Visualization of nitric oxide production and intracellular calcium in juxtamedullary afferent arteriolar endothelial cells.
OBJECTIVE
Nitric oxide (NO) is an important signal transmitter with multiple haemodynamic functions in the kidney. Study of these is complicated by the difficulty in measuring NO directly or visualizing its production. Recently the synthesis of a group of new NO-sensitive fluorescent dyes, diaminofluoresceins (DAF), suitable for imaging applications has been reported. We attempted to use one DAF (DAF-2 DA) to investigate the relationship between endothelial calcium, NO production and afferent arteriolar reactivity.


METHODS
We used the isolated, perfused juxtamedullary nephron preparation (JMN) and loaded the afferent arteriolar endothelium with Fura-2 AM and DAF-2 DA (4,5-diaminofluorescein-2-diacetyl). After in vitro calibration of the imaging system, we measured Fura-2 and DAF-2 fluorescence in single endothelial cells of afferent arterioles (AA) perfused at a pressure of 100 mmHg.


RESULTS
Carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO) (10-3 m), a specific NO scavenger, decreased DAF-2 fluorescence in the endothelium by 16.1% and the mid-afferent arteriolar diameter by 10.2%, and increased endothelial calcium by 17.8%. Nomega-nitro-l-arginine methyl ester (l-NAME) (10-4 m) decreased fluorescence intensity of DAF-2 by 18.6%, increased cellular calcium level by 19.7% and constricted the vessels by 11.6%. Addition of carbachol (10-4 m) increased average DAF-2 fluorescence by 22.8% and endothelial calcium concentration by 28.9%, whereas the arteriolar diameter remained essentially unchanged. Carbachol failed to increase DAF-2 fluorescence when administered after l-NAME pre-treatment.


CONCLUSIONS
We conclude that endothelial NO homeostasis is an important determinant of AA reactivity and suggest that DAF are suitable for real-time imaging of afferent arteriolar NO production in the isolated, perfused JMN and may be used in combination with calcium-sensitive fluorophores. We have found that NO reduction by carboxy-PTIO or l-NAME increases endothelial calcium, suggesting involvement of calcium signalling in an autocrine NO production feedback in the endothelium. This method should help to further clarify the role of endothelial NO in renal haemodynamics.
Pittner
J
J
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Liu
R
R
Brown
R
R
Wolgast
M
M
Persson
A E G
AE
D000818:Animals; D001160:Arterioles / Q000378:metabolism; D001565:Benzoates / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D002217:Carbachol / Q000494:pharmacology; D004727:Endothelium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D005453:Fluorescence; D016166:Free Radical Scavengers; D007093:Imidazoles / Q000494:pharmacology; D007202:Indicators and Reagents; D007679:Kidney Medulla / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009399:Nephrons / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley
C414689:4,5-diaminofluorescein diacetate; D001565:Benzoates; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D007093:Imidazoles; D007202:Indicators and Reagents; C079393:1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole; D009569:Nitric Oxide; D002217:Carbachol; D002118:Calcium; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester