Nitric oxide (NO) synthesized by vascular endothelial cells is a potent vasodilator substance. The actions of NO extend well beyond its vasodilatory properties, and increasingly, NO has been recognized as an important signal for intercellular and intracellular communication. Recently, NO has been implicated in the regulation of vascular and bloodbrain barrier permeability. NO has also been shown to modulate ion channels in excitable cells, thus affecting neuronal firing. We report the results of patch-clamp experiments that show a modulatory action of NO as well as cGMP and cAMP on a hyperpolarization-activated current (Iha) carried by both Na+ and K' ions in blood-brain barrier endothelial cells. lha was recorded in cells dialyzed with 0.2 mmol/L GTP-y-S to inhibit a large inwardly rectifying potassium current. This ionic current and its modulation by NO may play a role in the regulation of the transport of ions, nutrients, and other molecules to the brain and serve as an integral part of the blood-brain barrier. The modulation of Iha by a cyclic guanosine nucleotide may also explain previous reports suggesting a role for NO in the regulation of blood-brain barrier function. (Circ Res. 1994;75:528-538.) Key Words * ion homeostasis * membrane permeability * cyclic nucleotides * hyperpolarization A recisely regulated extracellular ionic environ-A ment in the mammalian central nervous system is vital for normal neuronal function. Capillary and arteriolar endothelial cells (ECs) control the movements of ions and water between blood and brain interstitial fluids; in particular, they regulate the movement of Na+ and K' across the blood-brain barrier,1'2 preventing excessive potassium accumulation in the cerebrospinal fluid (CSF) and maintaining Na+ concentrations sufficiently high for action potential generation. A Na4,K4-ATPase localized on the abluminal side of brain capillaries3,4 and an amiloride-sensitive cation channel,5 acting synergistically with the Na4-Cl-transporter,6 provide a direct path for transendothelial Na+ transport. Potassium transport is primarily directed from the CSF to the blood so that the blood-brain barrier must play a critical role in brain potassium homeostasis.1'4 7Despite the importance of blood-brain barrier function, little is known of the regulation of these ionic fluxes or their modulation by hormones or transmitters.Endothelium-derived relaxing factor (EDRF) has been identified as nitric oxide (NO) synthesized from the guanidino group of L-arginine.89 The continuous release of NO by the endothelium and the resulting activation of the guanylate cyclase signaling cascade have been shown to impact on the permeability of ECs to solutes and larger molecules.10-3 In addition, cGMP caused a decrease in the electrical resistance of in vitro blood-brain barrier endothelial monolayers."4The main voltage-dependent ionic current expressed in ECs belongs to the family of inwardly rectifying Received January 4, 1994; accepted May 20, 1994 potassium currents15-7; thus, ECs are consider...
Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4+ T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4+ T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4+ T cells belonged to the CD45RO+ memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4+ T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4+ T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4+ T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.
K(+) dilate and constrict cerebral vessels in a dose-dependent fashion. Modest elevations of abluminal K(+) cause vasodilatation, whereas larger extracellular K(+) concentration ([K(+)](out)) changes decrease cerebral blood flow. These dilations are believed to be mediated by opening of inward-rectifier potassium channels sensitive to Ba(2+). Because BaCl(2) also blocks ATP-sensitive K(+) channels (K(ATP)), we challenged K(+) dilations in penetrating, resistance-size (<60 mmu) rat neocortical vessels with the K(ATP) channel blocker glibenclamide (1 microM). Glibenclamide reduced K(+) responses from 138 +/- 8 to 110 +/- 0.8%. K(+) constrictions were not affected by glibenclamide. The Na(+)-K(+)-pump inhibitor ouabain (200 microM) did not significantly change resting vessel diameter but decreased K(+) dilations (from 153 +/- 9 to 99 +/- 2%). BaCl(2) blocked K(+) dilations with a half-maximal dissociation constant of 2.9 microM and reduced dilations to the specific K(ATP) agonist pinacidil with equal potency. We conclude that, in resistance vessels, K(+) dilations are mediated by K(ATP); we hypothesize that [K(+)](out) causes activation of Na(+)-K(+) pumps, depletion of intracellular ATP concentration, and subsequent opening of K(ATP). This latter hypothesis is supported by the blocking effect of ouabain.
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