Extracellular Potassium Concentration Research Articles

Mild functional effects of a novel GFAP mutant allele identified in a familial case of adult-onset Alexander disease

Alexander disease is a neurological genetic disorder characterized by progressive white-matter degeneration, with astrocytes containing cytoplasmic aggregates, called Rosenthal fibers, including the intermediate filament glial fibrillary acidic protein (GFAP). The age of onset of the disease defines three different forms, infantile, juvenile and adult, all due to heterozygous GFAP mutations and characterized by a progressive less severe phenotype from infantile to adult forms. In an Italian family with a recurrent mild adult onset of Alexander disease, we have identified two GFAP mutations, coupled on a same allele, leading to p.[R330G; E332K]. Functional studies on this complex allele revealed less severe aggregation patterns compared to those observed with p.R239C GFAP mutant, associated with a severe Alexander disease phenotype. Moreover, in addition to confirming the involvement of the ubiquitin-proteasome system in cleaning cells from aggregates and a dominant effect of the novel mutant protein, in cells expressing the mild p.[R330G; E332K] mutant we have observed that indirect alphaB-crystallin overexpression, induced by high extracellular potassium concentration, could completely rescue the correct filament organization while, under the same experimental conditions, in cells expressing the severe p.R239C mutant only a partial rescue effect could be achieved.

A Discrete-Time Control Algorithm Applied to Closed-Loop Pacing of HL-1 Cardiomyocytes

Electrical stimulation represents a useful tool for electrophysiologic investigation of electrically excitable cells such as cardiomyocytes. The stimulation threshold and electrophysiologic response to precisely timed pulses yields valuable information regarding physiologic processes. However, determining these parameters accurately, while simultaneously resolving time-dependent or transient effects has been difficult or impossible with previous methods. This paper presents a discrete-time algorithmic controller used for closed-loop electrical stimulation of HL-1 clonal cardiomyocytes cultured on, and stimulated using, a planar microelectrode array. We introduce the temporal error-controlled algorithm (TECA), that is well-suited to control using capture fraction, a low data rate, highly quantized feedback parameter describing stimulation efficacy. HL-1 cardiomyocytes were electrically stimulated and resulting parameters were used to develop a representative model of partial capture, enabling extensive analysis of the algorithm. The performance of this approach is compared via computer simulation to a previously introduced conditional convergence algorithm to quantify its performance and relative advantages. Operation of the TECA is demonstrated by tracking the real-time biological response of stimulation threshold to a rapid increase in extracellular potassium concentration in four independent cell cultures. This work enables the use of stimulation threshold as a real-time, continuously monitored parameter with considerable utility in cardiac pharmacology, electrophysiology, and cell-based biosensing.

Potentiometric platform for the quantification of cellular potassium efflux

Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.

REGULATION OF PULMONARY VASOCONSTRICTION BY AGONISTS AND CAVEOLAE

Sustained pulmonary vasoconstriction contributes to the elevated pulmonary vascular resistance observed in pulmonary arterial hypertension. A rise in cytosolic Ca2 + in pulmonary artery smooth muscle cells (PASMCs) is major trigger for pulmonary vasoconstriction. One family of drugs currently being pursued as a potential treatment for pulmonary hypertension are the statins, which act by depleting cholesterol and reducing the number of caveolae. This study aimed at investigating the role of caveolae, membrane receptors, and ion channels (that are potentially located in the caveolae) in agonist-mediated pulmonary vasoconstriction in order to gain a greater understanding of the signaling mechanisms involved in the regulation of pulmonary vascular tone. Chronic treatment of PASMCs with the cholesterol-depleting agent, methyl-β -cyclodextrin (Mβ CD), significantly reduced the number of cholesterol rich caveolae regions in the membrane. This disruption of cholesterol in caveolae significantly inhibited pharmacomechanical (induced by phenylephrine), but not electromechanical (induced by elevated extracellular potassium concentration), rat pulmonary artery contraction. These results indicate that receptors may functionally colocalize in caveolae in PASMCs and coordinate to regulate pulmonary vascular tone.

Extracellular Acidification and Hyperkalemia Induce Changes in HERG Inhibition by Ibutilide

Background: A high incidence of proarrhythmia has been reported with ibutilide, especially in patients with underlying heart diseases. Our previous studies have shown that extracellular acidosis and hyperkalemia attenuate the HERG-inhibitory effect of proarrhythmic drugs, e.g. quinidine, but have little impact on the less-proarrhythmic drug amiodarone. We hypothesized that ibutilide would behave like quinidine in the presence of extracellular acidosis and hyperkalemia. Methods and Results: HERG was expressed on Xenopus oocytes, and the two-electrode voltage clamp technique was employed. Our results showed that ibutilide was a potent HERG inhibitor. When extracellular solution contained 5 mM KCl and pH was 7.4, the IC₅₀ of ibutilide was 0.9 ± 0.1 µM. The inhibitory effect of ibutilide was attenuated when extracellular pH decreased to 6.2. There was a significant difference in current inhibition by ibutilide at pH 7.4 versus pH 6.2 (p < 0.01). When the extracellular potassium concentration was increased from 5 to 10 mM, ibutilide produced less current inhibition, and the IC₅₀ was increased to 2.0 ± 0.1 µM. Conclusion: Extracellular acidosis and hyperkalemia attenuate the HERG-inhibitory effect of ibutilide. The differences in HERG inhibition between acidic and hyperkalemic regions compared to normal regions in the myocardium may result in heterogeneity in repolarization, which may contribute to the proarrhythmic toxicity of ibutilide.

Differential expression of Na+/K+-ATPase alpha-subunits in mouse hippocampal interneurones and pyramidal cells.

The sodium pump (Na+/K+-ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump alpha subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD-65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different alpha subunit isoforms. GFP-positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of alpha-subunits of the Na+/K+-ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3-isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1-isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.

Cardiac Glycosides Regulate Endothelial Tissue Factor Expression in Culture

Tissue factor (TF) plays an important role in acute coronary syndromes and stent thrombosis. This study investigates whether Na(+)/K(+)-ATPase regulates TF expression in human endothelial cells. Ouabain inhibited tumor necrosis factor (TNF)-alpha-induced endothelial TF protein expression; maximal inhibition occurred at 10(-5) mol/L, reached more than 70%, and was observed throughout the 5 hours stimulation period. The decrease in protein expression was paralleled by a reduced TF surface activity. Similarly, lowering of extracellular potassium concentration inhibited TNF-alpha-induced TF protein expression. In contrast, ouabain did not affect TNF-alpha-induced expression of full-length TF mRNA for up to 5 hours of stimulation; instead, expression of alternatively-spliced TF mRNA was upregulated after 3 and 5 hours of stimulation. Ouabain did not affect TNF-alpha-induced activation of the MAP kinases p38, extracellular signal-regulated kinase (ERK), and c-Jun terminal NH(2) kinase; activation of Akt and p70S6 kinase remained unaltered as well. Similar to the MAP kinases, ouabain did not affect TNF-alpha-induced degradation of IkappaB-alpha. Ouabain had no effect on TF protein degradation. Na(+)/K(+)-ATPase is required for protein translation of endothelial TF in culture. This observation provides novel insights into posttranscriptional regulation of TF expression.

Conditional Knock-Out of Kir4.1 Leads to Glial Membrane Depolarization, Inhibition of Potassium and Glutamate Uptake, and Enhanced Short-Term Synaptic Potentiation

During neuronal activity, extracellular potassium concentration ([K+]out) becomes elevated and, if uncorrected, causes neuronal depolarization, hyperexcitability, and seizures. Clearance of K+ from the extracellular space, termed K+ spatial buffering, is considered to be an important function of astrocytes. Results from a number of studies suggest that maintenance of [K+]out by astrocytes is mediated by K+ uptake through the inward-rectifying Kir4.1 channels. To study the role of this channel in astrocyte physiology and neuronal excitability, we generated a conditional knock-out (cKO) of Kir4.1 directed to astrocytes via the human glial fibrillary acidic protein promoter gfa2. Kir4.1 cKO mice die prematurely and display severe ataxia and stress-induced seizures. Electrophysiological recordings revealed severe depolarization of both passive astrocytes and complex glia in Kir4.1 cKO hippocampal slices. Complex cell depolarization appears to be a direct consequence of Kir4.1 removal, whereas passive astrocyte depolarization seems to arise from an indirect developmental process. Furthermore, we observed a significant loss of complex glia, suggestive of a role for Kir4.1 in astrocyte development. Kir4.1 cKO passive astrocytes displayed a marked impairment of both K+ and glutamate uptake. Surprisingly, membrane and action potential properties of CA1 pyramidal neurons, as well as basal synaptic transmission in the CA1 stratum radiatum appeared unaffected, whereas spontaneous neuronal activity was reduced in the Kir4.1 cKO. However, high-frequency stimulation revealed greatly elevated posttetanic potentiation and short-term potentiation in Kir4.1 cKO hippocampus. Our findings implicate a role for glial Kir4.1 channel subunit in the modulation of synaptic strength.

Can latent heat safely warm blood? – in vitro testing of a portable prototype blood warmer

BackgroundTrauma/retrieval patients are often in shock and hypothermic. Treatment of such patients usually involves restoring their blood volume with transfusion of blood (stored at 2°C – 6°C) and/or crystalloids or colloids (stored at ambient temperature). Rapid infusion of these cold fluids can worsen or even induce hypothermia in these patients. Warming of intravenous fluids at accident sites has traditionally been difficult due to a lack of suitable portable fluid warmers that are not dependent on mains electrical or battery power. If latent heat, the heat released when a liquid solidifies (an inherently temperature limiting process) can warm intravenous fluids, portable devices without a reliance on electrical energy could be used to reduce the incidence of hypothermia in trauma patients.MethodsRapid infusion of red cells into patients was timed to sample typical clinical flow rates.An approved dry heat blood warmer was compared with a prototype blood warmer using a supercooled liquid latent heat storage material, to warm red cells whilst monitoring inlet and outlet temperatures. To determine the effect of warming on red cell integrity compared to the normal storage lesion of blood, extracellular concentrations of potassium, lactate dehydrogenase and haemoglobin were measured in blood which had been warmed after storage at 2°C – 6°C for 1 to 42 days.ResultsA prototype latent heat fluid warmer consistently warmed red cells from approximately 4°C to approximately 35°C at typical clinical flow rates. Warming of stored blood with latent heat did not affect red cell integrity more than the approved dry heat blood warmer.ConclusionUsing latent heat as an energy source can satisfactorily warm cold blood or other intravenous fluids to near body temperature, without any adverse affects.

Matching native electrical stimulation by graded chemical stimulation in isolated mouse adrenal chromaffin cells

Adrenal chromaffin cells release multiple transmitters in response to sympathetic stimulation. Modest cell firing, matching sympathetic tone, releases small freely soluble catecholamines. Elevated electrical firing rates matching input under sympathetic stress results in release of catecholamines as well as semi-soluble vaso- and neuro-active peptides packaged within the dense core of the secretory granule. This activity-dependent differential transmitter release has been shown to rely on a mechanistic shift in the mode of exocytosis through the regulated dilation of the secretory fusion pore between granule and cell surface membranes. However, biochemical description of the mechanism regulating fusion pore dilation remains elusive. In the experimental setting, electrical stimulation designed to mimic sympathetic input, is achieved through single-cell voltage-clamp. While precise, this approach is incompatible with biochemical and proteomic analysis, both of which require large sample sizes. We address this limitation in the current study. We describe a bulk chemical stimulation paradigm calibrated to match defined electrical activity. We utilize calcium and single-cell amperometric measurements to match extracellular potassium concentrations to physiological electrical stimulation under sympathetic tone as well as acute stress conditions. This approach provides larger samples of uniformly stimulated cells for determining molecular players in activity-dependent differential transmitter release from adrenal chromaffin cells.

Two-pore-domain potassium channels contribute to neuronal potassium release and glial potassium buffering in the rat hippocampus

Two-pore-domain potassium (K2P) channels have been suggested to be involved in neuronal K + release and glial K + uptake. We studied effects of the K2P channel blockers quinine (200 or 500 μM), quinidine (500 μM), and bupivacaine (200 μM) on stimulus-induced and iontophoretically induced transient increases of the extracellular potassium concentration ([K +] o) in area CA1 of rat hippocampal slices, always in presence of AMPA/kainate and NMDA receptor antagonists. Increases in [K +] o evoked by repetitive alvear stimulation (20 Hz) were blocked by quinine and quinidine but amplitudes of population spikes were only modestly reduced. Bupivacaine suppressed both rises in [K +] o and population spikes. In contrast, iontophoretically induced rises in [K +] o were moderately augmented by quinine and quinidine while bupivacaine had no effect. Barium at concentrations of 2 mM which should block both potassium inward rectifier (Kir) and some K2P channels doubled iontophoretically induced rises in [K +] o also in presence of quinine, quinidine, and bupivacaine. The data suggest that quinine/quinidine-sensitive K2P channels mediate K + release from neurons and possibly contribute to glial K + buffering.

Slow potassium dynamics and seizure evolution

Seizures involve dynamics on a wide range of temporal scales, from spike times on the order of milliseconds to the large depolarizations seen in single cells that can last several tens of seconds. At the longest time scales, these events modify the cellular environment, altering oxygen, potassium, sodium and other electrolyte concentrations to produce a durable but transient modification of the network dynamics. In order to investigate these slow dynamics we have developed a highly simplified model that monitors the changes in ionic concentrations in and around highly active cells, while disregarding the fast dynamics responsible for action potential generation. We model the time-dependent potassium concentration in and around a cell resulting from flow through voltage-gated channels, pumps, and the surrounding glial network. The flow through voltage-gated channels is determined by time-averaging simulated potassium currents in a Hodgkin-Huxley conductance-based neuron. The current is a function of both intra- and extracellular potassium concentrations and responds to changes in the concentration gradient over a duration that is long compared to the time associated with spiking events. On the other hand, this response time, which can be as slow as a fraction of a second, is still short compared to the lifetime of a network seizure and can be considered instantaneous. Therefore we disregard the response time and approximate the model as a pair of differential equations which are amenable to a complete phase plane analysis. We report on the results of this phase plane analysis and show comparisons with results from in vitro experiments.

Rhythms of high-grade block in an ionic model of a strand of regionally ischemic ventricular muscle

Electrical alternans, a beat-to-beat alternation in the electrocardiogram or electrogram, is frequently seen during the first few minutes of acute myocardial ischemia, and is often immediately followed by malignant cardiac arrhythmias such as ventricular tachycardia and ventricular fibrillation. As ischemia progresses, higher-order periodic rhythms (e.g., period-4) can replace the period-2 alternans rhythm. This is also seen in modelling work on a two-dimensional (2-D) sheet of regionally ischemic ventricular muscle. In addition, in the experimental work, ventricular arrhythmias are overwhelmingly seen only after the higher-order rhythms arise. We investigate an ionic model of a strand of ischemic ventricular muscle, constructed as a 3-cm-long 1-D cable with a centrally located 1-cm-long segment exposed to an elevated extracellular potassium concentration ([K +] o). As [K +] o is raised in this “ischemic segment” to represent one major effect of ongoing ischemia, the sequence of rhythms {1:1→2:2 (alternans)→2:1} is seen. With further increase in [K +] o, one sees higher-order periodic 2 N: M rhythms {2:1→4:2→4:1→6:2→6:1→8:2→8:1}. In a 2 N: M cycle, only M of the 2 N action potentials generated at the proximal end of the cable successfully traverse the ischemic segment, with the remaining ones being blocked within the ischemic segment. Finally, there is a transition to complete block {8:1→2:0→1:0} (in an n:0 rhythm, all action potentials die out within the ischemic segment). Changing the length of the ischemic segment results in different rhythms and transitions being seen: e.g., when the ischemic segment is 2 cm long, the period-6 rhythms are not seen; when it is 0.5 cm long, there is a 3:1 rhythm interposed between the 2:1 and 1:0 rhythms. We discuss the relevance of our results to the experimental observations on the higher-order rhythms that presage reentrant ischemic ventricular arrhythmias.

