α1A-Adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro

Several lines of evidence suggest that cochlear blood flow is under the control of the sympathetic nervous system and that this control is mediated via alpha-adrenergic receptors. The goal of the present study was to determine whether alpha-adrenergic receptors mediate vasoconstriction of the spiral modiolar artery and, if so, to determine which subtype dominates this response. Vascular diameter was measured with video microscopy in the isolated superfused spiral modiolar artery in vitro. The diameter of the spiral modiolar artery under control conditions was 61 +/- 2 microm (n = 60). Spontaneous vasomotion was observed in most specimens. Addition of norepinephrine to the superfusate caused a phasic vasoconstriction and an increase in the amplitude of vasomotion. These effects were limited to the vicinity of arteriolar branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction occurred with EC50 of (1.9 +/- 0.4) x 10(-5) M (n = 44) and the vascular diameter was maximally reduced by a factor of 0.87 +/- 0.01 (n = 29). Neither the phasic nature nor the EC50 of the norepinephrine-induced vasoconstrictions was altered in the presence of the beta2-adrenergic receptor antagonist 10(-5) M ICI118551 or the nitric oxide synthase inhibitor 10(-4) M NOARG. In contrast, the alpha2-adrenergic receptor antagonist 10(-7) M yohimbine and the alpha1-adrenergic receptor antagonist 10(-9) and 10(-8) M prazosin caused a significant shift in the dose-response curve. The affinity constants (K(DB)) for yohimbine and prazosin were (5+/-2) x 10(-8) M (n=4) and (2.0+/-0.7) x 10(-10) M (n=18), respectively. The alpha1A-adrenergic receptor antagonist 10(-8) M 5-methyl urapidil and the alpha1D-adrenergic receptors antagonist 5 x 10(-6) M BMY7378 caused a significant shift in the dose-response curve. The K(DB) values for 5-methyl urapidil and for BMY7378 were (2.7 +/- 0.7) x 10(-10) M (n = 8) and (4.4 +/- 2.7) x 10(-7) M (n = 8), respectively. Further, total RNA was isolated from microdissected spiral modiolar arteries and the presence of transcripts for alpha1-adrenergic receptor subtypes was determined by reverse transcription polymerase chain reaction (RT-PCR). Primers specific for gerbil alpha1-adrenergic receptor subtypes were developed using RNA from rat and gerbil brain. Analysis of RNA extracted from the spiral modiolar artery revealed RT-PCR products of the appropriate size for the alpha1A-adrenergic receptor, however, no evidence for the alpha1B- and alpha1D-adrenergic receptor was found. Further, analysis of RNA extracted from blood, which was a contaminant of the microdissected spiral modiolar arteries, revealed no RT-PCR products. Sequence analysis of the RT-PCR product of the alpha1A-adrenergic receptor from the spiral modiolar artery confirmed its identity. Identity between the 175 nt gerbil sequence fragment and the known rat, mouse and human alpha1A-adrenergic receptor sequences was 90.9, 92.0 and 85.2%, respectively. These observations demonstrate that the spiral modiolar artery contains alpha1A-adrenergic receptors which mediate vasoconstriction at branch points.