Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells.
It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.
Motais
R
R
Laboratoire Jean Maetz, Département de Biologie du Commissariat à l'Energie Atomique, Villefranche-sur-Mer, France.
Borgese
F
F
Scheuring
U
U
Garcia-Romeu
F
F
D000818:Animals; D002352:Carrier Proteins / Q000097:blood*; D002712:Chlorides / Q000097:blood; D000242:Cyclic AMP / Q000097:blood / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007474:Ion Exchange; D011433:Propranolol / Q000494:pharmacology; D012964:Sodium / Q000097:blood; D017923:Sodium-Hydrogen Exchangers; D014337:Trout
D002352:Carrier Proteins; D002712:Chlorides; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D011433:Propranolol; D000242:Cyclic AMP; D005976:Glutaral