Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 May 22.
Published in final edited form as: Hear Res. 2011 Nov 10;283(0):144–150. doi: 10.1016/j.heares.2011.11.002

Molecular and pharmacological characteristics of the gerbil α1a-adrenergic receptor

Kelly M Witt a, Charles S Bockman a, Herbert K Dang a, Daniel D Gruber b, Philine Wangemann c, Margaret A Scofield a
PMCID: PMC4030439  NIHMSID: NIHMS337965  PMID: 22101021

Abstract

The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α1A–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 α1a-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α1a-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α1a-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 α1a-receptors, respectively. We transiently transfected the α1a-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [3H]prazosin binding. Unlabeled prazosin had a Ki of 0.89 ± 0.1 nM. The α1A-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had Ki values of 4.9 ± 1 and 1.0 ± 0.1 nM, respectively. BMY-7378, an α1D-adrenergic receptor-selective antagonist, bound with low affinity (260 ± 60 nM). The 91.6% amino acid sequence identity and Kis of the cloned gerbil α1a-adrenergic receptor are similar to those of the human α1a-adrenergic receptor clone. These results show that the gerbil α1a-adrenergic receptor is representative of the human α1a-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.

Keywords: Alpha1-adrenergic receptor subtype, Gerbil cochlea, Spiral modiolar artery

1. Introduction

The cochlea is the hearing component of the inner ear. It translates mechanical energy from movement of middle ear bones into nerve impulses, which are ultimately transmitted to the brain where sound is perceived. The cochlea receives its blood supply from the spiral modiolar artery and the cochlear branch of the vestibulocochlear artery. Cochlear viability depends on adequate blood flow through these arteries (Seidman et al., 1999). For example, experimental interruption of blood flow to the inner ear results in ischemia-induced damage to the cochlea (Billett et al., 1989; Iwasaki et al., 1997; Koga et al., 2003), and disturbances in cochlear blood flow are implicated in sudden sensorineural hearing loss, presbyacusis and noise-induced hearing loss in humans (Nakashima et al., 2003; Seidman et al., 1999; Shikowitz, 1991).

α1–Adrenergic receptors mediate constriction of various blood vessels and are therapeutic targets in the treatment of several cardiovascular diseases. By convention, native α1–adrenergic receptor subtypes are denoted by uppercase letters, while cloned α1–adrenergic receptor subtypes are indicated with lowercase letters (Hieble et al., 1995). α1–Adrenergic receptors are subdivided into α1A-, α1B- and α1D-adrenergic receptor subtypes based on their molecular structure and pharmacological characteristics. Hence, subtype-selective antagonists, which have varying affinities for each subtype, can be used to distinguish the α1A- from the α1B- from the α1D-adrenergic receptor. For example, at α1A-receptors, 5-methylurapidil has a higher affinity than BMY-7378; while at α1B-receptors, 5-methylurapidil and BMY-7378 have similar affinities; and at α1D-receptors, BMY-7378 has a higher affinity than 5-methylurapidil (Bockman et al., 2004; Goetz et al., 1995; Gross et al., 1988).

Because cochlear function is particularly sensitive to ischemic conditions, it is important to understand the mechanisms that regulate vascular tone of arteries supplying the cochlea. The spiral modiolar artery is innervated by norepinephrine-containing sympathetic nerves (Carlisle et al., 1990), and vasoconstriction of the spiral modiolar artery is mediated by α1-adrenergic receptors (Gruber, 1998). In addition, α1-adrenergic receptors have been shown to regulate cochlear blood flow (Ohlsen et al., 1991). Thus, α1-adrenergic receptors on the spiral modiolar artery may be important in the pathophysiology of sensorineural hearing loss caused by impaired cochlear blood flow.

Consistent blood flow measurements are possible in the gerbil because of its large ear parts and thin cochlear bone (Mom et al., 1999), and the vascular anatomy of gerbil and human cochleae are similar. Consequently, the gerbil is widely used as a model of cochlear blood flow (Nakashima et al., 2003). It is therefore important to understand the regulatory mechanisms controlling gerbil cochlear vasculature. Previously, we reported that reverse transcription-polymerase chain reaction identified mRNA for only the α1A-adrenergic receptor subtype in gerbil spiral modiolar arteries (SMA). In addition, functional characterization of the α1-subtype on the SMA showed that 5-methylurapidil blocked norepinephrine-induced contraction with relative high affinity, indicating the α1A-adrenergic receptor subtype regulates contraction of the SMA (Gruber et al., 1998).

It is important to know whether or not the gerbil α1A-adrenergic receptor subtype is typical of α1A-subtypes from other species, particularly human, because of its role in a key model of cochlear blood flow. Thus, we first cloned the gerbil α1a-adrenergic receptor to determine its molecular properties, and then compared its deduced amino acid sequence to those of cloned α1a-adrenergic receptors from other species. Secondly, we used radioligand binding to directly determine the pharmacological characteristics of the expressed gerbil α1a-receptor, and then compared the affinities (Ki values) to those of other mammalian α1a/A-adrenergic receptors.

2. Materials and Methods

2.1 Tissue isolation and total RNA extraction

Animal experiments were carried out in accordance with the European Commission Directive 86/609. Gerbils were anesthetized with pentobarbital (50 mg/kg, i.p.) and then decapitated with approval by Creighton University's Institutional Animal Care and Use Committee. Spiral modiolar arteries (SMA) were removed, frozen in liquid nitrogen and stored at −70°C. Total RNA was extracted with TRIzol Reagent (Invitrogen).

2.2 Amplification of gerbil α1a-adrenergic receptor cDNA by RT-PCR

Approximately 1 μg of SMA total RNA was reverse transcribed in 20 μl containing 20 mM Tris HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 25 pmol antisense gene-specific primer, 20 units RNasin, 1 mM dNTP and 50 units murine leukemia virus reverse transcriptase and incubated for 50 min at 42°C, followed by 5 min at 99°C in a PerkinElmer 480 DNA Thermal Cycler. The polymerase chain reaction (PCR) was performed on 10 μl of the reverse-transcribed cDNA reaction containing 25 pmol of sense primer and 2.5 units of Platinum Pfx DNA polymerase (Invitrogen). PCR conditions included denaturation for 5 min at 95°C, followed by 39 one-minute cycles at 95°C, 1 min at 58°C, 3 min at 72°C and a final extension of 10 min at 72°C. RT-PCR products were visualized on a 3% agarose gel stained with ethidium bromide and documented with the 1000 gel documentation system (Bio-Rad, Hercules, CA).

Table 1 lists oligonucleotide sense and antisense primers used in RT-PCR. Primer designs were based on those previously described (Scofield et al., 1995) and the third cytoplasmic loop of the gerbil α1a-adrenergic receptor (Gruber et al., 1998). In addition, a degenerative primer was used to amplify the 5′ region of the gerbil α1a-adrenergic receptor, and a 3′ RACE (FirstChoice RLM-RACE Kit, Ambion, Austin, TX) with gerbil-specific primers from regions already sequenced was used to obtain the C-terminal sequence. PCR and 3′ RACE products were then cloned into the pCRII cloning vector using a TA Cloning Kit (Invitrogen). Clones were sequenced (ABI model 373, Life Technologies Corporation, Carlsbad, CA) and analyzed using the Wisconsin Package Version 10.1 software (Genetics Computer Group, Madison, WI).

Table 1.

Sequence and species specificity of oligonucleotide primers. Y=C/T, K=T/G, and R=A/G.

Primer Sequence Species
UP1 5′-GTAGCCAAGAGAGAAAGCCG-3′ Rat/ Mouse
DN1 5′-CAACCCACCACGATGCCCAG-3′ Rat
UP2 5′-ATGGTGYTTCTYTCKGRAAA-3′ Rat/Mouse/Human/Cow
DN2 5′-GCAGCAGACCTGCAAAAAG-3′ Rat
UP3 5′-AGGGATCGGCCAGGATTACA-3′ Rat/ Gerbil
DN3 5′-ATCAGCAGGACCTAGCGTCAA-3′ Gerbil
UP4 5′-ATGGTGTTTCTTTCGGAAAATGC-3′ Gerbil
UP-EcoRI 5′-GAATTCGAATTCGCCACCATGGTGTTTCTTTCGGAAAA-3′ Gerbil
DN-NotI 5′-GCGGCCGCGCGGCCGCCTAGACTTCCTCCCCGTTTTCG-3′ Gerbil
Open in a new tab

2.3 Cloning of full length gerbil α1a-adrenergic receptor cDNA into an expression vector

The full length gerbil α1a-adrenergic receptor from total RNA was amplified by RT-PCR with primers UP4 and DN3 (Figure 1, Table 1) as described above, except Platinum Pfx DNA polymerase (Invitrogen) with 2.5 mM MgSO4 was used. Restriction digest sites and a Kozak sequence were then added by performing PCR with the sense primer UP-EcoRI containing two EcoR I digest sites and a Kozak sequence GCCACC, and the antisense primer DN-NotI containing two Not I digest sites (Table 1) (Kozak, 1987). The PCR product was cloned into the pcDNA3.1+ vector (Invitrogen) using restriction digest sites EcoR I and Not I. The DNA was sequenced and analyzed.

