Assessment of gold nanoparticle effect on prostate cancer LNCaP cells.

UNLABELLED: In recent years gold nanoparticles (AuNPs) have received considerable attention for various biomedical applications including diagnostics and targeted drug delivery. However, more research is still needed to characterize such aspects of their use in clinical oncology as permeability, retention and functional effect on tumor cells. AIMS: This study was designed to describe the effect of non-functionalized AuNPs on LNCaP prostate cancer cells growth. MATERIAL AND METHODS: LNCaP cells were cultured in RPMI-1640 medium containing AuNPs covered by polyvinylpyrrolidone of average size 26.4 nm (10.0 µg/ml). Counts of cells were calculated and their morphology was examined. RESULTS: AuNPs conglomerates have been visualized in cultured cells. After 4-day incubation in presence of AuNPs significant retardation of LNCaP cells growth was observed both in 5α-dihydrotestosterone stimulated and non-stimulated cultures. No morphological changes of live LNCaP cells were seen in any experiment. CONCLUSION: Given absence of morphological changes in live cells and dribble and relatively constant numbers of dead cells, it was concluded that inhibitory effect of AuNPs on LNCaP cells growth was caused by alterations of proliferation.

Modulation by testosterone of an endogenous hERG potassium channel current.

hERG (human ether-a-go-go-related gene) potassium (K+) channels are expressed in a range of tissue types including neuroblastoma cells and the heart, in which hERG K+ current is important for action potential repolarization. Whilst gender differences in cardiac repolarization and the QT interval of the cardiac electrocardiogram are well-established, comparatively little is known about regulation of hERG channels by sex hormones. In this study, whole-cell patch-clamp recordings were made at 37 degrees C from SH-SY5Y human neuroblastoma cells to investigate modulation of endogenous hERG K+ channel current (I(hERG)) by testosterone. Acutely applied testosterone at a physiologically relevant concentration (10 nM) produced a modest (approximately 13-15 %) increase in I(hERG) amplitude, whilst a high concentration (1 microM) slightly decreased I(hERG). The stimulatory effect of testosterone was inhibited by the androgen receptor antagonist flutamide (10 microM) and the PI-3 kinase inhibitor wortmannin (1 microM). Chronic (24 h) application of testosterone also augmented IhERG via flutamide-sensitive receptor activation, without modulation of the current's voltage-dependence. These results demonstrate for the first time that testosterone can stimulate (hERG) K+ channels via activation of classical androgen receptors and implicate PI-3 kinase in the acute response.

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells.

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.

Direct modulation of volume-regulated anion channels by Ca(2+) chelating agents.

Ca(2+) chelating agents are widely used in biological research for Ca(2+) buffering. Here we report that BAPTA, EDTA and HEDTA produce fast, reversible, voltage-dependent inhibition of swelling-activated Cl(-) current (I(Cl,swell)) in LNCaP prostate cancer epithelial cells that is unrelated to their Ca(2+) binding. BAPTA was the most effective (maximal blockade 67%, IC(50)=70 microM, at +100 mV) followed by EDTA and HEDTA. I(Cl,swell) blockade by EDTA was pH-dependent. BAPTA blocked I(Cl,swell) also in other cell types. We conclude that Ca(2+) chelating agents block I(Cl,swell) by acting directly on the underlying channel, and that the negative charge of the free chelator form is critical for the blockade.

Testosterone-mediated modulation of HERG blockade by proarrhythmic agents.

Diverse drugs from many therapeutic classes exert cardiotoxic side effects by inducing torsades de pointes (TdP), a life threatening cardiac arrhythmia, which often results from drug interaction with HERG (human ether-a-go-go related gene) encoded K(+) channels, that generate an I(Kr) component of the delayed rectifier cardiac K(+) current. Men are known to be at a lower risk for drug-induced TdP than women suggesting a role of sex steroid hormones, androgens and estrogens, in modulation of drug sensitivity of cardiac K(+) channels, particularly those encoded by HERG. Here by using neuroleptic agents haloperidol, pimozide, and fluspirilene, all of which can induce TdP, and a steroid hormone-sensitive system Xenopus oocytes for HERG channels expression we show that testosterone is able to reduce HERG-blocking potency of neuroleptics. Haloperidol, pimozide, and fluspirilene inhibited HERG current with IC(50) of 1.36, 1.74, and 2.34 microM, and maximal block of 73%, 76% and 65%, respectively. The action of these neuroleptics was voltage-dependent, most consistent with an open-channel blocking mechanism. Pretreatment of HERG-expressing oocytes with 1 microM testosterone increased the IC(50) values to 2.73, 2.08, and 5.04 microM, reduced the maximal block to 65%, 59%, and 64%, and strongly diminished voltage-dependence of the blockade. Testosterone treatment per se produced about a 35% reduction of HERG current compared with untreated oocytes. Our data suggest that androgens may protect against the arrhythmogenic actions of some cardiotoxic drugs.

