Pharmacological characterization of the P2X(7) receptor on human macrophages using the patch-clamp technique.

Whole-cell patch-clamp recordings were made from macrophages derived from human monocytes that had been cultured for 5-7 days. The P2X agonists ATP (100 microM) and 2',3'-(4-benzoyl)-benzoyl ATP (BzATP, 100 microM) induced inward currents. A second application of the agonists was characterized by strong desensitisation of the maximum current. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a non-specific P2X antagonist, and 1-( N, O- bis[5-isoquinolinesulphonyl]- N-methyl- L-tyrosyl)-4-phenylpiperazine (KN62), a potent P2X(7) antagonist at the human receptor, both reduced the ATP-induced inward current. KN62 also inhibited the BzATP-induced current. The P2X(7) antagonist Coomassie Brilliant Blue G (BBG), believed to be potent at the human but even more so potent at the rat receptor, did not reduce the BzATP-induced inward current significantly. These results indicate that the native P2X(7) receptor subtype is expressed in human macrophages and that this receptor subtype is involved in the ATP-mediated inward current. Our experiments suggest that other P2X receptors also appear to be involved in the ATP-mediated current in human monocyte-derived macrophages.

Identification of inwardly rectifying potassium channels in bovine retinal and choroidal endothelial cells.

Ion channels were studied using the whole-cell patch clamp technique in bovine retinal and choroidal microvascular endothelial cells (MVEC) cultured under the same conditions. The two types of MVEC expressed inward currents at hyperpolarizing voltage steps and showed small outward currents at depolarizing steps. The extrapolated reversal potentials of the inward currents were near to the potassium equilibrium potential. Cs(+) and the K(+) channel blocker TEA reduced the amplitudes of the currents indicating the selectivity and permeability for potassium. This was confirmed by changes of outside K(+) concentration shifting the I-V curves to the right. RT-PCR studies revealed the presence of mRNA of Kir2.1, an inwardly rectifying K(+) channel, in retinal and choroidal MVEC. The profile of the small outward currents is related to the Kv family but not identical with the Kv1.4 subtype.

Voltage-dependent potassium channels in cytokeratin-positive and cytokeratin-negative microvascular endothelial cells of the corpus luteum.

We have recently described cytokeratin-positive (CK(+)) and cytokeratin-negative (CK(-)) microvascular endothelial cells (MVECs) in the bovine corpus luteum. The two phenotypes show a different expression and release of adhesion molecules and cytokines. Since secretion of mediators is specifically regulated by the electrophysiological membrane parameters, this report will compare voltage-dependent potassium (K(+)) channels in the two cell types cultured under the same conditions. CK(+) and CK(-) MVECs derived from the microvascular bed of one organ differ in their K(+)-channel characteristics. In CK(-) MVECs, an inwardly rectifying K(+) current was discovered, showing the characteristics of the Kir2.1. CK(+) MVECs displayed a voltage-dependent K(+) current that activates rapidly on depolarization and inactivates very slowly, and is associated with a member of the Kv family. The mRNA for Kir2.1 was identified by RT-PCR in CK(-) and CK(+) MVECs, but there was no evidence of Kv1.4 mRNA in either of them. The function of Kir2.1 in CK(-) MVECs might be induced during cultivation, whereas CK(+) MVECs appear to be more resistant to environmental conditions and do not express an inward current.