Voltage-dependent K؉ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K ؉ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-␣ mimicked LPS effects, and studies with TNF-␣ receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-␣-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response.

Abstract-Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O 2 -sensitive K ϩ current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 ␣ subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO 2 ϭϷ30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 ␣ subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E M ) of PA VSMCs (Ϸ28% at Ϫ40 mV). Coexpression of Kv1.2 and Kv1.5 ␣ subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials (Ϸ65% at Ϫ40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 ␣ subunits can assemble to form an O 2 -sensitive heteromeric channel, and (3) Key Words: Kv channel Ⅲ hypoxia Ⅲ pulmonary artery Ⅲ heteromeric H ypoxia induces constriction of the small pulmonary resistance arteries, a process known as hypoxic pulmonary vasoconstriction (HPV). 1 This constrictor response is the opposite of that which occurs in resistance vessels of the systemic circulation. In these vessels, hypoxia results in vasodilation. 2 In the fetus, HPV contributes to high pulmonary arterial resistance by diverting blood flow through the ductus arteriosus. 3 In the adult, HPV reduces blood flow through atelectatic or underventilated areas of the lung where ventilation is not adequate for oxygenation. 3 In this way, short-term HPV is an essential mechanism that helps match ventilation to perfusion, diverting blood flow away from poorly ventilated regions of the lung to maximize arterial saturation. 4 However, when hypoxia becomes more generalized, as seen in patients suffering from either long-term obstructive lung diseases or high altitude exposure, 4,5 the subsequent pulmonary vasoconstriction causes an increase in pulmonary arterial pressure that can lead to the development of pulmonary hypertension.In pulmonary arterial (PA) vascular smooth muscle cells (VSMCs), voltage-gated potassium (Kv) channels play an important role in setting the resting membrane potential (E M ϭϷϪ55 mV) and, consequently, vascular tone. 6 -8 It is thought that hypoxia reversibly inhibits Kv channels and, thereby, regulates vasoconstriction. 7,9 -12 This hypothesis is supported by the observation that hypoxia inhibits whole-cell K ϩ currents and ca...

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To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon gamma (IFN-gamma) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-gamma only induced the concentrative N1 and N2 systems. IFN-gamma also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.