Figure 1.

Figure 1

Open in a new tab

Oligonucleotide primers and the corresponding PCR products. Upstream sense primers are UP1, UP2, UP3 and UP4; and downstream antisense primers are DN1, DN2 and DN3. Primer locations with respect to their complementary cDNA sequence are indicated by boxes. Primer sequences and species specificity are listed in Table 1. PCR products, their size and respective locations on the gerbil α1a-adrenergic receptor gene are indicated by arrows.

2.4 Transfection of recombinant gerbil α1a-adrenergic receptor

COS-1 cells in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum were grown to confluence in T-75 cell culture flasks at 37°C in a humidified 95% air, 5% CO2 incubator. Recombinant gerbil α1a-adrenergic receptor was transiently transfected into several flasks of COS-1 cells using FuGENE 6 Reagent with a 2:1 ratio according to the manufacturer's protocol (Roche, Indianapolis, IN). To confirm transfection of the recombinant gerbil α1a-adrenergic receptor, receptor expression (fmol/mg protein) was quantified by [3H]prazosin binding (See Subsection 2.5). We determined specific binding (total minus nonspecific binding in the presence of 10 μM phentolamine) of a single concentration of [3H]prazosin and then calculated receptor expression according to the law of mass action. α1a-Adrenergic receptor expression in the transfected COS-1 cells was 560 ± 95 fmol/mg protein, n = 3.

2.5 Cell membrane preparation and radioligand binding

Cell membrane preparation and radioligand binding were performed as described previously (Bockman et al., 2004). Briefly, 48 hr after transfection, cells were rinsed with phosphate-buffered saline, scraped and then harvested by centrifugation at 4°C for 15 minutes at 1000g. The pellet was suspended in ice-cold 50 mM Tris buffer (pH 7.4), and then homogenized twice at 22,000 rpm for 10 seconds. The homogenate was centrifuged at 4°C for 20 minutes at 30,000g. After resuspending the membrane pellet in Tris buffer, it was washed twice by centrifugation and stored at −70°C. The pellet was resuspended in 50 mM Tris buffer and homogenized prior to analysis. Protein concentrations were determined by the Bradford assay (Pierce, Rockford, USA) (Bradford, 1976).

Membranes expressing transfected α1a-adrenergic receptor were incubated with [3H]prazosin in the presence of increasing concentrations of unlabeled drugs for 30 min at 37°C. Membrane suspensions were separated by vacuum filtration through GF/B glass microfiber filters and then rinsed three times with 5 ml of ice-cold 50 mM Tris buffer. Radioactivity retained on the filters was counted by liquid scintillation spectroscopy. Radioligand binding data were analyzed by nonlinear least-squares curve fitting to determine IC50 values. Ki values were calculated from IC50 values (Cheng and Prusoff, 1973).

2.6 Spiral Modiolar Artery Contraction and Chloroethylclonidine Inactivation of α1A-Adrenergic receptors

Concentration-response curves for norepinephrine in causing contraction of spiral modiolar arteries were obtained as previously described (Gruber et al., 1998). In the current report, the diameters of artery segments were between 55 and 67 μm at rest. Norepinephrine caused a mean maximal reduction in the vascular diameter of 12 ± 2%.

Chloroethylclonidine (CEC) irreversibly inactivates α1A –adrenergic receptors, which results in a reduction in their receptor number. α1A-Adrenergic receptors mediating contraction of the spiral modiolar artery were inactivated by pretreatment with CEC as formerly described (Xiao et al., 1998). For the present study, control concentration-response curves for norepinephrine-induced contraction were generated and subsequently washed out for 30 min. Arteries were then incubated with 100 μM CEC for 2, 5, 7 or 10 min, followed by 45 min of extensive washing to remove any unbound CEC. Concentration- response curves to norepinephrine were then repeated. Concentration-response curves were analyzed using non-linear least squares curve fitting to determine the concentration of norepinephrine causing 50% of the maximal response (EC50). Maximal contraction and EC50 values before and after CEC treatment were compared using a Student's t-test with a value of P < 0.05 accepted as a significant difference between groups. Values for maximal contraction and EC50 are means ± S.E.

2.7 Materials

BMY-7378 dihydrochloride, chloroethylclonidine dihydrochloride, 5-methylurapidil, (−)-norepinephrine bitartrate, phentolamine methanesulfonate, prazosin hydrochloride, and WB-4101 hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO). [7-methoxy-3H]prazosin ([3H]prazosin) (70–87 Ci/mmol) was obtained from PerkinElmer (Boston, MA). EcoR I and Not I were purchased from Invitrogen (Carlsbad, CA) and New England Biologicals (Ipswich, MA), respectively.

3. Results and Discussion

3.1 Molecular characteristics of the gerbil α1a-adrenergic receptor clone

In the present study, the gerbil α1a-adrenergic receptor was cloned from total RNA by RTPCR using gene-specific primers. Previously, we amplified a 175-nucleotide sequence from the third cytoplasmic loop of the gerbil α1a-adrenergic receptor in spiral modiolar arteries (Gruber et al., 1998). In addition to these primers directed against the third cytoplasmic loop, we used formerly described primers (Scofield et al., 1995) based on α1a-adrenergic receptor sequences from other species to clone portions of the gerbil α1a receptor (Table 1, Figure 1). Consequently, the 5′ region was cloned with a degenerate primer (UP2) from the open reading frame start site based on consensus sequences for the α1a-adrenergic receptor from rat, mouse, human and cow. The RT-PCR product generated from the UP2 and DN2 primers is shown in Figure 2.

Figure 2.

Figure 2

Open in a new tab

RT-PCR products from gerbil total RNA were amplified with indicated sense (UP) and antisense (DN) primers from Table 1. Lane 1: 215 bp product of primers UP1+DN1. Lane 2: 1305 bp product of primers UP2+DN2. Lane 3: 100 bp DNA ladder. Lane 4: 219 bp product of primers UP3+DN3. Lane 5: 1453 bp product of primers UP4+DN3.

The 3′ region of the gerbil α1a-adrenergic receptor was amplified by a 3′ RACE using the gene-specific primer UP3. The complete cDNA sequence was amplified by RT-PCR with primers UP4 and DN3, and the resulting PCR product (Figure 2) was cloned and sequenced (Table 2). The cDNA clone of the complete gerbil α1a-receptor consisted of 1404 nucleotides with a 1401 bp open reading frame, which had a 93.0, 93.1 and 88.1% identity compared to the cDNA nucleotide sequences of rat, mouse and human α1a-adrenergic receptors, respectively. The coding region encoded a 467 amino acid peptide with an overall amino acid composition exhibiting a high degree of sequence homology with those of α1a-adrenergic receptors from three other species. The deduced amino acid sequence of the full-length gerbil α1a receptor was 96.8, 96.4 and 91.6% identical to the amino acid sequences of rat, mouse and human α1a-receptors, respectively (Table 3).

Table 2.

Nucleotide and amino acid sequences of the gerbil α1a-adrenergic receptor (accession number HM754402).

graphic file with name nihms-337965-t0001.jpg
Open in a new tab

Table 3.

Comparison of amino acid sequences of gerbil (accession number HM754402), rat (U071126), mouse (AF031431), and human (U02569) α1a-adrenergic receptors.

graphic file with name nihms-337965-t0002.jpg
Open in a new tab

Proposed membrane spanning domains are underlined. Conserved amino acids are shaded.

α1-Adrenergic receptor subtypes are members of the G protein-coupled family of signaling proteins and mediate the actions of the sympathetic neurotransmitter norepinephrine. α1-Adrenergic receptors consist of seven transmembrane domains containing three cytoplasmic loops. The amino acid sequences of the seven transmembrane domains are highly conserved among species (Zhong and Minneman, 1999). In our study (Table 3), alignment of the amino acid residues of the gerbil α1a-receptor with those of cloned α1a-receptors from various species revealed greater than 95% identity of the seven membrane spanning domains between gerbil, rat, mouse and human. These results are consistent with the structural homology of the transmembrane domains of the α1a-receptor subtype from various species. In contrast, sequence differences commonly occur in the third cytoplasmic loop (Zhong and Minneman, 1999). In the present report, alignment of the amino acid sequences between species showed an additional glycine residue in the third cytoplasmic loop of the gerbil α1a-receptor (Table 3).

3.2 Pharmacological characteristics of the gerbil α1a-adrenergic receptor clone

The recombinant gerbil α1a-adrenergic receptor was transfected into COS-1 cells, and then directly examined in membranes by radioligand binding with [3H]prazosin (Figure 3). The pharmacological characteristics of the gerbil α1a- adrenergic receptor were determined and compared to those of other mammalian α1a/A-adrenergic receptors (Table 4). For example, affinities (Ki values) of α1-adrenergic receptor antagonists were obtained from competition curves for inhibition of [3H]prazosin binding by prazosin (nonselective), WB-4101 and 5-methylurapidil (α1A-selective) and BMY-7378 (α1D-selective) (Figure 3). Ki values in nM were 0.89 ± 0.09 for prazosin, 1.0 ± 0.1 for WB-4101, 4.9 ± 1 for 5-methylurapidil and 260 ± 60 for BMY-7378. The rank order of affinity values: prazosin > WB-4101 > 5-methylurapidil > BMY-7378 and the absolute affinity values are typical for the α1a-adrenergic receptor subtype across species. Table 4 compares our Ki values for these antagonists in the gerbil with published Ki values in human, mouse and rat and indicates the similarity between the pharmacological characteristics of the gerbil and human α1a-adrenergic receptor subtypes.

Figure 3.

Figure 3

Open in a new tab

Mean competition binding curves showing α1-adrenergic receptor antagonist inhibition of [7-methoxy-3H]prazosin (3H-prazosin) binding in membranes from COS-1 cells transiently transfected with gerbil α1a-adrenergic receptor. For each concentration of antagonists, [3H]prazosin binding is expressed as a percentage of the specific binding in the absence of any drug.

Table 4.

Comparison of affinities of assorted α1-adrenergic receptor antagonist drugs in various species.

Drug Gerbil Human Mouse Rat
Prazosin 0.89±0.09 0.13 – 0.21 0.042 – 0.48 0.26 – 0.69
WB-4101 1.0±0.1 0.23 – 1.3 0.72 – 1.4 0.063 – 2.1
5-methylurapidil 4.9±1 3.2 – 30 0.24 – 2.8 0.16 – 3.2
BMY-7378 260±60 250 95 – 720 160 – 260
Open in a new tab

Affinities are expressed as Ki values in nM. Affinities of drugs for the gerbil α1a-adrenergic receptor are mean values ± S.E. of at least 3 separate experiments on tissues from individual animals. For human, mouse and rat, affinities are ranges of values from a collection of radioligand binding studies of recombinant α1a- and native endogenous α1A-adrenergic receptors (Bockman et al., 2004; Bruchas et al., 2008; Goetz et al., 1995; Hirasawa et al., 1993; Lomasney et al., 1991; Morrow et al., 1986; Patel et al., 2001; Porter et al., 1992; Schwinn et al., 1995; Waugh et al., 2001; Weinberg et al., 1994; Xiao et al., 1998; Yang et al., 1998).

3.3 Effect of chloroethylclonidine on the α1A–adrenergic receptor- mediated vascular response of spiral modiolar arteries

Figure 4 illustrates mean concentration-response curves for norepinephrine in causing contraction of gerbil spiral modiolar arteries before and after pretreatment with 100 μM chloroethylclonidine (CEC) for 2, 5, 7 or 10 min. CEC pretreatment significantly inhibited maximal contractile responses to norepinephrine in a time-dependent manner. Norepinephrine-induced maximal contraction was reduced by 34 ± 13% at 2 min, 62 ± 10% at 5 min, 67 ± 5% at 7 min and 80 ± 4% at 10 min compared to the control maximal response, P < 0.05. These results are consistent with the kinetics of inactivation by CEC of α1A –adrenergic receptors (Xiao et al., 1998). Conversely, CEC pretreatment for 2, 5, 7 or 10 min had no effect on EC50 values of norepinephrine-induced contraction compared with the control EC50, P > 0.05 (See Figure 4 legend for mean EC50 values).

Figure 4.

