Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices.

Differences in vascular nitric oxide and endothelium-derived hyperpolarizing factor bioavailability in blacks and whites.

OBJECTIVE: Abnormalities in nitric oxide (NO) bioavailability have been reported in blacks. Whether there are differences in endothelium-derived hyperpolarizing factor (EDHF) in addition to NO between blacks and whites and how these affect physiological vasodilation remain unknown. We hypothesized that the bioavailability of vascular NO and EDHF, at rest and with pharmacological and physiological vasodilation, varies between whites and blacks. APPROACH AND RESULTS: In 74 white and 86 black subjects without known cardiovascular disease risk factors, forearm blood flow was measured using plethysmography at rest and during inhibition of NO with N(G)-monomethyl-L-arginine and of K(+) Ca channels (EDHF) with tetraethylammonium. The reduction in resting forearm blood flow was greater with N(G)-monomethyl-L-arginine (P=0.019) and similar with tetraethylammonium in whites compared with blacks. Vasodilation with bradykinin, acetylcholine, and sodium nitroprusside was lower in blacks compared with whites (all P<0.0001). Inhibition with N(G)-monomethyl-L-arginine was greater in whites compared with blacks with bradykinin, acetylcholine, and exercise. Inhibition with tetraethylammonium was lower in blacks with bradykinin, but greater during exercise and with acetylcholine. CONCLUSIONS: The contribution to both resting and stimulus-mediated vasodilator tone of NO is greater in whites compared with blacks. EDHF partly compensates for the reduced NO release in exercise and acetylcholine-mediated vasodilation in blacks. Preserved EDHF but reduced NO bioavailability and sensitivity characterizes the vasculature in healthy blacks. CLINICAL TRIAL REGISTRATION URL: http://clinicaltrials.gov/. Unique identifier: NCT00166166.

Disease and region-related cardiac fibroblast potassium current variations and potential functional significance.

AIMS: Fibroblasts, which play an important role in cardiac function/dysfunction, including arrhythmogenesis, have voltage-dependent (Kv) currents of unknown importance. Here, we assessed the differential expression of Kv currents between atrial and ventricular fibroblasts from control dogs and dogs with an atrial arrhythmogenic substrate caused by congestive heart failure (CHF). METHODS AND RESULTS: Left atrial (LA) and ventricular (LV) fibroblasts were freshly isolated from control and CHF dogs (2-week ventricular tachypacing, 240 bpm). Kv currents were measured with whole-cell voltage-clamp, mRNA by quantitative polymerase chain reaction (qPCR) and fibroblast proliferation by (3)H-thymidine incorporation. Robust voltage-dependent tetraethylammonium (TEA)-sensitive K(+) currents (IC50 ∼1 mM) were recorded. The morphologies and TEA responses of LA and LV fibroblast Kv currents were similar. LV fibroblast Kv-current densities were significantly greater than LA, and Kv-current densities were significantly less in CHF than control. The mRNA expression of Kv-channel subunits Kv1.5 and Kv4.3 was less in LA vs. LV fibroblasts and was down-regulated in CHF, consistent with K(+)-current recordings. Ca(2+)-dependent K(+)-channel subunit (KCa1.1) mRNA and currents were less expressed in LV vs. LA fibroblasts. Inhibiting LA fibroblast K(+) current with 1 mmol/L of TEA or KCa1.1 current with paxilline increased proliferation. CONCLUSIONS: Fibroblast Kv-current expression is smaller in CHF vs. control, as well as LA vs. LV. KCa1.1 current is greater in LA vs. LV. Suppressing Kv current with TEA enhances fibroblast proliferation, suggesting that Kv current might act to check fibroblast proliferation and that reduced Kv current in CHF may contribute to fibrosis. Fibroblast Kv-current remodelling may play a role in the atrial fibrillation (AF) substrate; modulating fibroblast K(+) channels may present a novel strategy to prevent fibrosis and AF.

Glutamate transporter type 3 regulates mouse hippocampal GluR1 trafficking.

BACKGROUND: Rapid trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to the plasma membrane is considered a fundamental biological process for learning and memory. GluR1 is an AMPAR subunit. We have shown that mice with knockout of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, have impaired learning and memory. The mechanisms for this impairment are not known and may be via regulation of AMPAR trafficking. METHODS: Freshly prepared 300µm coronal hippocampal slices from wild-type or EAAT3 knockout mice were incubated with or without 25mM tetraethylammonium for 10min. The trafficking of GluR1, an AMPAR subunit, to the plasma membrane and its phosphorylation were measured. RESULTS: Tetraethylammonium increased the trafficking of GluR1 and EAAT3 to the plasma membrane in the wild-type mouse hippocampal slices but did not cause GluR1 trafficking in the EAAT3 knockout mice. Tetraethylammonium also increased the phosphorylation of GluR1 at S845, a protein kinase A (PKA) site, in the wild-type mice but not in the EAAT3 knockout mice. The PKA antagonist KT5720 attenuated tetraethylammonium-induced GluR1 phosphorylation and trafficking in the wild-type mice. The PKA agonist 6-BNz-cAMP caused GluR1 trafficking to the plasma membrane in the EAAT3 knockout mice. In addition, EAAT3 was co-immunoprecipitated with PKA. CONCLUSIONS: These results suggest that EAAT3 is upstream of PKA in a pathway to regulate GluR1 trafficking. GENERAL SIGNIFICANCE: Our results provide initial evidence for the involvement of EAAT3 in the biochemical cascade of learning and memory.

Non-specific inhibitors of aquaporin-4 stimulate S100B secretion in acute hippocampal slices of rats.

Aquaporin-4 (AQP-4) is the principal brain water channel and is predominantly expressed in astrocytes suggesting its dynamic involvement in water homeostasis in brain tissue. Due to the co-localization of AQP-4 and inward rectifier K(+) channels Kir 4.1, a functional coupling between these proteins has been proposed. AQP-4 has a putative role in the physiopathology of brain disorders including epilepsy and trauma. S100B is a calcium-binding protein expressed and secreted by astrocytes, and commonly used as a parameter of astroglial activation. Here, we investigate a possible link between AQP-4 activity (and Kir 4.1) and S100B secretion in hippocampal slices of rats of different ages using non-specific inhibitors of AQP-4 (AZA, acetazolamide and TEA, tetraethylammonium) and Kir 4.1 (barium chloride). We found that blockade of AQP-4 with TEA and AZA produced an increase in S100B secretion in young rats, compatible with an astroglial activation observed in many conditions of brain injury. On the other hand, BaCl(2) induced Kir 4.1 inhibition caused a decrease in S100B secretion. Both channels, AQP-4 and Kir 4.1, exhibited a similar ontogenetic profile, in spite of the functional uncoupling, in relation to S100B secretion. Moreover, we found a significant increase in the S100B secretion basal levels with the increasing of animal age and the incubation with high levels of potassium resulted in a decrease of S100B secretion in 30 and 90-day old rats. These data, together with previous observations from gap junctions and glutamate transport of astrocytes, contribute to characterize the operational system involving astroglial activation, particularly on S100B secretion, in brain disorders.

[The role of fast and slow potassium currents in electrical activity of amphibian myelinated nerve fibres].

The paper reviews the information about the role of fast and slow potassium currents in electrical activity of amphibian myelinated nerve fibres. It demonstrates the importance of discovering of fast and slow potassium currents and their following pharmacological separation (by potassium channels blockers 4-aminopyridine and tetraethylammonium) in investigation of mechanisms of biological potentials generation. The information about the existence of fast and slow potassium channels in the nerve membrane and about the properties of 4-aminopyridine and tetraethylammonium action served as a base for determination the nature of biological potentials and discovering the mechanism of potential-dependent action of 4-aminopyridine that for tens of years suffered from the lack of adequate explanation.

Pharmacology of currents underlying the different firing patterns of spinal sensory neurons and interneurons identified in vivo using multivariate analysis.

The operation of neuronal networks depends on the firing patterns of the network's neurons. When sustained current is injected, some neurons in the central nervous system fire a single action potential and others fire repetitively. For example, in Xenopus laevis tadpoles, primary-sensory Rohon-Beard (RB) neurons fired a single action potential in response to 300-ms rheobase current injections, whereas dorsolateral (DL) interneurons fired repetitively at 10-20 Hz. To investigate the basis for these differences in vivo, we examined drug-induced changes in the firing patterns of Xenopus spinal neurons using whole cell current-clamp recordings. Neuron types were initially separated through cluster analysis, and we compared results produced using different clustering algorithms. We used these results to develop a predictive function to classify subsequently recorded neurons. The potassium channel blocker tetraethylammonium (TEA) converted single-firing RB neurons to low-frequency repetitive firing but reduced the firing frequency of repetitive-firing DL interneurons. Firing frequency in DL interneurons was also reduced by the potassium channel blockers 4-aminopyridine (4-AP), catechol, and margatoxin; 4-AP had the greatest effect. The calcium channel blockers amiloride and nimodipine had few effects on firing in either neuron type but reduced action potential duration in DL interneurons. Muscarine, which blocks M-currents, did not affect RB neurons but reduced firing frequency in DL interneurons. These results suggest that potassium currents may control neuron firing patterns: a TEA-sensitive current prevents repetitive firing in RB neurons, whereas a 4-AP-sensitive current underlies repetitive firing in DL interneurons. The cluster and discriminant analysis described could help to classify neurons in other systems.

Characterization of ionic currents and electrophysiological properties of goldfish somatotropes in primary culture.

Growth hormone release in goldfish is partly dependent on voltage-sensitive Ca(2+) channels but somatotrope electrophysiological events affecting such channel activities have not been elucidated in this system. The electrophysiological properties of goldfish somatotropes in primary culture were studied using the whole-cell and amphotericin B-perforated patch-clamp techniques. Intracellular Ca(2+) concentration ([Ca(2+)]i) of identified somatotropes was measured using Fura-2/AM dye. Goldfish somatotropes had an average resting membrane potential of -78.4 ± 4.6 mV and membrane input resistance of 6.2 ± 0.2 GΩ. Voltage steps from a holding potential of -90 mV elicited a non-inactivating outward current and transient inward currents at potentials more positive than 0 and -30 mV, respectively. Isolated current recordings indicate the presence of 4-aminopyridine- and tetraethylammonium (TEA)-sensitive K(+), tetrodotoxin (TTX)-sensitive Na(+), and nifedipine (L-type)- and ω-conotoxin GVIA (N-type)-sensitive Ca(2+) channels. Goldfish somatotropes rarely fire action potentials (APs) spontaneously, but single APs can be induced at the start of a depolarizing current step; this single AP was abolished by TTX and significantly reduced by nifedipine and ω-conotoxin GVIA. TEA increased AP duration and triggered repetitive AP firing resulting in an increase in [Ca(2+)]i, whereas TTX, nifedipine and ω-conotoxin GVIA inhibited TEA-induced [Ca(2+)]i pulses. These results indicate that in goldfish somatotropes, TEA-sensitive K(+) channels regulate excitability while TTX-sensitive Na(+) channels together with N- and L-type Ca channels mediates the depolarization phase of APs. Opening of voltage-sensitive Ca(2+) channels during AP firing leads to increases in [Ca(2+)]i.

Glial cell-derived neurotrophic factor enhances synaptic communication and 5-hydroxytryptamine 3a receptor expression in enteric neurons.

BACKGROUND & AIMS: Glial cell-derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system during embryogenesis. We have observed the presence of Gdnf transcripts in the gastrointestinal tract of adult mice, and its early up-regulation after inflammation. We therefore investigated the effects of GDNF on enteric neuronal function in vitro. METHODS: Primary neuronal cultures were established from isolated myenteric plexi, and characterized by immunostaining and Ca(2+) imaging. Gene expression of several ion channels was analyzed by quantitative polymerase chain reaction (PCR) and the electrophysiologic properties of the neurons were studied by patch clamp. RESULTS: GDNF enhanced synaptogenesis and intercellular communication in primary myenteric neuronal cultures. Expression profiling revealed that GDNF exposure results in an up-regulation of Htr3a expression in the cultures and a similar increase was observed in inflamed colonic tissue where Gdnf expression was also increased. The increased Htr3a expression was accompanied by a functional increase in the response of neurons to acute challenge with 5-hydroxytryptamine (5-HT). GDNF treatment also caused inhibition of delayed rectifying voltage-gated potassium (Kv) currents, which correlated with the up-regulation of Htr3a and 5-HT-induced responses. Furthermore, pharmacologic blockade of Kv channels mimicked the effect of GDNF by increasing Htr3a expression as well as enhancing 5-HT-induced responses in the cultured myenteric neurons. CONCLUSIONS: GDNF promotes synaptic communication in cultured myenteric neurons. It also up-regulates 5-HT(3a)-receptor expression via modulation of Kv channel activity. Up-regulation of Gdnf after gastrointestinal inflammation might play an important role in the pathophysiology of gastrointestinal diseases.

Age-related differences in the beta-adrenergic vasodilator response in rat aortic rings.

Mechanisms underlying age-dependent changes in vasodilator responses to beta-adrenergic drugs are poorly understood. The aim of the current study was to compare responses to isoproterenol (a non-selective beta-adrenergic receptor agonist) in phenylephrine or KCl precontracted aortic rings from 3 week and 3 month old male Wistar rats. Both the mechanism and the subtype of beta-adrenergic receptor underlying the response to isoproterenol in the both age groups were examined. Endothelial removal, pre-contraction with KCl (40 mM), pre-treatment with tetraethylammonium or with N(omega)-Nitro-L-arginine methyl ester inhibited the vasodilator response to isoproterenol only in aortic rings from older rats. The inhibition was total when TEA and L-NAME were administered together. In both age groups the response to isoproterenol was unaffected by the beta1-adrenergic antagonist CGP20712A, but was significantly inhibited by ICI 118551 (a beta2-adrenergic-antagonist) and to a greater extent by SR 59230A (a non-selective beta 3-adrenergic antagonist), the inhibition being more evident in the older rats. Unlike younger rats, in older animals the response to isoproterenol was partially dependent on endothelial nitric oxide and on K+ channels. In both age groups, beta2- and beta3-, but not beta1-adrenergic receptors were involved. The degree of relative participation of beta2 and beta3 adrenergic receptors may change with age and explain the differences in response to isoproterenol.

Low-voltage-activated potassium channels underlie the regulation of intrinsic firing properties of rat vestibular ganglion cells.

Individual primary vestibular afferents exhibit spontaneous activity the regularity of which can vary from regular to irregular. Different aspects of vestibular responsiveness have been associated with this dimension of regularity of resting discharge. Isolated rat vestibular ganglion cells (VGCs) showed heterogeneous intrinsic firing properties during sustained membrane depolarization: some neurons exhibited a strong adaptation generating just a single or a few spikes (phasic type), whereas other neurons showed moderate adaptation or tonic firing (tonic type). Tonic discharging VGCs were rare at postnatal days 5-7 and increased up to approximately 60% of neurons during postnatal 2-3 wk. To explore the major factors responsible for the discharge regularity of primary vestibular afferents, we investigated the contribution of K+ channels to the firing properties of isolated rat VGCs. Phasic firing became tonic firing in the presence of 4-aminopyridine or alpha-dendrotoxin, indicating that Kv1 potassium channels control the firing pattern of the phasic VGCs. Tetraethylammonium decreased the number of spikes during step current stimuli in all types. Blockade of Ca2+-activated K+ channels decreased the number of spikes in tonic VGCs. Our results suggest that Kv1 channels are critical both in determining the pattern of spike discharge in rat vestibular ganglion neurons and in their proportional change during maturation.

4-aminopyridine-induced contracture in frog ventricle is due to calcium released from intracellular stores.

The aim of the study is to demonstrate the presence of intracellular calcium store in frog ventricle based on contractures induced by 4-aminopyridine in calcium-free media. Frog-ventricular strips were subjected to field stimulation at 0.2 Hz and the force of contraction was recorded after stabilization. The preparation was then kept quiescent for some time in solutions with different sodium concentrations, containing 0 or 1 mmol/L calcium. Caffeine, 4-aminopyridine (4-AP), or tetraethylammonium chloride was then added. Frog skeletal muscle preparations were used as positive controls for the caffeine experiments. Frog ventricular preparations did not develop contractures (sustained contractions) in the presence of caffeine (25 mmol/L), while frog skeletal muscle preparations developed caffeine-induced contractures. However, 4-AP (16 mmol/L) was able to induce contractures in quiescent frog ventricular preparations, even when they were superfused with calcium-free solution. 4-AP contractures in frog ventricle were seen in the presence of nifedipine also. Amplitude of 4-AP evoked contractures in frog ventricle were much larger in low sodium (30 mmol/L) and sodium-free (sodium substituted by lithium) solutions than in normal sodium solution, suggesting that the route of extrusion of the cytosolic calcium (released from intracellular stores by 4-AP) is the sodium calcium exchanger, which gets reversed in low sodium solutions. Tetraethylammonium chloride (TEA) was not able to induce contractures in frog ventricle suggesting that the contracture evoked by 4-AP is not due to its potassium channel blocking effect. In quiescent frog skeletal muscle preparations, caffeine as well as 4-AP induced contractures in calcium-free solutions. We therefore conclude that there is a caffeine-insensitive, 4-AP sensitive intracellular calcium store in the frog ventricle.

Bursting in substantia nigra pars reticulata neurons in vitro: possible relevance for Parkinson disease.

Projection neurons of the substantia nigra reticulata (SNr) convey basal ganglia (BG) processing to thalamocortical and brain stem circuits responsible for movement. Two models try to explain pathological BG performance during Parkinson disease (PD): the rate model, which posits an overexcitation of SNr neurons due to hyperactivity in the indirect pathway and hypoactivity of the direct pathway, and the oscillatory model, which explains PD as the product of pathological pattern generators disclosed by dopamine reduction. These models are, apparently, incompatible. We tested the predictions of the rate model by increasing the excitatory drive and reducing the inhibition on SNr neurons in vitro. This was done pharmacologically with bath application of glutamate agonist N-methyl-d-aspartate and GABA(A) receptor blockers, respectively. Both maneuvers induced bursting behavior in SNr neurons. Therefore synaptic changes forecasted by the rate model induce the electrical behavior predicted by the oscillatory model. In addition, we found evidence that Ca(V)3.2 Ca(2+) channels are a critical step in generating the bursting firing pattern in SNr neurons. Other ion channels involved are: hyperpolarization-activated cation channels, high-voltage-activated Ca(2+) channels, and Ca(2+)-activated K(+) channels. However, although these channels shape the temporal structure of bursting, only Ca(V)3.2 Ca(2+) channels are indispensable for the initiation of the bursting pattern.

Role of BK channels in testosterone-induced relaxation of the aorta in spontaneously hypertensive rats.

The previous data indicated that the testosterone (Tes)-induced relaxation of thoracic aorta is greater in spontaneously hypertensive rats (SHR) than in normotensive rats (Wistar-Kyoto rats; WKY) and that there were differences between SHR and WKY in the functions of K(ATP), K(v), and K(Ca) channels. The present study was carried out to ascertain the mechanisms of the Tes-induced relaxation. Indomethacin (30 muM) pretreatment suppressed the Tes-induced relaxation. Following noradrenalin (NA)-induced vasoconstriction, the relaxation induced by Tes was significantly attenuated by endothelium removal in SHR (not in WKY), but the dilatory effect of Tes following KCl-induced vasoconstriction was not attenuated by endothelium removal. After tetraethylammonium (K(Ca) channel inhibitor) or iberiotoxin (large conductance, Ca(2+) activated BK channel inhibitor) pretreatment, the Tes-induced relaxation was attenuated in SHR, but not in WKY. This attenuation in SHR was not observed after endothelium removal. The above results suggest that the relaxation induced by Tes following NA-induced vasoconstriction in SHR results from hyperpolarization due to BK channel opening.

Calcimimetic, AMG 073, induces relaxation on isolated rat aorta.

Calcimimetics are a class of compounds that positively modulate the calcium-sensing receptor (CaR) by allosterically increasing the affinity of the receptor for extracellular Ca(2+). In this study we have investigated the effects of the clinically used calcimimetic, AMG 073, on contractility of the rat aorta by wire myography. AMG 073 elicited a concentration-dependent vasodilatation of the precontracted aorta. Inhibition of endothelium function by L-NAME and indomethacin reduced AMG 073-induced relaxation of the vessel precontracted with phenylephrine, but not with 125 mM K(+). The vasodilatory effect could be mediated by the CaR or/and a direct action on the ion channels. Intriguingly, CaR agonists, neomycin and gadolinium, did not have any effect on the contractility of the aorta. Immunohistochemical staining of the aorta with two CaR specific antibodies demonstrated the presence of the CaR protein, predominantly in endothelial and adventitial layers.

Transport of organic cations across the blood-testis barrier.

Throughout the mammalian spermatogenic pathway, differentiating spermatogenic cells remain in close contact with somatic Sertoli cells, and this has been considered to be essential for the proliferation, differentiation, and survival of spermatogenic cells. It is thought that Sertoli cells form tight junctions to protect developing spermatogenic cells against harmful agents and provide several nutrients for spermatogenesis from the blood stream. Accordingly, Sertoli cells should regulate the movement of various nutritious and xenobiotic compounds by selective membrane transporters. However, the information on membrane transporters in Sertoli cells is limited. In the present study, we characterized the transport systems of organic cations in Sertoli cells. Uptake of [14C]tetraethylammonium (TEA) was measured by primary-cultured rat Sertoli cells. Initial uptake of TEA was concentration dependent, and an Eadie-Hofstee plot indicated the involvement of two saturable transport systems. The apparent Km values of high- and low-affinity components were comparable to those of previously known organic cation transporter (OCT) or organic cation/carnitine transporter (OCTN). In addition, OCT1, OCT3, OCTN1, and OCTN2 were expressed in Sertoli cells. In conclusion, multiple organic cation transporters, OCTs and OCTNs are expressed in Sertoli cells and the cells regulate transport of cationic compounds to protect and/or maintain the spermatogenesis in testis as the blood-testis barrier.

Changes in stretch-induced tone induced by intracellular acidosis in rabbit basilar artery: effects on BKCa channel activity.

It is known that myogenic reactivity is a fundamental determinant of the relative constancy of blood flow through the cerebral artery. It is also known that acute alteration of pH significantly affects the cerebral circulation and, therefore, we investigated the effect of mechanism of action of intracellular acidosis on myogenic tone in rabbit basilar artery. Myogenic tone was developed by imposed stretch of basilar artery and intracellular acidosis induced by the bath application of 20 mmol/L sodium acetate. Sodium acetate caused a biphasic increase in myogenic tone. The initial component reached a peak quickly and then fell slowly to a lower steady-state significantly above basal tone. The sodium acetate-induced increase in myogenic tone was completely inhibited by elimination of external Ca2+, or treatment of nifedipine, but not with gadolinium or NPPB. TEA (5 mmol/L) and iberiotoxin (100 nmol/L) inhibited the sodium acetate-induced increase in myogenic tone. In inside-out patch-clamp recordings, decreasing pH of the mock intracellular solution from 7.4 to 6.9 markedly inhibited BKCa currents. Several inhibitors involved in Ca2+ sensitization pathways, 10(-6) mol/L Y-27632, 5 x 10(-7) mol/L calphostin C and 10(-5) mol/L PD98059 had no effect on the sodium acetate-induced increase in myogenic tone. These results suggest that intracellular acidosis increases stretch-induced myogenic tone in rabbit basilar artery. Furthermore, voltage-dependent Ca2+ influx plays a key role in intracellular acidosis-induced increase in myogenic tone and may be mediated, at least in part, by inhibition of BKCa.

Sodium-activated potassium current in guinea pig gastric myocytes.

This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.

The antiestrogen tamoxifen activates BK channels and stimulates proliferation of MCF-7 breast cancer cells.

In the present study, we investigated the effect of the antiestrogen compound tamoxifen on BK channels by the use of the patch-clamp technique. The perfusion of 10 nM tamoxifen significantly increased the magnitude of a voltage-dependent K+ current by 22.6 +/- 10.6% (n = 23). The effect of tamoxifen was always obtained in the first minute, peaked at 5.9 +/- 2.2 min (n = 23), and was abolished by the perfusion of tetraethylammonium (0.5 mM), charybdotoxin (50 nM), or iberiotoxin (100 nM). The stimulatory effect of 10 nM tamoxifen was the same at low (50 nM) and high (700 nM) internal calcium concentration and was not additive to that of 17-beta-estradiol (E2) or its membrane-impermeant form, beta-estradiol 6-(O-carboxymethyl)oxime:bovine serum albumin. Furthermore, the effect of tamoxifen was still recorded in the presence of the selective estrogen receptor antagonist faslodex (ICI-182,780; 1 microM). At the single-channel level, tamoxifen significantly increased the open probability of the BK channel by 46.2 +/- 10.1% (n = 4) without changing its unitary conductance. Moreover, we show here that the stimulation of BK channel activity by tamoxifen is involved in MCF-7 cell proliferation. Taken together, these results permitted us to identify the BK channel as the molecular target of tamoxifen that probably acts at the same extracellular molecular level as E2. The site of action of tamoxifen is probably the channel itself or the auxiliary beta subunits.

Sodium-activated Potassium Current in Guinea pig Gastric Myocytes

This study was designed to identify and characterize Na+ -activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+] degree). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]degree (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+ - and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+ -selective, since reversal potentials (E(rev)) of various concentrations of [K+]degree-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microgram), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes.