Molecular and pharmacological characteristics of the gerbil α(1a)-adrenergic receptor.

The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α(1A)-adrenergic receptor subtype mediates vasoconstriction of the gerbil spiral modiolar artery. Although the gerbil cochlea is often used as a model in hearing research, the molecular and pharmacological characteristics of the cloned gerbil α(1a)-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α(1a)-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α(1a)-adrenergic receptors. The cDNA clone contained 1404 nucleotides, which encoded a 467 amino acid peptide with a deduced sequence having 96.8, 96.4 and 91.6% identity to rat, mouse and human α(1a)-receptors, respectively. We transiently transfected the α(1a)-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [(3)H]prazosin binding. Unlabeled prazosin had a K(i) of 0.89±0.1nM. The α(1A)-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had K(i) values of 4.9±1 and 1.0±0.1nM, respectively. BMY-7378, an α(1D)-adrenergic receptor-selective antagonist, bound with low affinity (260±60nM). The 91.6% amino acid sequence identity and K(i)s of the cloned gerbil α(1a)-adrenergic receptor are similar to those of the human α(1a)-adrenergic receptor clone. These results show that the gerbil α(1a)-adrenergic receptor is representative of the human α(1a)-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.

Mutations in the transmembrane domain M3 generate spontaneously open orphan glutamate δ1 receptor.

Glutamate delta-1 receptors (GluRδ1) are expressed in the adult hippocampus and inner ear and have recently been shown to be important for high-frequency hearing. Similar to the closest homolog glutamate delta-2 receptor (GluRδ2), no agonist-induced currents are observed from GluRδ1 receptors. In an effort to understand the function of the GluRδ1 subunit, we probed the conserved transmembrane 3 (TM3) region of the GluRδ1 subunit, where the GluRδ2 lurcher mutation is localized. Four mutations in the TM3 domain A650C, L652A, A654C, and F655A resulted in spontaneously open GluRδ1 channels suggesting that GluRδ1 receptors can form homomeric receptors. The leak currents were partially blocked by pentamidine but showed negligible inhibition by NASP. It has been demonstrated that extracellular Ca(2+) binds and stabilizes the ligand binding domain (LBD) dimer interface leading to potentiation of currents through GluRδ2(Lc) channels. We found that extracellular Ca(2+) potentiated the spontaneous currents through GluRδ1F655A suggesting that extracellular Ca(2+) may interact with the conserved residues at GluRδ1 LBD dimer interface. A recent study suggested that d-serine and glycine bind to the GluRδ2 LBD and reduce spontaneous currents through the GluRδ2(Lc) channels. d-Serine and glycine produced only a modest reduction of spontaneous currents through GluRδ1F655A and had no effect on the spontaneous current through GluRδ1L652A. However, spontaneous currents in a chimeric GluRδ1-δ2(Lc) were robustly inhibited by d-serine. These results suggest that the activation gate is conserved in GluRδ1 receptors. Moreover, the conformational changes induced by d-serine and extracellular Ca(2+) are conserved among GluRδ1 and GluRδ2 receptors.

Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium.

BACKGROUND: It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization. RESULTS: The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 microM) did not inhibit the effect of UTP (1 microM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization. CONCLUSION: The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.

Submandibular gland acinar cells express multiple alpha1-adrenoceptor subtypes.

We evaluated an acinar cell line (SMG-C10) cloned from rat submandibular glands as a possible model for alpha(1)-adrenoceptor regulation of submandibular function. alpha(1)-Adrenoceptors are subdivided into three subtypes called alpha(1A), alpha(1B), and alpha(1D), which can be distinguished from one another by their differential affinity values for subtype-selective alpha(1)-adrenoceptor antagonists. Thus, alpha(1)-adrenoceptor subtypes in SMG-C10 cells were characterized with reverse transcription-polymerase chain reaction (RT-PCR) and [(3)H]prazosin binding in side-by-side experiments with native submandibular glands. RT-PCR identified mRNAs for alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenoceptors in SMG-C10 cells and submandibular glands. The inhibition of [(3)H]prazosin binding by 5-methylurapidil (alpha(1A)-selective) was biphasic and fit best to a two-site binding model with 40 +/- 8% high (K(iH))- and 60 +/- 10% low (K(iL))-affinity binding sites in SMG-C10 cells, and 76% high- and 24% low-affinity binding sites in submandibular glands. Respective K(iH) and K(iL) values for 5-methylurapidil were 1.9 +/- 0.4 and 100 +/- 30 nM in SMG-C10 cells and 3.2 +/- 0.8 and 170 +/- 20 nM in submandibular glands. BMY-7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (alpha(1D)-selective)] bound with low affinity in SMG-C10 cells and submandibular glands with K(i) values of 81 +/- 20 and 110 +/- 20 nM, respectively. Chloroethylclondine, an irreversible alkylating agent selective for alpha(1B) adrenoceptors, reduced the density of [(3)H]prazosin binding sites by 42 and 26% in SMG-C10 and submandibular membranes, respectively. Thus, SMG-C10 cells and submandibular glands are similar in expressing receptor protein for alpha(1A)- and alpha(1B)-adrenoceptor subtypes, establishing SMG-C10 cells as a potential model for alpha(1)-adrenoceptor-mediated secretion.

Adrenergic receptor genes: cDNA and genomic library construction.

Adrenergic receptors mediate the central and peripheral actions of norepinephrine and epinephrine and are pharmacologically divided into three major types, alpha-1, alpha-2, and beta. These types are further subdivided into alpha-1A, alpha-1B, and alpha-1D; alpha-2A, alpha-2B, and alpha-2C; and beta-1, beta-2, and beta-3, respectively. Adrenergic receptor sequence information is presented in three tables with respect to species, subtype identification, GenBank accession number, source of the nucleic acid sequence, the presence of a 5' flanking region upstream of the transcription start site, and the nucleotides defined as introns, coding regions, or 3' and/or 5' untranslated but transcribed (UTR) regions. Sequences have been assigned to adrenergic subtype categories based on sequence comparison using either FASTA or denogram of Pileup from the GCG sequence analysis program rather than as described in the author definition line. Sequence information found in these tables can be important for probe development for screening libraries for isolating adrenergic receptor genes from species other than the most common species. Where commercial libraries for specific tissue or species needs are not available, we have described construction of genomic cosmid libraries or PCR-based synthesis of a cDNA library using a microgram of RNA.