Figure 4

Open in a new tab

Mean concentration-response curves for norepinephrine in causing contraction of gerbil spiral modiolar arteries before (Control) and after pretreatment with 100 μM chloroethylclonidine (CEC) for 2, 5, 7 or 10 minutes (Min). Contractile responses are normalized as the percent of maximal contraction to norepinephrine in the Control. Each curve represents mean responses of four to seven individual arteries each taken from different animals. CEC pretreatment significantly inhibited maximal contractile responses to norepinephrine compared to the control maximal response by 34 ± 13% (2 Min CEC), 62 ± 10% (5 Min CEC), 67 ± 5% (7 Min CEC) and 80 ± 4% (10 Min CEC), P < 0.05. Mean EC50 values for norepinephrine-induced contraction were 21 ± 3 μM (Control), 30 ± 6 μM (2 Min CEC), 29 ± 9 μM (5 Min CEC), 42 ± 10 μM (7 Min CEC) and 30 ± 10 μM (10 Min CEC). Mean EC50 values after CEC pretreatment were not different from the mean control EC50, P > 0.05.

Chloroethylclonidine (CEC) irreversibly inactivates α1A –adrenergic receptors, which results in a reduction in their receptor number (Bockman et al., 2004; Han et al., 1987; Xiao et al., 1998). We used CEC to inactivate α1A –adrenergic receptors and thus determine whether or not there is a receptor reserve for norepinephrine in causing α1A –adrenergic receptor-mediated contraction of the gerbil spiral modiolar artery. A receptor reserve is present when an agonist elicits a maximal response even after receptor inactivation results in reduction in receptor number (Furchgott, 1966; Ruffulo, 1982). In the present study, receptor inactivation with CEC resulted in inhibition of the maximal response to norepinephrine without a concomitant rightward shift of the concentration-response curve. Consistent with classical receptor theory, these data indicate that there is no receptor reserve for norepinephrine-induced contraction of the spiral modiolar artery (Ruffulo, 1982).

A receptor reserve is an important physiological mechanism regulating the response to neurotransmitters and other agonists (Ruffulo, 1982). For example, the presence of a receptor reserve provides a mechanism for a tissue to maintain its capacity to respond when disease or chronic drug treatment downregulates receptor number. In the present study, there was no α1A –adrenergic receptor reserve for norepinephrine-induced contraction. Thus, the contractile response of the spiral modiolar artery would be reduced by loss of receptor number, which may have important implications for cochlear blood flow. Although we did not examine cochlear blood flow, the demonstration of a lack of receptor reserve in an artery that is thought to contribute to cochlear blood flow adds to our understanding of the mechanisms controlling cochlear vasculature.

3.4 Conclusions

In addition to the rat, mouse and guinea pig, the gerbil is often used as a model in studies of the cochlear vasculature. The α1A –adrenergic receptor mediates vasoconstriction of the gerbil spiral modiolar artery, and the spiral modiolar artery regulates cochlear blood flow. However, the α1–adrenergic receptor subtype in the spiral modiolar artery has not been directly studied. Thus, we have cloned the recombinant gerbil α1a-adrenergic receptor and characterized its molecular and pharmacological properties. In summary, the 91.6% amino acid sequence identity and Kis of the cloned gerbil α1a-adrenergic receptor are similar to those of the human α1a-adrenergic receptor. These results show that the gerbil α1a-adrenergic receptor is typical of the human α1a-adrenergic receptor. Further, the results of the experiments with the inactivating agent, chloroethylclonidine, which show a lack of receptor reserve, indicate that norepinephrine-induced contraction of the spiral modiolar artery is tightly regulated by a limited number of α1A –adrenergic receptors. Thus, the gerbil spiral modiolar artery provides a useful model for further examining the role of α1A –adrenergic receptors in sensorineural hearing loss caused by impaired cochlear blood flow.

Research Highlights

  • gerbil and human α1a-adrenergic receptors share 91.6% amino acid sequence identity

  • Kis of gerbil and human α1a-adrenergic receptor are similar

  • gerbil α1a-adrenergic receptor is representative of a human α1a-adrenergic receptor

  • gerbil spiral modiolar artery is a model of α1A receptors and cochlear blood flow

Abbreviations

CEC

Chloroethylclonidine

5-MU

5-methylurapidil

RT-PCR

reverse transcription-polymerase chain reaction

RACE

rapid amplification of cDNA ends

SMA

spiral modiolar artery

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Billett TE, Thorne PR, Gavin JB. The nature and progression of injury in the organ of Corti during ischemia. Hear Res. 1989;41:189–97. doi: 10.1016/0378-5955(89)90010-5. [DOI] [PubMed] [Google Scholar]
  2. Bockman CS, Bruchas MR, Zeng W, O'Connell KA, Abel PW, Scofield MA, Dowd FJ. Submandibular gland acinar cells express multiple α1-adrenoceptor subtypes. J Pharmacol Exp Ther. 2004;311:364–72. doi: 10.1124/jpet.104.066399. [DOI] [PubMed] [Google Scholar]
  3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bruchas MR, Toews ML, Bockman CS, Abel PW. Characterization of the α1-adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland acinar cells. Eur J Pharmacol. 2008;578:349–58. doi: 10.1016/j.ejphar.2007.09.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlisle L, Aberdeen J, Forge A, Burnstock G. Neural basis for regulation of cochlear blood flow: peptidergic and adrenergic innervation of the spiral modiolar artery of the guinea pig. Hear Res. 1990;43:107–13. doi: 10.1016/0378-5955(90)90219-f. [DOI] [PubMed] [Google Scholar]
  6. Cheng YC, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol. 1973;22:3099–108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  7. Furchgott RF. The use of β-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonists complexes. In: Harper A, Simmonds B, editors. Advances in Drug Research. Vol. 3. Academic Press; London: 1966. pp. 21–55. [Google Scholar]
  8. Goetz AS, King HK, Ward SD, True TA, Rimele TJ, Saussy DL., Jr. BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors. Eur J Pharmacol. 1995;272:R5–6. doi: 10.1016/0014-2999(94)00751-r. [DOI] [PubMed] [Google Scholar]
  9. Gross G, Hanft G, Rugevics C. 5-Methyl-urapidil discriminates between subtypes of the α1-adrenoceptor. Eur J Pharmacol. 1988;151:333–5. doi: 10.1016/0014-2999(88)90819-9. [DOI] [PubMed] [Google Scholar]
  10. Gruber DD, Dang H, Shimozono M, Scofield MA, Wangemann P. α1A-adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro. Hear Res. 1998;119:113–24. doi: 10.1016/s0378-5955(98)00036-7. [DOI] [PubMed] [Google Scholar]
  11. Han C, Abel PW, Minneman KP. Heterogeneity of alpha 1-adrenergic receptors revealed by chlorethylclonidine. Mol Pharmacol. 1987;32:505–10. [PubMed] [Google Scholar]
  12. Hieble JP, Bylund DB, Clarke DE, Eikenburg DC, Langer SZ, Lefkowitz RJ, Minneman KP, Ruffolo RR., Jr. International Union of Pharmacology. X. Recommendation for nomenclature of α1-adrenoceptors: consensus update. Pharmacol Rev. 1995;47:267–70. [PubMed] [Google Scholar]
  13. Hirasawa A, Horie K, Tanaka T, Takagaki K, Murai M, Yano J, Tsujimoto G. Cloning, functional expression and tissue distribution of human cDNA for the alpha 1C-adrenergic receptor. Biochem Biophys Res Commun. 1993;195:902–9. doi: 10.1006/bbrc.1993.2130. [DOI] [PubMed] [Google Scholar]
  14. Iwasaki S, Mizuta K, Gao J, Wu R, Hoshino T. Focal microcirculation disorder induced by photochemical reaction in the guinea pig cochlea. Hear Res. 1997;108:55–64. doi: 10.1016/s0378-5955(97)00045-2. [DOI] [PubMed] [Google Scholar]
  15. Koga K, Hakuba N, Watanabe F, Shudou M, Nakagawa T, Gyo K. Transient cochlear ischemia causes delayed cell death in the organ of Corti: an experimental study in gerbils. J Comp Neurol. 2003;456:105–11. doi: 10.1002/cne.10479. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15:8125–48. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lomasney JW, Cotecchia S, Lorenz W, Leung WY, Schwinn DA, Yang-Feng TL, Brownstein M, Lefkowitz RJ, Caron MG. Molecular cloning and expression of the cDNA for the α1A-adrenergic receptor. The gene for which is located on human chromosome 5. J Biol Chem. 1991;266:6365–9. [PubMed] [Google Scholar]
