Effect of efflux of guanosine 3',5' cyclic monophosphate (cGMP) on the regulation of intracellular levels of cGMP in the inner medullary collecting duct.
Guanosine 3',5' cyclic monophosphate (cGMP) acts as a second messenger in the inner medullary collecting duct (IMCD) where it inhibits sodium transport; therefore, it is important to investigate processes that regulate intracellular cGMP levels. We hypothesized that efflux of cGMP is a major mechanism in this process. IMCDs were isolated from rat kidneys and exposed to atrial natriuretic peptide (ANP) for 0, 3, and 20 min in buffer with or without isobutyl methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor. Extracellular and intracellular cGMP levels were measured by radioimmunoassay. After cGMP production was stimulated by addition of ANP (10(-7) M), cGMP efflux was 3.29 +/- 0.60 fmol/microgram.min at 3 min (P = 0.016) and 0.51 +/- 0.25 fmol/microgram.min at 20 min (NS). Intracellular cGMP peaked at 3 min at 26.66 +/- 4.84 fmol/microgram (P = 0.017) and decreased to 12.98 +/- 2.76 fmol/microgram at 20 min (NS). Since PDEs were inhibited, these data suggest that efflux regulates intracellular cGMP. Efflux was correlated with intracellular cGMP levels (r = 0.97). After 3 min of stimulation with 10(-9) M ANP, efflux was 2.0 +/- 0.3 fmol/microgram.min, while intracellular cGMP content was 13.8 +/- 3.6 fmol/microgram. With 10(-8) M ANP, efflux was 3.5 +/- 0.7 fmol/microgram.min, while intracellular content was 20.5 +/- 7.6 fmol/microgram; and at 10(-7) M ANP, efflux was 5.1 +/- 0.6 fmol/microgram.min and intracellular content was 26.6 +/- 8.0 fmol/microgram. By 20 min, efflux and intracellular levels had returned to control values. Finally, we measured, efflux and PDE activity in the absence of IBMX. Efflux was approximately 15% of PDE activity (N = 7). We conclude that cGMP efflux is concentration-dependent and, under some circumstances, may be an important regulator of intracellular cGMP levels in isolated IMCDs.
Stoos
B A
BA
Department of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI 48202, USA.
Garvin
J L
JL
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D006152:Cyclic GMP / Q000032:analysis / Q000378:metabolism*; D007679:Kidney Medulla / Q000737:chemistry / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D009320:Atrial Natriuretic Factor; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine