Activation of a Cl-dependent K flux by cAMP in pig red cells

Abstract

Activation of a Cl-dependent K flux by adenosine 3',5'-cyclic monophosphate (cAMP) was characterized in pig red cells, a cell type that lacks both the Ca-activated K channel and the Na-K-Cl cotransport pathway. As in other red cells, both Cl-dependent K efflux and K influx are stimulated on cell swelling. Although pig red cells fail to respond to beta-adrenergic stimuli, it is possible to raise the intracellular cAMP content by preincubating cells in the presence of 1 mM cAMP. The Cl-dependent K flux was compared in cells having a basal cAMP content of approximately 0.29 nmol/g hemoglobin vs. cAMP-loaded cells having approximately 8.4 nmol cAMP/g hemoglobin. Loading with cAMP stimulated both Cl-dependent K efflux and influx of hypotonically swollen cells. In maximally swollen cells whose volume was increased by approximately 17%, the Cl-dependent Rb influx occurs with a maximum velocity (Vmax) of 17.9 +/- 3.2 mumol.g hemoglobin (Hb)-1.h-1 and Km for Rb of 22.9 +/- 4.1 mM. In cAMP-loaded cells, both Vmax and Km were increased to 59.8 +/- 8.5 mumol.g Hb-1.h-1 and 63.1 +/- 8.8 mM, respectively. The Cl-dependent Rb influx is much larger in young cells than in old cells. However, both cell types respond to cAMP activation. Whereas cAMP and its analogues, 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate are stimulatory, AMP and guanosine 3',5'-cyclic monophosphate (cGMP) are not. These findings suggest that, like other ion transport systems, the Cl-dependent K flux of pig red cells is endowed with the capacity to respond to cAMP.

Volume-activated Na/H exchange activity in fetal and adult pig red cells: Inhibition by cyclic AMP

“…As in fetal cells, adult cells lost Na but not K upon shrinkage and lost K but not Na upon enlargement. The results of a detailed investigation on K flux of hypotonically swollen cells have been presented elsewhere (Kim et al, 1989). We found that the K flux is chloride dependent and stimulated by increased cyclic AMP content.…”

“…However, we (Sergeant &Kim, 1985;Kim et al, 1989) and others (Garay, 1982) have shown that the cyclic AMP content of erythrocytes can still be raised far in excess of basal levels by incubation of cells with exogenously added cAMP. Since pig erythrocytes can also be loaded with cyclic nucleotides, it was of interest to determine whether cyclic nucleotides play a role in the control of the Na/H exchanger.…”

“…Pig erythrocytes were loaded with cyclic AMP and cyclic GMP by the method described elsewhere (Sergeant & Kim, 1985;Kim et al, 1989). Briefly, cells were incubated with 1 mM cyclic nucleotides in order to increase the intracellular cyclic nucleotide content.…”

“…Moreover, cell swelling activates the ouabain-resistant chloride-dependent K flux. Increased cyclic AMP content, which can be accomplished by incubation of ceils with exogenously added cyclic AMP, results in an augmentation of ouabain-resistant chloride-dependent K flux in normal and swollen cells (Kim et al, 1989).…”

Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells

“…Kinetic manifestations of the volume-sensitive biochemical control processes have been described by several investigators (2, 10, 17-19, 24, 25, 27). For example, transition times ranging from 5 to 25 min have been reported for activation of volume-sensitive ion flux pathways in red blood cells (RBCs) from an array of vertebrate species (10,18,20,27,34). Delays in activation of these pathways reflect the temporal characteristics of the volume-sensitive biochemical processes responsible for transporter regulation.…”