Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.
1. Effects on rod phototransduction following manipulation of retinal CO2-HCO3- and H+ fluxes were studied in dark-adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction-pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3-] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3- across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM-sodium bicarbonate to replace half the buffer of a nominally CO2-HCO3(-)-free (12 mM-phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM-AAA into bicarbonate-containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA-bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate-AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA-containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity completely. The decay proceeded regardless of whether bicarbonate and AAA were present or not. 8. Rods that had lost their photosensitivity in DIDS recovered almost fully when the pH of the DIDS perfusate was raised to 8.5. They also recovered when DIDS was washed out with bicarbonate Ringer solution at constant pH (7.5). 9. It is proposed that all our treatments ultimately modulate the intracellular pH of the rods which is determined by the relative rates of H+ leakage and HCO3- transport into the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Donner
K
K
Department of Zoology, University of Helsinki, Finland.
Hemilä
S
S
Kalamkarov
G
G
Koskelainen
A
A
Shevchenko
T
T
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000086:Acetazolamide / Q000494:pharmacology; D000557:Ambystoma; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D003623:Dark Adaptation / Q000502:physiology; D004596:Electroretinography / Q000187:drug effects; D010775:Photic Stimulation; D010786:Photoreceptor Cells / Q000187:drug effects / Q000502:physiology*; D011896:Rana temporaria
D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D003432:Cross-Linking Reagents; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D002256:Carbonic Anhydrases; D006152:Cyclic GMP; D000086:Acetazolamide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid