title: β-Adrenergic receptor stimulation increases surface NKCC2 expression in rat thick ascending limbs in a process inhibited by phosphodiesterase 4.
abstract: The thick ascending limb of the loop of Henle (THAL) reabsorbs ∼30% of the filtered NaCl in a process mediated by the apical Na-K-2Cl cotransporter NKCC2. Stimulation of β-adrenergic receptors in the THAL enhances NaCl reabsorption and increases intracellular cAMP. We found that intracellular cAMP stimulates NKCC2 trafficking to the apical membrane via protein kinase A (PKA). Several cAMP-specific phosphodiesterases (PDE) have been identified in rat THALs, and PDE4 decreases cAMP generated by β-adrenergic stimulation in other cells. However, it is not known whether β-adrenergic receptors activation stimulates NKCC2 trafficking. Thus we hypothesized that β-adrenergic receptor stimulation enhances THAL apical membrane NKCC2 expression via the PKA pathway and PDE4 blunts this effect. THAL suspensions were obtained from Sprague-Dawley rats, and surface NKCC2 expression was measured by surface biotinylation and Western blot. Incubation of THALs with the β-adrenergic receptor agonist isoproterenol at 0.5 and 1.0 μM increased surface NKCC2 by 17 ± 1 and 29 ± 5% respectively (P < 0.05). Preventing cAMP degradation with 3-isobutyl-methylxanthine (IBMX; a nonselective phosphodiesterase inhibitor) enhanced isoproterenol-stimulated surface NKCC2 expression to 51 ± 7% (P < 0.05 vs. isoproterenol). The β-adrenergic receptor antagonist propranolol or the PKA inhibitor H-89 completely blocked isoproterenol + IBMX-induced increase on surface NKCC2, while propranolol or H-89 alone had no effect. Selective inhibition of PDE4 with rolipram (20 μM) potentiated the effect of isoproterenol on surface NKCC2 and increased cAMP levels. We concluded that β-adrenergic receptor stimulation enhances surface NKCC2 expression in the THALs via PKA and PDE4 blunts this effect.
authors.affiliation: Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, MI 48202, USA.
authors.forename: Mohammed Z
authors.initials: MZ
authors.lastname: Haque
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Caceres
authors.forename: Pablo A
authors.initials: PA
authors.lastname: Ortiz
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000187:drug effects / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008138:Loop of Henle / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D023421:Models, Animal; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000187:drug effects* / Q000502:physiology*; D012965:Sodium Chloride / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000187:drug effects / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D010726:Phosphodiesterase Inhibitors; D011943:Receptors, Adrenergic, beta; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D013449:Sulfonamides; D012965:Sodium Chloride; D011433:Propranolol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine