natriuretic peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential (E m) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. We measured changes in E m using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were preincubated in the potentiometric dye DiBAC4(3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K ϩ channel blockers, tetraethylammonium or iberiotoxin, the nonspecific cation channel blocker, La 3ϩ , or by depletion or repletion of extracellular Ca 2ϩ or K ϩ , respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of PKG or by small interfering RNA (siRNA)-mediated knockdown of natriuretic peptide receptor-B (NPR-B). CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-bromo-cGMP, and attenuated by both the endothelial nitric oxide synthase (eNOS) inhibitor, N -nitro-L-arginine methyl ester (L-NAME), and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Presence of iberiotoxinsensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating large-conductance calcium-activated potassium channels mediated by the activation of NPR-B, PKG, eNOS, and sGC.large-conductance calcium-activated potassium channels; ion channel C-TYPE NATRIURETIC PEPTIDE (CNP) is a highly conserved member of the natriuretic peptide family and an important, but controversial, regulator of vascular tone and blood pressure. CNP consists of 22 amino acid residues and shares a 17-amino acid disulfide ring structure with atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) (29). CNP functions as a paracrine hormone in both the pulmonary and systemic circulations and is expressed and secreted by vascular endothelial cells (17,35,37). Like ANP and BNP, CNP has been demonstrated to relax blood vessels in a number of vascular beds (8,14,20,39,40). Furthermore, CNP appears to have both anti-inflammatory (18, 26, 33) and anti-mitogenic (12, 18, 26) properties. Thus CNP may serve a potentially useful therapeutic agent, especially in the pulmonary circulation, where it has been found to attenuate both bleomycin-induced lung fibrosis and monocrotaline-induced pulmonary hypertension (18, 26). However, although the physiological effects of CNP are intriguing, the signaling mechanisms that underlie many of its vascular activities remain incompletely described.The physiological ...
SUMMARY1. The effects of some neutral clinical and experimental general anaesthetics on the resting potential of normal squid axons and squid axons exposed to tetrodotoxin and 3,4-diaminopyridine have been studied.2. Depolarizations of 1-4 mV were produced by all the anaesthetics at 'clinical' concentrations in the normal axon. Larger depolarizations (5-11 mV) were produced by the same anaesthetic concentrations in axons exposed to tetrodotoxin and 3,4-diaminopyridine.3. The conductance of axons exposed to tetrodotoxin and either tetraethylammonium or 3,4-diaminopyridine in zero Na+, 430 mM-K+ artificial sea water was examined by voltage clamp and AC bridge techniques.4. The evidence that this conductance is due predominantly to K+ is discussed. 5. Pre-pulse protocols under voltage clamp have been used to show that part of this conductance arises from the incompletely blocked delayed rectifier.6. Substantial reductions in this conductance are produced by anaesthetics at clinical' concentrations.7. It is concluded that there is a component of the K+ conductance of the resting squid axon other than the Hodgkin-Huxley delayed rectifier which is extremely sensitive to anaesthetics and which to an appreciable extent determines the resting potential.
SUMMARY1. The effects of 'clinical' concentrations of some general anaesthetics on the minimum stimulus required to produce an action potential in the squid giant axon have been examined as a function of time from exposure to the anaesthetic. The resting potential in these experiments was also monitored.2. The minimum stimulus varied with time in different ways for different anaesthetics. For chloroform, diethyl ether, n-pentanol, halothane and cyclopropane the stimulus initially declined, reached a minimum after about 3 min and then recovered to near-normal values at 10-15 min. For n-pentane, cyclopentane and, to a lesser extent methoxyflurane, the stimulus often declined to such low values that the axon exhibited spontaneous action potentials which persisted until the anaesthetic was removed. For one substance, the experimental local anaesthetic diheptanoyl phosphatidylcholine, the stimulus increased considerably over the 10-15 min required to reach the steady state. In all instances the axons reverted to normal behaviour after removal of the anaesthetic although the time course by which they did so was more variable than for the initial exposure.3. For all anaesthetics the resting potential changed in the positive direction monotonically by ca. 1-5 mV and reached a steady state in approximately 3 min. On removal of the anaesthetic the resting potential returned to normal, also monotonically.4. The voltage-gated Na+ and K+ currents were significantly affected even at the low anaesthetic concentrations used. Estimates of the changes in the HodgkinHuxley parameters were obtained partly by direct experiment and partly from results previously obtained for higher anaesthetic concentrations.5. The time dependencies of the minimum stimuli have been accounted for semiquantitatively in terms of the resting potential changes and the voltage shifts in the Na+ current steady-state activation, and the time dependencies respectively of these two parameters.6. Quantitative calculations of the resting potential changes for comparison with experiment have been made based on the changes in K+ conductance determined in the preceding paper (Haydon, Requena & Simon, 1988) and changes in the Hodgkin-Huxley parameters of the Na+ and delayed-rectifier K+ currents.7. Calculations of the minimum stimulus in the steady state have been made from the experimental resting potential changes and from the anaesthetic-affected Hodgkin-Huxley parameters. Good agreement with the experimental stimuli was found, especially in the prediction of high and low values.
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