Na(+)-K+(NH4+)-2Cl- cotransport in medullary thick ascending limb: control by PKA, PKC, and 20-HETE

Am J Physiol. 1996 Aug;271(2 Pt 1):C455-63. doi: 10.1152/ajpcell.1996.271.2.C455.


Cell pH was monitored in suspensions of medullary thick ascending limbs (MTALs) of rat kidney to determine possible effects of various transduction pathways on apical Na(+)-K+ (NH4+)-2Cl- cotransport, the activity of which was measured as the bumetanide-sensitive component of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8-Bromoadenosine 3',5'-cyclic monophosphate stimulated cotransport activity through activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), since the cAMP effect was abolished by N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89); stimulation by cAMP (P < 0.02) was observed even when other Na+, Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2-carboxylate, and barium, which indicates that cotransport was directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated cotransport activity (P < 0.03), which was abolished by protein kinase C (PKC) blockade by staurosporine. In contrast, cotransport activity was reduced (P < 0.001) by arachidonic acid or 20-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic acid or ionomycin was abolished by econazole and SKF-525A that inhibit cytochrome P-450-dependent monoxygenase, which produces 20-HETE from arachidonic acid in the MTAL, and the ionomycin effect was prevented when phospholipase A2 (PLA2) was blocked by 4-bromophenacyl bromide or oleyloxyethyl phosphorylcholine. The results demonstrate that MTAL apical Na(+)-K+(NH4+)-2Cl- cotransport is stimulated by PKA and PKC and inhibited by 20-HETE that may be produced after a rise in [Ca2+]i through PLA2 activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ammonia / antagonists & inhibitors
  • Ammonia / metabolism*
  • Animals
  • Arachidonic Acid / pharmacology
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / metabolism*
  • Cyclic AMP / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / physiology*
  • Hydroxyeicosatetraenoic Acids / pharmacology*
  • Kidney Medulla
  • Loop of Henle / drug effects
  • Loop of Henle / metabolism*
  • Male
  • Membrane Proteins / metabolism
  • Protein Kinase C / physiology*
  • Rats
  • Rats, Sprague-Dawley
  • Sodium-Potassium-Chloride Symporters


  • Carrier Proteins
  • Hydroxyeicosatetraenoic Acids
  • Membrane Proteins
  • Sodium-Potassium-Chloride Symporters
  • Arachidonic Acid
  • Ammonia
  • 20-hydroxy-5,8,11,14-eicosatetraenoic acid
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C