Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (I sc ) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. I sc under control conditions was 1090 ± 21 μA/cm2 (n= 213) in gerbil SMC and 2001 ± 95 μA/cm2 (n= 6) in murine SMC. Direct stimulation of adenylate cyclase with 10-5 m forskolin but not with 10−5 m 1,9-dideoxy-forskolin resulted in an increase in the I sc by a factor of 1.14 ± 0.01 (n= 6). The vasopressin-receptor agonist 10−8 m Arg8-vasopressin had no significant effect on I sc in gerbil and murine SMC. The β-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated I sc with an EC 50 of (6 ± 2) × 10−7 m (n= 28), (3 ± 1) × 10−6 m (n= 40) and (7 ± 2) × 10−6 m (n= 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC 50 of (5 ± 2) × 10−7 m (n= 8). The β-antagonist 10−4 m propanolol completely inhibited 2 × 10−5 m isoproterenol-induced stimulation of I sc . The β-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I sc with a K DB of 1 × 10−7 m (pK DB = 6.96 ± 0.15, n= 14), 1 × 10−7 m (pK DB = 7.01 ± 0.14, n= 15), 2 × 10−9 m (pK DB = 8.73 ± 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a K DB of 1 × 10−10 m (pK DB = 9.94 ± 0.55, n= 9). RT-PCR of total RNA isolated from SV using primers specific for the β1-, β2- and β3-adrenergic receptors revealed products of the predicted sizes for the β1- and β2- but not the β3-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K+ secretion in SMC is under the control of β1-adrenergic receptors but not β2-adrenergic or vasopressin-receptors and that the β1-subtype is the primary β-adrenergic receptor in SV although SV contains transcripts for both β1- and β2-adrenergic receptors.
The Journal of Membrane Biology – Springer Journals
Published: Jun 1, 2000
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