvement in the anaerobic process are of evolutional interest. The corresponding gene, CYP 55, of the fungus Fusarium oxysporum was isolated and sequenced. The P-450nor gene contained 7 introns that consisted of 49 to 55 bp. The presence of the introns suggested that horizontal transfer of the gene from bacteria to the fungus, if it has occurred, was an early event in evolution. Besides a TATA box, several inverted repeats were found in the 5'-upstream flanking region. One inverted repeat exhibited the same sequence as the binding site of FNR (fumarate and nitrate reduction), a DNA-binding, O2 (dioxygen)-sensor protein that positively regulates expressions of many hypoxic genes in Escherichia coli and other bacteria. The result suggested that the expression of P450nor is regulated in response to oxygen tension by an FNR-like system. Northern blot analyses showed that both nitrate and nitrite are the actual inducers of P-450nor and that its expression is predominantly regulated at the transcriptional level. These results raise the interesting possibility that the expression of the fungal denitrification system is regulated, as in the case of bacterial nitrate respiration, by a set of mechanisms, i.e., a combination of an FNR-like system and a system responding to nitrate/nitrite.

Expression of the fungal cytochrome P-450nor cDNA in Escherichia coli.

We succeeded in expressing the unique cytochrome P-450 (P-450), nitric oxide reductase (P-450nor), in Escherichia coli by utilizing P-450nor cDNA and the expression vector pKK233-2 or pUC19. The expression was confirmed by Western blot analysis and by detecting the unique nitric oxide reductase activity. The expressed protein was recovered in the soluble fraction of transformed cells, supporting that P-450nor is the first soluble P-450 of eukaryote. The results also provided conclusive evidence that the enzymatic reaction depends solely on P-450nor without support of any other electron transferring components.