Subtypes of low voltage-activated Ca2+ channels in laterodorsal thalamic neurons: possible localization and physiological roles.

The macroscopic, low-voltage-activated (LVA or T-type) Ca2+ current in isolated associative (or local-circuit) neurons from the laterodorsal thalamic nucleus of 14-17-day old rats was dissected into two components ("fast" and "slow"), corresponding to the activation of two LVA channel subtypes, based on the difference in the kinetics of inactivation and recovery from inactivation. The steady-state activation and inactivation properties of the channel subtypes endowed slow channels with a substantial window current, whereas fast channels had almost no such current. Fast channels were almost 2 times more sensitive to 30 microM nifedipine (78% inhibition), 10 microM flunarizine (92% inhibition) and 1 microM La3+ (87% inhibition), but about 1.8-fold less sensitive to 100 microM Ni2+ (32% inhibition) than slow channels (40%, 52%, 46% and 56% inhibition respectively). Both channels were almost equally sensitive to 100 microM amiloride (58% and 51% inhibition of fast and slow channels respectively). Comparison of the fast and slow LVA Ca2+ current amplitudes and densities between enzymatically isolated and intact (in brain slices) neurons suggest a predominant localization of the fast channels in soma and the proximal dendrites that remain intact during isolation procedure, whereas the slow channels are more evenly distributed with some preference to the distal areas. These data, together with our previous studies, support the notion of two LVA Ca2+ channel subtypes in associative thalamic neurons and suggest a role for the slow channels in providing the constant Ca2+ influx necessary for the outgrowth of the neurites and for the fast channels in the generation of low-threshold Ca2+ spikes and bursting activity.

Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line.

Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl(-) was the primary charge carrier (termed I(Cl,swell)). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br(-) approximately I(-) > Cl(-) > F(-) > methanesulfonate >> glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC conductance was determined at 9.6 +/- 1.8 pS. Conventional Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) and DIDS (100 microM) inhibited whole cell I(Cl,swell) in a voltage-dependent manner, with the block decreasing from 39.6 +/- 9.7% and 71.0 +/- 11. 0% at +50 mV to 26.2 +/- 7.2% and 14.5 +/- 6.6% at -100 mV, respectively. Verapamil (50 microM), a standard Ca(2+) antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I(Cl,swell) (genistein with an IC(50) of 2.6 microM and lavendustin A by 60 +/- 14% at 1 microM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 microM) stimulated I(Cl,swell) by 54 +/- 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.

Divalent cation selectivity of the subtypes of low voltage-activated Ca2+ channels in thalamic neurons.

LVA Ca2+ current in isolated associative neurons from the laterodorsal thalamic nucleus of 14- to 17-day-old rats was dissected into two, 'fast' and 'slow', components based on the difference in the kinetics of inactivation. The selectivity of the channel responsible for the fast LVA current for Ca2+, Sr2+ and Ba2+ (I(Ca):I(Sr):I(Ba) = 1.0:1.23:0.94) as well as the shifts of the I-V produced by these ions were found to be almost identical to those observed for LVA channels in other preparations. The channel responsible for the slow LVA current showed selectivity more characteristic of HVA Ca2+ channels (I(Ca):I(Sr):I(Ba) = 1.0:2.5:3.4), although the ability of Ca2+, Sr2+ and Ba2+ to shift its voltage dependence remained the same as for the fast channel.

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger.

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.

Suppression of mammalian K+ channel family by ebastine.

Nonsedating H1 receptor (H1-R) antagonists exert variable effects on QT interval, most likely mediated through modulation of cardiac K+ channels. We examined the effects of a novel H1-R antagonist, ebastine, on a family of K+ currents in isolated rat and guinea pig ventricular cardiomyocytes as well as on HERG-induced rapidly delayed rectifier K+ current (I(Kr)) in Xenopus laevis oocytes. The effect of ebastine was compared with that of two other H1-R antagonists, terfenadine and loratadine, with and without reported cardiotoxicity, respectively. In guinea pig ventricular myocytes, ebastine at concentrations approximating those found in plasma under certain conditions suppressed in a voltage-independent manner the I(Kr) (Kd = 0.14 microM, maximum block 74%) more effectively than the slowly delayed rectifier K+ current (I(Ks)) (Kd = 0.8 microM, maximum block 60%). Ebastine also suppressed I(Kr) in HERG-expressing X. laevis oocytes with the Kd value of 0.3 microM and a maximal block of 46% at 3 microM. The block of the rapidly activating delayed rectifier channel in rat myocytes (Iped) (Kd = 1.7 microM, maximum block 58%) had a small voltage dependence. Ebastine only minimally suppressed rat transient K+ current (Ito) (Kd = 1.1 microM, maximum block 10%). The drug was also not a very potent blocker of the inwardly rectifier K+ current (I(K1)) of rat and guinea pig (15 +/- 3% block at 3 microM). At concentrations of <100 nM, ebastine produced negligible effect on all K+ currents. We conclude that ebastine blocks various cardiac K+ channels with different potencies. The group of delayed rectifier K+ currents appeared to be most susceptible to ebastine with the order of sensitivity of I(Kr) > I(Ks) > Iped. Ebastine-induced inhibition of all K+ current types was always weaker than that observed with similar concentrations of terfenadine.

Beta-adrenoceptor-coupled Gs protein facilitates the activation of cAMP-dependent cardiac Cl- current.

Here a comparison is made between adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- current (ICl) density and activation time course in response to beta-adrenoceptor stimulation with isoproterenol and adenylyl cyclase activation with forskolin. Saturating concentrations of isoproterenol and forskolin failed to activate an ICl in guinea pig atrial as well as in rat and frog ventricular cardiomyocytes. In guinea pig ventricular cardiomyocytes, step application of 1 microM isoproterenol induced an ICl of -0.89 +/- 0.32 pA/pF (holding potential -40 mV, temperature 22 +/- 1 degrees C). ICl activation started after 3 +/- 1 s, was complete within 44 +/- 9 s, and was abolished after cell dialysis with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. Stimulation with increasing concentrations of forskolin (0.01-10 microM) increased ICl density and accelerated ICl activation. With 1 microM forskolin, ICl density was maximal (-0.57 +/- 0.30 pA/pF) but significantly smaller than that achieved with 1 microM isoproterenol. Although ICl density could not be further augmented by forskolin > 1 microM, current activation (latency 28 +/- 8 s, full activation after 112 +/- 8 s with 1 microM forskolin) was further accelerated by 3 and 10 microM forskolin. However, ICl activation with 10 microM forskolin was still slower than that with 1 microM isoproterenol. A low isoproterenol concentration (1 nM), which did not activate ICl by itself, accelerated the 1 microM forskolin-induced activation of ICl by 35%; this speeding up was abolished after cell dialysis with guanosine 5'-O-(2-thiodiphosphate). ICl deactivation after the washout of 1 microM forskolin or 1 microM isoproterenol followed a similar time course. After stimulation with 10 microM forskolin or 1 microM forskolin + 1 microM isoproterenol, but not with 1 microM forskolin + 1 nM isoproterenol, the decay of ICl was significantly delayed. These results indicate that both cAMP-dependent and cAMP-independent G protein pathways contribute to the regulation of guinea pig ventricular ICl.

Glass-funnel technique for the recording of membrane currents and intracellular perfusion of Xenopus oocytes.