Antidromic propagation of action potentials in branched axons: implications for the mechanisms of action of deep brain stimulation

Electrical stimulation of the central nervous system creates both orthodromically propagating action potentials, by stimulation of local cells and passing axons, and antidromically propagating action potentials, by stimulation of presynaptic axons and terminals. Our aim was to understand how antidromic action potentials navigate through complex arborizations, such as those of thalamic and basal ganglia afferents-sites of electrical activation during deep brain stimulation. We developed computational models to study the propagation of antidromic action potentials past the bifurcation in branched axons. In both unmyelinated and myelinated branched axons, when the diameters of each axon branch remained under a specific threshold (set by the antidromic geometric ratio), antidromic propagation occurred robustly; action potentials traveled both antidromically into the primary segment as well as "re-orthodromically" into the terminal secondary segment. Propagation occurred across a broad range of stimulation frequencies, axon segment geometries, and concentrations of extracellular potassium, but was strongly dependent on the geometry of the node of Ranvier at the axonal bifurcation. Thus, antidromic activation of axon terminals can, through axon collaterals, lead to widespread activation or inhibition of targets remote from the site of stimulation. These effects should be included when interpreting the results of functional imaging or evoked potential studies on the mechanisms of action of DBS.

A linear system of partial differential equations modeling the resting potential of a heart with regional ischemia

Ischemic ST-segment shift has been modeled using scalar stationary approximations of the bidomain model. Here, we propose an alternative simplification of the bidomain equations: a linear system modeling the resting potential, to be used in determining ischemic TP shift. Results of 2D and 3D simulations show that the linear system model is much more accurate than the scalar model. This improved accuracy is important if the model is to be used for solving the inverse problem of determining the size and location of an ischemic region. Furthermore, the model can provide insight into how the resting potential depends on the variations in the extracellular potassium concentration that characterize ischemic regions.

NOISE CONTROLLED SYNCHRONIZATION IN POTASSIUM COUPLED NEURAL MODELS

The paper applies biologically plausible models to investigate how noise input to small ensembles of neurons, coupled via the extracellular potassium concentration, can influence their firing patterns. Using the noise intensity and the volume of the extracellular space as control parameters, we show that potassium induced depolarization underlies the formation of noise-induced patterns such as delayed firing and synchronization. These phenomena are associated with the appearance of new time scales in the distribution of interspike intervals that may be significant for the spatio-temporal oscillations in neuronal ensembles.

Effects of the GABAA receptor antagonists bicuculline and gabazine on stimulus‐induced sharp wave‐ripple complexes in adult rat hippocampus in vitro

