This article concerns the identification of different types of voltage-gated Na ؉ channels and of muscarinic and purinergic receptors that are expressed in human erythroid precursor cells and red cell ghosts. We analyzed, by RT-PCR, RNA that was extracted from purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes in 7 to 14 days. These extracts were free of white cell and platelet contamination. Two types of voltage-gated, tetrodotoxin-sensitive Na ؉ channels were found. These were Nav1.4 and Nav1.7, the former known to be present in skeletal muscle and the latter in peripheral nerve. By using a pan Na ؉ channel antibody and Western blotting, an immunoreactive channel was detected in ghosts of human red blood cells, consistent with the expression of these two channels. The transcripts for four of the five known subtypes of muscarinic receptors were also identified, including subtypes M2, M3, M4, and M5, whereas subtype M1 was not found. Expression was also detected for the purinergic type receptors P2X1, P2X4, P2X7, and P2Y1 whereas types P2Y2, P2Y4, and P2Y6 were not found. We also searched for but did not find transcripts for hBNP-1, a type 1b human brain sodium phosphate cotransporter, and cystic fibrosis transmembrane conductance regulator (CFTR). Implications regarding the presence of these different types of channels and receptors in human red blood cells and their functional significance are discussed.

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na+ and Ca2+ pumps. The formation of the Na+- and Ca2+-dependent phosphointermediates of both types of pumps (ENa-P and ECa-P) that conventionally can be labeled with trace amounts of [γ-3P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [γ-3P]ATP in the pool. Running the pumps forward with either Na+ or Ca2+ removes pool ATP and allows the normal formation of labeled ENa-P or ECa-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10–15 minutes. We observed that when unlabeled “caged ATP” is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of ENa-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na+ (without Mg2+) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of 45Ca by the Ca2+ pump, analogous to the Na+ pump uptake of 22Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na+ and Ca2+ pumps.

1 The e ect of bradykinin on the Na + -K + pump of airway smooth muscle was investigated by measuring ouabain-sensitive 86 Rb + uptake in cultured guinea-pig tracheal smooth muscle cells. 2 Bradykinin induced a concentration-dependent increase in ouabain-sensitive 86 Rb + uptake, with an EC 50 of 3 nM (pD 2 =8.50+0.10). Stimulation was not a ected by indomethacin (1 mM) suggesting that it is not mediated by cycloxygenase products of arachidonic acid. 3 The B 1 receptor agonists Lys-des-Arg 9 -bradykinin and des-Arg 9 -bradykinin had no e ect on ouabain-sensitive 86 Rb + uptake. In contrast, the B 1 and B 2 receptor agonist Lys-bradykinin induced a concentration-dependent increase in ouabain-sensitive 86 Rb + uptake with an EC 50 of 6 nM (pD 2 =8.21+0.20). 4 The B 1 receptor antagonist des-Arg 10 -HOE 140 (1 mM) had no e ect on bradykinin-stimulated ouabain-sensitive 86 Rb + uptake. The B 2 receptor antagonists HOE 140 and WIN 64338 antagonized bradykinin-stimulated ouabain-sensitive 86 Rb + uptake with pK B values (7log M) of 8.20+0.08 and 8.11+0.20 respectively. 5 Reducing extracellular Na + from 146 mM to 11 mM caused a 53.5% decrease in basal ouabainsensitive 86 Rb + uptake and abolished bradykinin-induced uptake. Two inhibitors of the Na + -H + exchanger, methylisobutyl-amiloride (MIA; 1 ± 100 mM) and ethylisopropyl-amiloride (EIPA; 0.1 ± 10 mM), inhibited bradykinin-stimulated ouabain-sensitive 86 Rb + uptake without a ecting basal uptake. 6 These results suggest that bradykinin increases Na + -K + pump activity of guinea-pig tracheal smooth muscle via stimulation of B 2 receptors and activation of the Na + -H + exchanger.