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Results for “patch clamp”

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PubMed2001

Hormonally-regulated expression of voltage-operated Ca(2+) channels in osteocytic (MLO-Y4) cells.

Voltage operated calcium channels (VOCCs) are important in stimulus-response coupling in osteoblasts. We have investigated the expression of VOCCs in the mouse osteocyte cell line, MLO-Y4. Using the whole-cell patch clamp technique we were unable to detect any VOCC currents (n = 436) even in the presence of the L-type VOCC agonist Bay K 8644 (n = 350). Reverse transcription polymerase chain reaction (RT-PCR), using primers to detect alpha(1C), alpha(1D), and alpha(1G) VOCC subunits (all of which are expressed in primary osteoblasts), did not generate detectable products with mRNA from MLO-Y4 cells. However, after treatment with physiological levels of hormones, VOCC alpha(1) subunit mRNAs were detected in MLO-Y4 cells. PTH, 17beta-estradiol, and dexamethasone-treatment induced expression of L-type (alpha(1C), alpha(1D)) subunit transcripts. ATP-treatment induced expression of T-type (alpha(1G)) transcripts. Using whole-cell patch clamp we detected VOCC currents in 5-10% of cells after treatment. Current characteristics (L- or T-type) were consistent with the transcript expressed.

Adenosine Triphosphate↗

PubMed1997

Involvement of both calcium influx and calcium mobilization in growth hormone-induced [Ca2+]i increases in Chinese hamster ovary cells.

This study reports rapid effects of growth hormone (GH) on the intracellular free calcium concentration ([Ca2+]i) in Chinese hamster ovary (CHO) cells stably expressing rabbit GH receptor. [Ca2+]i was measured by spectrofluorimetric methods in single cells and membrane Ca2+ currents by patch clamp techniques in the whole-cell configuration. In individual CHO cells, bathed in a standard saline solution containing 2 mM Ca2+, basal [Ca2+]i was 191 +/- 27 nM (mean +/- S.D.; n=83). Short term administration of GH (100 ng/ml, 30 s) induced a [Ca2+]i increase in 54% of cells tested (n = 398 of 743). Responses were clearly heterogeneous. Maximum calcium increase varied from 16 to 853 nM and time to peak varied from 4 to 320 s. On examination of the [Ca2+]i increases, it was possible to define two different types of calcium responses to GH. Experimental manipulations of extracellular and intracellular calcium concentrations demonstrated that GH-induced calcium increases involved both calcium influx and calcium mobilization. Calcium influx, a long lasting, small amplitude (63 +/- 34 nM) response, was observed in 121 out of 398 cells (30%) whereas calcium mobilization, a transient, large amplitude (263 +/- 175 nM) response, was observed in 277 out of 398 cells (70%). Moreover, patch clamp data show that influx did not involve the dihydropyridine-sensitive calcium channels.

Animals↗

PubMed1997

Extracellular nucleotides activate non-selective cation and Ca(2+)-dependent K+ channels in rat osteoclasts.

1. Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 microM) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3. The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 +/- 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to -21 +/- 3 mV, indicating that ATP activates a non-selective cation current, consistent with involvement of P2X receptors. 4. The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I-V relationship, and reversed at -71 +/- 4 mV. The reversal potential shifted 51 mV per 10-fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5. In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca(2+)-free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br-A23187) to elevate [Ca2+]i, IK activated when [Ca2+]i exceeded approximately 400 nM, with half-maximal activation at 580 +/- 50 nM. 6. In cell-attached patches, ATP activated a channel with a conductance of 48 +/- 6 pS, that reversed director, near EK. Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7. These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non-selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca(2+)-dependent K+ channel.

Adenosine Triphosphate↗

PubMed2002

Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons.

Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.

Action Potentials↗

PubMed2002

Novel class of potent 4-arylalkyl substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling.

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.

Animals↗

PubMed1996

cAMP stimulates fluorescent bile acid uptake into hepatocytes by membrane hyperpolarization.

Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acids. The purpose of this study was to determine to what extent these two phenomena are linked. Fluorescent bile acid analogues (FBA) were used to probe bile acid transport into whole cell patch-clamped hepatocytes. Na(+)-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysine (C-NBD-L), an FBA with a net charge of -1, was shown to be electrogenic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA with a net charge of -2, was neutral. Incubation of hepatocytes with 8-bromo-cAMP (8-BrcAMP; 100 microM) increased the uptake rate of the electrogenically transported FBA by 25% (P = 0.002), but had no effect on the uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepatocytes by 6-8 mV. To determine if hyperpolarization is responsible for the cAMP-induced increase in uptake rate, cAMP was directly introduced into hepatocytes during whole cell patch clamp under voltage-clamp conditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was terminated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP had no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-mV hyperpolarization increased the uptake rate of C-NBD-L by 23%. We therefore conclude that short-term cAMP-induced stimulation of fluorescent bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.

