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

1996 ◽  
Vol 271 (2) ◽  
pp. C455-C463 ◽  
Author(s):  
H. Amlal ◽  
C. Legoff ◽  
C. Vernimmen ◽  
M. Paillard ◽  
M. Bichara
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Medullary Thick Ascending Limb ◽  
Kinase C ◽  
Dependent Protein ◽  
Cytochrome P 450

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.

ANG II controls Na+-K+( NH 4 + )-2Cl−cotransport via 20-HETE and PKC in medullary thick ascending limb

1998 ◽  
Vol 274 (4) ◽  
pp. C1047-C1056 ◽  
Author(s):  
Hassane Amlal ◽  
Christian LeGoff ◽  
Catherine Vernimmen ◽  
Manoocher Soleimani ◽  
Michel Paillard ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Diacylglycerol Lipase ◽  
Medullary Thick Ascending Limb ◽  
Kinase C ◽  
Cell Ph ◽  
Cytochrome P 450

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na+-K+([Formula: see text])-2Cl−cotransport. ANG II at 10−16to 10−12 M inhibited 30–50% ( P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10−12 M ANG II level cotransport activity; eventually, 10−6 M ANG II stimulated 34% cotransport activity ( P < 0.003). Inhibition by 10−12M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10−12 M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10−6 M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus ≤10−12 M ANG II inhibits via 20-HETE, whereas ≥5 × 10−11 M ANG II stimulates via PKC Na+-K+([Formula: see text])-2Cl−cotransport; all ANG II effects involve AT1 receptors and PLC activation.

cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

1988 ◽  
Vol 255 (2) ◽  
pp. F292-F300 ◽  
Author(s):  
S. M. Gapstur ◽  
S. Homma ◽  
T. P. Dousa
Keyword(s):  
Protein Kinase ◽  
Binding Proteins ◽  
Rat Kidney ◽  
Photoaffinity Labeling ◽  
Type I ◽  
Thick Ascending Limb ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

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