Hippocampal sharp wave-ripple-complexes (SPW-Rs) are characterized by slow field potential transients superimposed by ripples with a frequency of approximately 200 Hz. In epileptic humans and rats frequencies of such transient network oscillations can reach up to 500 Hz potentially due to loss of functional inhibition. Therefore, we investigated whether GABA(A) receptor antagonists increased ripple frequency during SPW-Rs. Within area CA3, SPW-Rs were induced by repeated stimulation of stratum radiatum in area CA1 of adult Wistar rat hippocampal slices. Intracellular recordings showed that in approximately 50% of recorded CA3 pyramidal cells SPW-Rs were accompanied by compound excitatory postsynaptic potentials (EPSPs) of approximately 10 mV superimposed by up to four action potentials (APs). The remaining cells responded with a compound inhibitory postsynaptic potential (IPSP) during SPW-Rs. The GABA(A) receptor antagonists bicuculline (BMI) or gabazine (SR-95531) led to a transition of SPW-Rs into prolonged bursts with a significant increase in amplitude and duration reminiscent of recurrent epileptiform discharges (REDs). Ripple frequencies increased from approximately 190 Hz to approximately 300 Hz. In naïve slices SR-95531 and BMI also evoked REDs with similar incidence and high frequency ripple frequencies of approximately 240 Hz. Elevations in extracellular potassium concentration during REDs were approximately 20-fold higher than those observed during SPW-Rs. Intracellular recordings revealed bursts that were characterized by a large (> 25 mV) prolonged depolarization superimposed by up to 40 APs in close synchrony with extracellularly recorded ripples. Our results suggest that the generation of high frequency ripples, which are also observed in epileptic humans and rats, could indicate a loss of functional inhibition.

A comparative study of reducing the extracellular potassium concentration in red blood cells by washing and by reduction of additive solution

Extracellular potassium concentration (K(+)) increases in the supernatant of whole and packed red blood cell units (pRBCs) with duration of refrigerated storage in citrate-phosphate-dextrose-adenine (CPDA-1) and additive solution (AS). Studies have shown that to avoid hyperkalemia, washed pRBCs are preferred if relatively fresh pRBCs are not available. To determine whether a simpler procedure, AS reduction, results in lowering of K(+) in pRBCs comparable to that achieved by washing, the K(+) levels by both methods were compared. Pre- and post-K(+) levels were measured in 6 washed and 11 AS-reduced pRBC units. Each unit was weighed, hematocrit was determined, K(+) was measured, and total K(+) was calculated. Washed units were 3 to 21 and AS-reduced units were 4 to 30 days old. Statistical analysis was performed with a t test. There was no significant difference (p > 0.35) in the initial K(+) between the two groups (mean +/- SD, 36.95 +/- 13.16 mEq/L before washing and 39.78 +/- 19.94 mEq/L before AS reduction). Washing and AS reduction both led to a significant decrease in K(+) levels (2.15 +/- 0.10 mEq/L after washing and 4.41 +/- 3.04 mEq/L after AS reduction, each p < 0.0005). Washing, however, was significantly better than AS reduction in reducing K(+) in stored pRBCs (p < 0.05). Washing pRBCs results in very low levels of K(+). AS reduction also significantly reduces K(+) levels. Selection of the method of K(+) reduction will depend on the stringency of K(+) reduction needed, the time constraints, and the availability of facilities and staff for washing.

Maintenance and termination of neocortical oscillations by dynamic modulation of intrinsic and synaptic excitability.

Mechanisms underlying seizure cessation remain elusive. The Lennox-Gastaut syndrome, a severe childhood epileptic disorder, is characterized by episodes of seizure with alternating epochs of spike-wave and fast run discharges. In a detailed computational model that incorporates extracellular potassium dynamics, we studied the dynamics of these state transitions between slow and fast oscillations. We show that dynamic modulation of synaptic transmission can cause termination of paroxysmal activity. An activity-dependent shift in the balance between synaptic excitation and inhibition towards more excitation caused seizure termination by favoring the slow oscillatory state, which permits recovery of baseline extracellular potassium concentration. We found that slow synaptic depression and change in chloride reversal potential can have similar effects on the seizure dynamics. Our results indicate a novel role for synaptic dynamics during epileptic neural activity patterns.

Urocortin Induces Endothelium-Dependent Vasodilatation and Hyperpolarization of Rat Mesenteric Arteries by Activating Ca2+-Activated K+ Channels

Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.