Animals↗

PubMed1999

Renal function and Ca(2+) currents after dye-labeling identification of renal sympathetic neurons.

The present study was performed to determine whether renal efferent sympathetic neurons could be identified using a retrograde neuronal tracer without compromising renal function and whether the labeling and identification procedure alters Ca(2+) currents and neuromodulation of those neurons. Renal sympathetic and superior cervical ganglion (SCG) neurons were labeled with the fluorescent retrograde tracer fast blue. Renal function studies made 1 wk after labeling revealed that renal hemodynamics and fluid and electrolyte excretion were similar between the dye-injected (left) kidney and the control (right) kidney under control conditions and after hemorrhage. After volume expansion, urine flow in the dye-injected kidney was slightly, but significantly, less than that of the control kidney, whereas urinary sodium excretion increased by approximately ninefold in both kidneys. Patch-clamp studies of SCG neurons in 10 mM external Ca(2+) revealed that peak currents were not affected by the presence of the dye or a 1-min exposure to ultraviolet (UV) light. Neither maximal norepinephrine-induced Ca(2+) current inhibition nor the sensitivity to norepinephrine was affected by the dye or 1-min UV exposure. Facilitation protocols revealed that G protein modulation of Ca(2+) currents remained intact in dye-labeled UV-exposed neurons. This study demonstrates that a retrograde fluorescent dye technique to identify renal sympathetic neurons does not compromise renal function and the presence of the dye label or UV exposure has no effect on Ca(2+) currents and neuromodulation in these neurons. Isolation of single identified renal sympathetic neurons coupled with patch-clamp techniques represents a tool to investigate the role of individual current systems in the modulation of excitability in these neurons, which play an important role in the control of renal hemodynamics and excretory function and in the pathogenesis of hypertension.

Amidines↗

PubMed2004

Direct myocardial depressant effect of naloxone: mechanical and electrophysiological actions in vitro.

BACKGROUND: Naloxone, an opioid antagonist, has a cardiovascular pressor effect and has been used in various types of shock states. The aim was to determine the non-opioid effect on direct cardiac muscle contractility and explore the mechanism using guinea pig right ventricular papillary muscles. METHODS: With institutional approval, isometric contractile force was measured in modified normal and 26 mM K+ Tyrode solution at various stimulation rates. The effects of naloxone on sarcoplasmic recticulum function were evaluated by measuring rested-state contraction in low Na+ (25 mM) Tyrode solution and rapid cooling contracture in modified normal Tyrode solution. Normal and slow action potentials (APs) were measured using conventional microelectrode technique. Patch clamp study was performed to examine the direct effect on Ca2+ current in cardiac ventricular myocytes. RESULTS: Naloxone (50, 100, and 200 microM) caused a concentration-dependent depression of peak force and maximal rate of force development. Modest depression, approximately 20%, was shown in rested-state contraction in low Na+ Tyrode solution. Naloxone (100 microM) modestly depressed the rapid cooling contracture to 80 +/- 3% of baseline, accompanied by prolongation of time to peak contracture by approximately 37%. In 26 mM K+ Tyrode solution, naloxone (100 microM) markedly and selectively depressed the late force development. While naloxone (100 microM) did not alter the amplitude and dV/dt-max in normal and slow APs at 0.25 Hz, AP duration was prolonged significantly. In patch clamp experiment, naloxone (50 microM) depressed Ca2+ current by approximately 50%. CONCLUSIONS: The direct myocardial depressant effect of naloxone appeared to be in part caused by depression of Ca2+ influx through cardiac membrane. Sarcoplasmic reticulum function appeared to be modestly depressed.

Action Potentials↗

PubMed2004

Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed.

BACKGROUND: Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. METHODS: Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. RESULTS: hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. CONCLUSION: Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.

Adult↗

PubMed2001

Membrane orientation of droplets prepared from Chara corallina internodal cells.

It is generally accepted that the membrane surrounding droplets from characean cells originates from the tonoplast, but there is some uncertainty regarding droplet membrane sidedness. This issue was addressed directly by combining two different droplet isolation methods and the patch clamp technique. Neutral red accumulation was used to demonstrate the presence of H(+)-transport over the membrane and to predict membrane orientation. Two types of droplet populations with differently oriented membranes could be formed in an iso-osmotic bath solution. Cytoplasmic droplets (cytosolic side of the tonoplast inside) contained cytoplasm, while the second type of droplet population contained vacuolar sap (vacuolar droplets, vacuolar side of the tonoplast inside). Smaller vesicels also appeared inside the droplets, with an apparently inversely oriented membrane. Confocal laser scanning microscopy indirectly demonstrated that, at least with one of the droplet isolation methods, the plasma membrane entirely remains in the internodal cell after intracellular perfusion. Both types of droplet populations allowed the formation of excised patches and single-channel measurements by the patch clamp technique. Properties of anion channels in the tonoplast could be used to prove the predicted membrane orientation, knowing that Ca2+ can only activate these channels from the cytosolic side. These results provide useful data for studies addressing ligand-binding, block and modulation, organization and interaction of proteins within the membrane or with other regulatory factors, where it is important to control membrane orientation.

Cell Membrane↗

PubMed2005

Stimulation of cannabinoid (CB1) and prostanoid (EP2) receptors opens BKCa channels and relaxes ocular trabecular meshwork.

Prostanoids and cannabinoids have ocular hypotensive and neuroprotective properties. The effect of the prostanoid AH13205 (EP2), the thromboxane-mimetic U46619, the cannabinoid (CB) agonists WIN55212-2 and CP 55,940, endothelin-1 (ET-1) and 8-bromo-cAMP on the membrane currents of trabecular meshwork (TM) cells were measured using the patch-clamp technique and compared to their effects on TM contractility. Previous studies show relaxation of TM to AH 13205 and other substances that elevate cAMP, while U46619 and endothelin-1 contract TM. This study shows that after contraction (100%) with carbachol (10(-6)m), the CB agonist CP 55,940 dose-dependently reduced contractility to 83+/-4% (n=9) (10(-6)m) and 61+/-10%, (n=7) (10(-5)m). In the presence of both the CB1 antagonist AM251 (10(-6)m) and CP 55,940 (10(-5)m), the contractile response to carbachol reached 84+/-3% (n=6) of the original level. In patch-clamp experiments, membrane permeable 8-bromo-cAMP (10(-4)m) had no effect on currents of TM cells. In contrast, AH 13205 and two cannabinoids reversibly enhanced outward current through high-conductance Ca(2+)-activated K(+) channels (BKCa, BK, maxi-K) to the following values (in % of the initial value at 100 mV): AH 13205 (10(-5)m): 200+/-28% (n=6), CP 55,940 (10(-6)m): 196+/-33% (n=7), CP 55,940 (10(-5)m): 484+/-113% (n=7), WIN55212-2 (10(-5)m): 205+/-41% (n=10). Iberiotoxin (10(-7)m) completely blocked these responses. The current response to CP 55,940 (10(-5)m) could be partially blocked by the CB1 antagonist AM251 (10(-6)m). Conversely, the contractile agents in this study either caused a transient reduction in outward current (ET-1(5x10(-8)m)) or had no effect (U46619 (10(-6)m)). We conclude that stimulation of EP2 and CB1 receptors in TM is coupled to the activation of BKCa channels via a non-diffusible second messenger cascade. This effect may contribute to the relaxant activity of EP2 and CB1 agonists in isolated TM strips, modulating ocular outflow.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

Effects of manidipine and nitrendipine enantiomers on the plateau phase of K+-induced intracellular Ca2+ increase in GH3 cells.

The aim of the present study was to investigate whether the chirality and type of substitution at position 3 of the dihydropyridine ring influences the pattern of voltage-gated Ca2+ channel blockade. For this purpose, the effect of R- and S-enantiomers of manidipine and nitrendipine, separated by chiral High-Pressure-Liquid-Chromatography columns, were investigated by fura-2 microfluorimetry during the plateau phase of the intracellular Ca2+ ([Ca2+]i) increase induced by 55 mM K+ and by patch-clamp recording of Ca2+ channel activity in GH3 cells. R- and S-enantiomers of both nitrendipine and manidipine produced a [Ca2+]i decay of the K+-induced plateau phase that followed a biexponential pattern with a 'fast' and a 'slow' phase. The S-configuration of both nitrendipine and manidipine produced a larger [Ca2+]i decrease during the 'fast phase', and a faster and smaller [Ca2+]i decrease in the 'slow phase' than did the R-enantiomers. The S- and R-enantiomers of manidipine, which possess a longer and more lipophilic side chain at position 3 of the dihydropyridine ring, induced a slower [Ca2+]i decrease than that observed with the respective nitrendipine enantiomers. Accordingly, patch-clamp experiments revealed that the S-enantiomers of both dihydropyridines displayed a faster onset of action and produced a greater blockade than the R-enantiomers. These results suggest that the enantiomeric configuration and a small side chain at position 3 of the dihydropyridine ring are factors in the chemical structure which influence the pattern of blockade of voltage-sensitive Ca2+ channels.