In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e., extra- and intracellular, compartments. The technique is fairly simple to use, provides a much higher clamp speed compared to the double-microelectrode voltage-clamp technique, and allows effective control of the composition of the intracellular milieu. To elucidate the performance of the technique with respect to various membrane currents we present data relating to the recording of Ca-channel currents expressed in X. laevis oocytes by means of mRNA extracted from the rat cerebellum and heart, as well as currents induced by cRNA for the skeletal muscle micro1 Na+ channel and the dog heart NCX1 Na+-Ca2+ exchanger. Due to effective elimination of intra- and extracellular Cl- it became possible to measure not only Ba2+ but also Ca2+ current through the expressed Ca channels, and to record the activity of the Na+-Ca2+ exchanger following dialysis of the oocyte with high-Ca2+ intracellular solutions. Corresponding currents showed properties identical to those obtained with other techniques, suggesting the adequacy of the glass-funnel technique for critical analysis of membrane ionic currents in Xenopus oocytes.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.

Characterization of hypothalamic low-voltage-activated Ca channels based on their functional expression in Xenopus oocytes.

Ca-channel currents expressed in Xenopus oocytes by means of messenger RNA extracted from rat thalamohypothalamic complex were studied using the double microelectrode technique. Currents were recorded in Cl(-)-free extracellular solutions with 40 mM Ba2+ as a charge carrier. In response to depolarizations from a very negative holding potential (Vh = -120 mV), inward Ba2+ current activated at around -80 mV, peaked at -30 to -20 mV and reversed at +50 mV indicating that it may be transferred through the low voltage-activated calcium channels. The time-dependent inactivation of the current during prolonged depolarization to -20 mV was quite slow and followed a single exponential decay with a time-constant of 1550 ms and a maintained component constituting 30% of the maximal amplitude. The current could not be completely inactivated at any holding potential. As expected for low voltage-activated current, steady-state inactivation curve shifted towards negative potentials. It could be described by the Boltzmann equation with half inactivation potential -78 mV, slope factor 15 mV and maintained level 0.3. Expressed Ba2+ current could be blocked by flunarizine with Kd = 0.42 microM, nifedipine, Kd = 10 microM, and amiloride at 500 microM concentration. Among inorganic Ca-channel blockers the most potent was La3+ (Kd = 0.48 microM) while Cd2+ and Ni2+ were not very discriminative and almost 1000-fold less effective than La3+ (Kd = 0.52 mM and Kd = 0.62 mM, respectively). Our data show that messenger RNA purified from thalamohypothalamic complex induces expression in the oocytes of almost exclusively low voltage-activated calcium channels with voltage-dependent and pharmacological properties very similar to those observed for T-type calcium current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.

The effect of permeant ions on single calcium channel activation in mouse neuroblastoma cells: ion-channel interaction.

1. Single low-threshold inactivating (LTI or T-type) Ca2+ channels of undifferentiated neuroblastoma cells (clone N1E-115) were investigated using the patch-clamp technique. 2. Single-channel conductance, gi, for Ca2+, Sr2+ or Ba2+ as a permeant cation was similar (7.2 pS). Mean channel open time, tau op, was also practically independent of the divalent ion species; it decreased from 0.7 to 0.3 ms between -40 and 0 mV. 3. Modification of the calcium channel selectivity by lowering the external Ca2+ concentration to 10(-8) M produced an increase in gi for Na+ and Li+ ions and a shift of potential-dependent characteristics in the hyperpolarizing direction. Voltage sensitivity and absolute values of tau op were also changed. These changes were dependent on both permeant monovalent ion type and concentration. 4. At high [Na+]o, tau op was almost potential independent (congruent to 0.3 ms). Decrease in [Na+]o and substitution of Li+ for Na+ increased tau op and the steepness of its potential dependency. 5. The divalent and monovalent cations that were tested had much smaller effect on the mean intraburst shut time, tau cl(f), which was nearly independent of membrane potential (congruent to 0.6 ms). By contrast, mean burst duration was strongly potential dependent and noticeably affected by permeant ion type. 6. All kinetic changes were analysed in terms of a four-state sequential model for channel activation. According to this model the channel enters the open state through three closed states. Transitions between closed states can be formally related to the transmembrane movement of two charged gating particles (m2 process). The interaction between ion flux and a sterical region of the Ca2+ channel selectivity filter may, depending on ion transfer rate and ionic radius, lead to a local increase of the dielectric constant, resulting in redistribution of the electric field and changes in potential dependency of tau op.

Membrane-delimited stimulation of heart cell calcium current by beta-adrenergic signal-transducing Gs protein.

