Cyclic adenosine 3',5'-monophosphate increases the open probability of potassium channels in activated human T cells.

In the present report we describe a cAMP-responsive K channel in activated human T cells. Single channel events were recorded using the patch-clamp technique in cell-attached-patch configuration. The channel was K selective, as determined by reversal potentials under different K gradients, and displayed voltage-independent gating. When the applied potential (Vp) was equal to zero, the conductance of the channel was 21.8 +/- 0.9 pS with 150 mM K in the electrode. Typical patches contained between two and seven channels that were relatively quiet, or silent, before agonist stimulation. Adenosine (20-30 microM) increased the average open time probability from 0.017 +/- 0.008 to 0.108 +/- 0.054 over a period of 108 s. Subsequent addition of the phosphodiesterase inhibitor Ro 20-1724 (0.5 mM) increased the probability of being in the open state to 1.155 +/- 0.407 over a period of 180 s. Channel kinetics were well described by assuming two open and two closed states. Exponential time constants for the open states were 0.51 +/- 0.06 and 4.34 +/- 0.31 ms, and closed state time constants were 0.58 +/- 0.05 and 10.1 3 +/- 2.32 ms. In addition, extracellular ATP (0.3-1.0 mM) decreased channel activity. Moreover, Rp-cAMP (0.5-1.0 mM), an antagonist that specifically blocks the ability of cAMP to bind and activate protein kinase A, failed to inhibit adenosine- and Ro 20-1724-induced increases in channel activity, implying a direct action of cAMP on channel gating.

Ethanol increases K+ conductance in human T-cells.

Ethanol increased the open probability of a K channel in cultured human T-cells. Single-channel currents were studied using the patch-clamp technique in cell-attached configuration. Ethanol increased the number of simultaneously active channels and subsequent current maxima at concentrations of 35 and 50 mM from control levels of 3.2 to 4.6 pA and 8.4 pA, respectively. Current responses were elicited by a 80 mV hyperpolarizing pulse following adjustment to the same resting potential. The channel was K-selective as determined by the reversal potentials under different Na-K gradients. When the pipette contained 155 mM K, single-channel currents had a slope conductance of 28 pS and a reversal potential of -4.3 mV. Baseline current levels often shifted in a negative direction during the application of ethanol, indicating a hyperpolarization of the membrane potential and an increase in channel activity.

A comparison of K+ channel characteristics in human T cells: perforated-patch versus whole-cell recording techniques.

Standard whole-cell records using the patch-clamp technique are obtained after rupturing the cell membrane just below the patch pipette. Inherent problems, such as the disruption of cellular architecture and the displacement of cytosol, are unavoidable. In the present report, a whole-cell recording technique which makes use of a monovalent cation ionophore, nystatin, was applied to lymphocytes. Nystatin-perforated patches allow electrical access to the cell interior while virtually blocking the diffusion of cellular constituents into the electrode. By comparing standard whole-cell and perforated-patch techniques we observed marked differences in: activation, inactivation, and deactivation kinetics; steady-state inactivation; and the conductance-voltage relationship of K+ currents in activated human T cells.

Regulation of human natural cytotoxicity by enkephalins and selective opiate agonists.

This study reports a bidirectional effect of the enkephalins and selective opiate receptor agonists on human natural killer (NK) cell activity. Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors and enriched for T lymphocytes and large granular lymphocytes (LGL) by passage over nylon wool columns. Nylon wool nonadherent cell populations were preincubated for 18 h in the presence of fetal bovine serum with and without methionine-enkephalin, leucine-enkephalin, dynorphin (fragment 1-13), [D-Ser2]-leucine-enkephalin-Thr (DSLET), and [D-Ala2,N Me-Phe4, Gly-ol5]-enkephalin (DAGO). NK activity was measured by a standard 51Cr release assay with radiolabeled K562 cells. The cytolytic capacity of low NK responder populations was enhanced by the endogenous opioids while the NK activity of high responder populations was suppressed. These results suggest an immunoregulatory action of opioid peptides on NK activity. This possibility was confirmed using a serum-free system in conjunction with recombinant interferon-alpha. In addition, the classic opioid receptor antagonist naloxone displayed both antagonist and direct immunomodulatory properties, which may indicate the presence of lymphocyte derived opioid peptides in the culture system.