Animals↗

PubMed1998

Actions of prostaglandin E2 on rat supraoptic neurones.

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2006

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Arabidopsis↗

PubMed2004

Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells.

PURPOSE: Hyposmolar perfusion of intact trabecular meshwork (TM) induces a decrease in its hydraulic conductivity (Lp). However, exposure to agents that elevate intracellular cAMP in TM cells increases Lp. Since volume of TM cells could directly influence porosity of the TM and hence Lp, this study has investigated changes in volume in response to acute hyposmotic shock (i.e. regulatory volume decrease or RVD) and elevated cAMP in cultured TM cells. METHODS: Bovine trabecular meshwork cells (BTMC), grown on glass coverslips and loaded with the fluorescent dye MQAE, were used to measure rapid changes in cell volume using the principle of dynamic fluorescence quenching. Activation of volume-regulated anion channels (VRAC) was assessed by measuring volume-sensitive Cl(-) currents (I(Cl,swell)) in the whole cell configuration of the patch clamp technique and by determining the swelling-induced enhancement in I(-) permeability using the halide-sensitivity of MQAE. Expressions of ClC (chloride channels of the ClC gene family), P-glycoprotein (Pgp), and cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels were examined by RT-PCR. Elevation of cAMP in response to forskolin was confirmed by determining the phosphorylation of cAMP response element-binding protein and activating transcription factor-1 (CREB, ATF-1), which form the downstream targets of protein kinase A. RESULTS: As a response to hyposmotic shock, there was an acute increase in cell volume but there was no robust RVD. Patch clamp experiments showed activation of a characteristic Cl(-) current in response to cell swelling. This Cl(-) current was inhibited by NPPB (100microM) and fluoxetine (50microM), both of which are known blockers of VRAC. Experiments, which used the halide-sensitivity of MQAE, also indicated a 9-fold increase in I(-) influx upon cell swelling (8.9+/-4.6; n=9), consistent with activation of a VRAC-like Cl(-) current. To examine whether RVD is limited by K(+) conductance, the swollen cells were exposed to gramicidin, which is known to induce cation channel activity. Such a maneuver led to secondary swelling with [Na(+)](o)=140mM but a rapid shrinkage [Na(+)](o)=8mM indicating that the RVD is limited by cationic conductance necessary for K(+) efflux. Exposure to forskolin, which resulted in CREB and ATF-1 phosphorylation, caused a reversible decrease in cell volume (14.5+/-5%; n=20) under isosmotic and hyposmotic conditions. RT-PCR analysis confirmed expression of ClC-2, ClC-5, and Pgp Cl(-) channels in bovine TM cells. However, ClC-3 and CFTR were not expressed. CONCLUSIONS: TM cells respond to acute hyposmotic shock in an osmometric manner, but their RVD is limited by K(+) conductance. The lack of CFTR expression and decrease in cell volume in response to forskolin concomitant with hyposmolarity suggest that elevated cAMP activates a K(+) conductance. Thus, the altered resistance to aqueous outflow in response to hyposmotic perfusion of the TM and elevated cAMP may be attributed to persistent cell swelling and cell shrinkage, respectively.

Animals↗

PubMed1998

The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats.

Whole-cell patch-clamp and extracellular field recordings were obtained from 450-microns-thick brain slices of infant rats (10-13 days postnatal) to determine the actions of corticotropin-releasing hormone on glutamate- and GABA-mediated synaptic transmission in the hippocampus. Synthetic corticotropin-releasing hormone (0.15 microM) reversibly increased the excitability of hippocampal pyramidal cells, as determined by the increase in the amplitude of the CA1 population spikes evoked by stimulation of the Schaffer collateral pathway. This increase in population spike amplitude could be prevented by the corticotropin-releasing hormone receptor antagonist alpha-helical (9-41)-corticotropin-releasing hormone (10 microM). Whole-cell patch-clamp recordings revealed that, in the presence of blockers of fast excitatory and inhibitory synaptic transmission, corticotropin-releasing hormone caused only a small (1-2 mV) depolarization of the resting membrane potential in CA3 pyramidal cells, and it did not significantly alter the input resistance. However, corticotropin-releasing hormone, in addition to decreasing the slow afterhyperpolarization, caused an increase in the number of action potentials per burst evoked by depolarizing current pulses. Corticotropin-releasing hormone did not significantly change the frequency, amplitude or kinetics of miniature excitatory postsynaptic currents. However, it increased the frequency of the spontaneous excitatory postsynaptic currents in CA3 pyramidal cells, without altering their amplitude and single exponential rise and decay time constants. Corticotropin-releasing hormone did not change the amplitude of the pharmacologically isolated (i.e. recorded in the presence of GABAA receptor antagonist bicuculline) excitatory postsynaptic currents in CA3 and CA1 pyramidal cells evoked by stimulation of the mossy fibers and the Schaffer collaterals, respectively. Current-clamp recordings in bicuculline-containing medium showed that, in the presence of corticotropin-releasing hormone, mossy fiber stimulation leads to large, synchronized, polysynaptically-evoked bursts of action potentials in CA3 pyramidal cells. In addition, the peptide caused a small, reversible decrease in the amplitude of the pharmacologically isolated (i.e. recorded in the presence of glutamate receptor antagonists) evoked inhibitory postsynaptic currents in CA3 pyramidal cells, but it did not significantly alter the frequency, amplitude, rise and decay time constants of spontaneous or miniature inhibitory postsynaptic currents. These data demonstrate that corticotropin-releasing hormone, an endogenous neuropeptide whose intracerebroventricular infusion results in seizure activity in immature rats, has diverse effects in the hippocampus which may contribute to epileptogenesis. It is proposed that the net effect of corticotropin-releasing hormone is a preferential amplification of those incoming excitatory signals which are strong enough to reach firing threshold in at least a subpopulation of CA3 cells. These findings suggest that the actions of corticotropin-releasing hormone on neuronal excitability in the immature hippocampus may play a role in human developmental epilepsies.

Action Potentials↗

PubMed1999

Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine, enhances L-type Ca2+ current and [Ca2+]i transient in cardiomyocytes.

This study investigates whether leukemia inhibitory factor (LIF), a potent cardiac hypertrophic cytokine, affects the L-type Ca2+ current (I(Ca,L)) and intracellular Ca2+ concentrations ([Ca2+]i) in cardiomyocytes. I(Ca,L) was recorded using a whole cell patch clamp configuration in guinea pig cardiomyocytes, and the [Ca2+]i transient was detected by use of Fluo-3 in rat cardiomyocytes. Cells were preincubated with LIF (1000 U/ml) for 15 min before whole cell recording. LIF increased I(Ca,L) by 41.8%. LIF synergistically increased I(Ca,L) with isoproterenol. Preincubation with H89 did not inhibit the LIF-induced increase in I(Ca,L), indicating that this phenomenon is PKA-independent. PD98059 completely inhibited the increase in I(Ca,L), and this effect was dose-dependent (IC50=3.6 micromol/l). Other signal transduction inhibitors including AG490, SB203580, chelerythrine, genistein, and KN62 did not affect the LIF-induced increase in I(Ca,L). Perforated patch clamp recording revealed that LIF maximally increased the I(Ca,L) by 25% at 15 min. LIF also increased the peak [Ca2+]i transient level by 63% at 15 min. PD98059 fully inhibited the increase in the [Ca2+]i transient. In conclusion, LIF increased I(Ca,L) and the [Ca2+]i transient in cardiomyocytes, and the Raf-1/MEK/ERK pathway might be involved in the modulation of this activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Inhibition of rat hippocampal excitability by the plant alkaloid 3-acetylaconitine mediated by interaction with voltage-dependent sodium channels.

The effects of the Aconitum alkaloid 3-acetylaconitine on neuronal activity were investigated in the slice preparation and on cultivated neurons of rat hippocampus by extracellular and patch-clamp recordings, respectively. 3-Acetylaconitine (0.01-1 microM) diminished the orthodromic and antidromic population spike in a concentration-dependent manner. The inhibitory action of the drug was preceded by a transiently enhanced excitability. The latency of onset of the inhibition was accelerated by increased stimulation frequency, whereas recovery during washout of the alkaloid was accelerated by decreased stimulation frequency. Moreover, the inhibitory effect of 3-acetylaconitine was evaluated in two different models of epileptiform activity induced either by blockade of GABA receptors by bicuculline (10 microM) or by a nominal Mg(2+)-free bathing medium. In accordance with the activity-dependent mode of action, this compound abolished the synaptically evoked population spikes in the presence of bicuculline or nominal Mg(2+)-free bathing medium, respectively. Whole-cell patch-clamp recordings revealed an interaction of 3-acetylaconitine with the voltage-dependent sodium channel. At a concentration of 1 microM, 3-acetylaconitine did not affect the peak amplitude of the sodium current, but shifted the current-voltage relationship in the hyperpolarized direction such that sodium currents were already activated at the resting potential.

Aconitine↗