A severalfold increase in calcium current (ICa) is a signal feature of the maximal beta-adrenergic response of the heart. It is generally ascribed to enhanced adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation of calcium (Ca) channels after beta-receptor activation of the guanosine nucleotide-binding (G) protein Gs, and Gs activation of the adenylyl cyclase cascade. We blocked phosphorylation pathways in guinea pig cardiomyocytes to unmask other possible ICa-stimulatory modes. In blocked cells, ICa increased by approximately 50% during 1) beta-receptor activation of Gs, 2) intracellular activation of Gs, and 3) intracellular application of preactivated Gs, We conclude that fast, membrane-delimited Gs modulation participates in the physiological regulation of cardiac ICa.

Whole-cell calcium current in guinea-pig ventricular myocytes dialysed with guanine nucleotides.

1. Whole-cell calcium current (ICa) was recorded in guinea-pig ventricular myocytes superfused with Na+,K(+)-free solution and dialysed with a substrate-free solution (minimum intracellular solution, MICS). A dual tight-seal pipette method was often used to permit pressure-enhanced dialysis of a test solution after a given pre-dialysis. 2. In dual-pipette experiments, test dialysates contained 100 mM-GTP-gamma-S (guanosine 5'-O-(3-thiotriphosphate] or 100 microM-GMP-PNP (guanyl-5'-imidodiphosphate). These non-hydrolysable analogues of guanosine triphosphate (GTP) enhanced ICa amplitude (+ 10 mV) by 20-40%. Dialysates containing 100 microM-GTP or GDP-beta-S (guanosine 5'-O-(2-thiodiphosphate] were ineffective, and pre-dialysis with GDP-beta-S blocked stimulation by GTP-gamma-S. 3. Non-hydrolysable GTP analogues slowed the inactivation of ICa and shifted the voltage eliciting maximum ICa by 5-10 mV in the negative direction. 4. ICa enhancement by GTP analogues was attributed to the activation of three GTP-binding regulatory (G) proteins (Gi, Gp and Gs). In single-pipette experiments, the inactivation of Gi by pre-treatment with pertussis toxin did not block enhancement, and a Gp-activating regimen (external acetylcholine-internal GTP) was without effect. Thus, it is probable that the effects of GTP analogues on ICa were primarily mediated by Gs activation. 5. PI-MICS dialysates contained phosphorylation-pathway inhibitors and were used to inhibit Ca2+ channel phosphorylation via the adenyl cyclase pathway. These were deemed effective since forskolin (1-5 microM) doubled ICa during control dialysis but was without effect after 8 min PI-MICS dialysis. However, 0.1 microM-isoprenaline increased ICa by 35% in myocytes totally unresponsive to forskolin, suggesting that beta-adrenergic receptor occupation can stimulate ICa even when the phosphorylation pathway is blocked. 6. After prolonged dialysis of myocytes with PI-MICS, ICa was still enhanced by pressure-assisted dialysis of 100 microM-GTP-gamma-S or GMP-PNP. We conclude that activated Gs has a direct effect on cardiac Ca2+ channels.

A dual-pipette technique that permits rapid internal dialysis and membrane potential measurement in voltage-clamped cardiomyocytes.

Guinea pig ventricular myocytes were voltage-clamped and dialysed using two glass patch pipettes (P1, P2) with tip openings of around 2 microns. A substantial improvement in the efficacy of dialysis from P2 was achieved by the application of positive pressure (15-30 cm H2O) to P2, and similar negative pressure to P1. Evidence of enhanced dialysis was obtained by measuring the effect on Ca channel current of P2 dialysates containing Ca, cAMP, GTP [gamma-S], trypsin, or the catalytic subunit of protein kinase A. Times to maximum response were 3-5 times shorter than those calculated or observed by others using a single-pipette method. The speeding-up was verified in comparative experiments with 100 microM GTP [gamma-S] dialysates; maximum stimulation of ICa occurred after 1.3-1.8 min with the dual-pipette method, versus 8.2 min with a single pipette. Other advantages of the dual-pipette method include the option of following a control dialysis from P1 with a test dialysis from P2, and the measurement of actual membrane potential. The disadvantages are that the rate of success is lower than with single-pipette experiments, and that smaller cardiomyocytes are difficult subjects.