  18. Mom T, Avan P, Bonfils P, Gilain L. A model of cochlear function assessment during reversible ischemia in the Mongolian gerbil. Brain Res Brain Res Protoc. 1999;4:249–57. doi: 10.1016/s1385-299x(99)00026-4. [DOI] [PubMed] [Google Scholar]
  19. Morrow AL, Creese I. Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4101 and [3H]prazosin binding. Mol Pharmacol. 1986;29:321–30. [PubMed] [Google Scholar]
  20. Nakashima T, Naganawa S, Sone M, Tominaga M, Hayashi H, Yamamoto H, Liu X, Nuttall AL. Disorders of cochlear blood flow. Brain Res Brain Res Rev. 2003;43:17–28. doi: 10.1016/s0165-0173(03)00189-9. [DOI] [PubMed] [Google Scholar]
  21. Ohlsen KA, Baldwin DL, Nuttall AL, Miller JM. Influence of topically applied adrenergic agents on cochlear blood flow. Circ Res. 1991;69:509–18. doi: 10.1161/01.res.69.2.509. [DOI] [PubMed] [Google Scholar]
  22. Patel S, Fernandez-Garcia E, Hutson PH, Patel S. An in vivo binding assay to determine central α1-adrenoceptor occupancy using [(3)H]prazosin. Brain Res Brain Res Protoc. 2001;8:191–8. doi: 10.1016/s1385-299x(01)00110-6. [DOI] [PubMed] [Google Scholar]
  23. Porter JE, Dowd FJ, Abel PW. Atypical alpha-1 adrenergic receptors on the rat parotid gland acinar cell. J Pharmacol Exp Ther. 1992;263:1062–7. [PubMed] [Google Scholar]
  24. Ruffulo RR. Important concepts of receptor theory. J Auton Pharmacol. 1982;2:277–295. doi: 10.1111/j.1474-8673.1982.tb00520.x. [DOI] [PubMed] [Google Scholar]
  25. Schwinn DA, Johnston GI, Page SO, Mosley MJ, Wilson KH, Worman NP, Campbell S, Fidock MD, Furness LM, Parry-Smith DJ, Peter B, Bailey DS. Cloning and pharmacological characterization of human Alpha-1 adrenergic receptors: sequence corrections and direct comparison with other species homologues. J Pharmacol Exp Ther. 1995;272:134–42. [PubMed] [Google Scholar]
  26. Scofield MA, Liu F, Abel PW, Jeffries WB. Quantification of steady state expression of mRNA for alpha-1 adrenergic receptor subtypes using reverse transcription and a competitive polymerase chain reaction. J Pharmacol Exp Ther. 1995;275:1035–42. [PubMed] [Google Scholar]
  27. Seidman MD, Quirk WS, Shirwany NA. Mechanisms of alterations in the microcirculation of the cochlea. Ann N Y Acad Sci. 1999;884:226–32. doi: 10.1111/j.1749-6632.1999.tb08644.x. [DOI] [PubMed] [Google Scholar]
  28. Shikowitz MJ. Sudden sensorineural hearing loss. Med Clin North Am. 1991;75:1239–50. doi: 10.1016/s0025-7125(16)30384-4. [DOI] [PubMed] [Google Scholar]
  29. Waugh DJ, Gaivin RJ, Zuscik MJ, Gonzalez-Cabrera P, Ross SA, Yun J, Perez DM. Phe-308 and Phe-312 in transmembrane domain 7 are major sites of α1-adrenergic receptor antagonist binding. Imidazoline agonists bind like antagonists. J Biol Chem. 2001;276:25366–71. doi: 10.1074/jbc.M103152200. [DOI] [PubMed] [Google Scholar]
  30. Weinberg DH, Trivedi P, Tan CP, Mitra S, Perkins-Barrow A, Borkowski D, Strader CD, Bayne M. Cloning, expression and characterization of human α adrenergic receptors α1a, α1b and α1c. Biochem Biophys Res Commun. 1994;201:1296–304. doi: 10.1006/bbrc.1994.1845. [DOI] [PubMed] [Google Scholar]
  31. Xiao L, Scofield MA, Jeffries WB. Molecular cloning, expression and characterization of cDNA encoding a mouse α1a-adrenoceptor. Br J Pharmacol. 1998;124:213–21. doi: 10.1038/sj.bjp.0701812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yang M, Reese J, Cotecchia S, Michel MC. Murine Alpha1-adrenoceptor subtypes. I. Radioligand binding studies. J Pharmacol Exp Ther. 1998;286:841–7. [PubMed] [Google Scholar]
  33. Zhong H, Minneman KP. α1-Adrenoceptor subtypes. Eur J Pharmacol. 1999;375:261–76. doi: 10.1016/s0014-2999(99)00222-8. [DOI] [PubMed] [Google Scholar]

RESOURCES