Internal Perfusion Research Articles

2-Aminoethoxydiphenyl borate modulates kinetics of intracellular Ca(2+) signals mediated by inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores in single pancreatic acinar cells of mouse.

Regulation of the kinetics of intracellular Ca(2+) signals with a novel, membrane-penetrable, inositol 1,4,5-trisphosphate (InsP(3)) receptor/Ca(2+) channel modulator, 2-amino-ethoxydiphenyl borate (2APB), has been investigated using patch-clamp, whole-cell recording to monitor Ca(2+)-activated Cl(-) currents in single isolated pancreatic acinar cells. 2APB itself fails to evoke a detectable current response but it dramatically changes the kinetics of agonist-induced Ca(2+) release from pulsatile spikes to long-lasting, huge Ca(2+) waves, suggesting that 2APB coordinates local Ca(2+) release to generate global Ca(2+) signals. The regulation by 2APB can be elicited by internal perfusion of InsP(3) in a concentration-dependent manner, indicating that this regulation is not mediated through membrane receptors or G protein signal transduction. The InsP(3) receptor blocker heparin, but not the ryanodine-sensitive receptor blockers ruthenium red or ryanodine, abolishes 2APB-mediated regulation of Ca(2+) release. This results also suggest that 2APB effects are mediated through InsP(3) receptors. 2APB substantially modifies single inward Cl(-) current pulse evoked by the photolytic release of caged InsP(3) but not by caged Ca(2+). These data indicate that 2APB-induced regulation is mediated neither by Ca(2+)-induced Ca(2+) release nor by affecting Cl(-) channel activity directly. We conclude that 2APB regulates the kinetics of intracellular Ca(2+) signals, represented as the change in the Ca(2+) oscillation patterns from brief pulsatile spikes to huge, long-lasting Ca(2+) waves. Moreover, this regulation seems to be mediated through InsP(3)-sensitive Ca(2+) pools. 2APB may act as a novel, useful pharmacological tool to study the genesis of intracellular Ca(2+) signals.

Characterization of a hyperpolarization-activated inward current in Cajal-Retzius cells in rat neonatal neocortex.

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current (I(h)) and to investigate whether I(h) contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I(h) in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to -90 mV activated a prominent inward current that was inhibited by 1 mM Cs(+) and was insensitive to 1 mM Ba(2+). The activation time constant for I(h) was strongly voltage dependent. In Na(+)-free solution, I(h) was reduced, indicating a contribution of Na(+). An analysis of the tail currents revealed a reversal potential of -45.2 mV, corresponding to a permeability coefficient (pNa(+)/pK(+)) of 0. 13. While an increase in the extracellular K(+) concentration ([K(+)](e)) enhances I(h), it was reduced by a [K(+)](e) decrease. This [K(+)](e) dependence could not be explained by an effect on the electromotive force on K(+) but suggested an additional extracellular binding site for K(+) with an apparent dissociation constant of 7.2 mM. Complete Cl(-) substitution by Br(-), I(-), or NO(3)(-) had no significant effect on I(h), whereas a complete Cl(-) substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I(h) by approximately 40%. The I(h) reduction observed in gluconate could be abolished by the addition of Cl(-). The analysis of the [Cl(-)](e) dependence of I(h) revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I(h) reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I(h) within 1 min after establishment of the whole cell configuration. An inhibition of I(h) by 1 mM Cs(+) was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I(h) with characteristic properties that does not contribute to the RMP.

Pressure polishing: a method for re-shaping patch pipettes during fire polishing

The resolution of patch-clamp recordings is limited by the geometrical and electrical properties of patch pipettes. The ideal whole-cell patch pipette has a blunt, cone-shaped tip and a low resistance. The best glasses for making patch pipettes are low noise, low capacitance glasses such as borosilicate and aluminasilicate glasses. Regrettably, nearly all borosilicate glasses form pipettes with sharp, cone-shaped tips and relatively high resistance. It is possible, however, to reshape the tip during fire polishing by pressurizing the pipette lumen during fire polishing, a technique we call ‘pressure polishing.’ We find that this technique works with pipettes made from virtually any type of glass, including thick-walled aluminasilicate glass. We routinely use this technique to make pipettes suitable for whole-cell patch-clamp recording of tiny neurons (1–3 μm in diameter). Our pipettes are made from thick-walled, borosilicate glass and have submicron tip openings and resistances <10 MΩ. Similar pipettes could be used to record from subcellular neuronal structures such as axons, dendrites and dendritic spines. Pressure polishing should also be useful in patch-clamp applications that benefit from using pipettes with blunt tips, such as perforated-patch whole-cell recordings, low-noise single channel recordings and experiments that require internal perfusion of the pipette.

G protein-mediated FMRFamidergic modulation of calcium influx in dissociated heart muscle cells from squid, Loligo forbesii.

The actions of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) on the L-type (ICa,L) and T-type (ICa,T) calcium currents were investigated in muscle cells dissociated from the heart of squid, Loligo forbseii. The heart muscle cells could be divided into type I and type II cells, on the basis of morphological differences in the dissociated myocytes. FMRFamide induced a substantial block of the L-type calcium current seen in type I cells; this inhibition was rapid, reversible and dose dependent (IC50 = 0.1 microM). FMRFamide induced an increase in the amplitude of the L-type calcium current in the type II heart muscle cells, but had no effect on the T-type calcium current in either type of dissociated heart muscle cell, even at concentrations much higher than those found to affect the L-type calcium current. Internal dialysis of isolated type I heart muscle cells with guanosine 5'-O-(3-thiotriphosphate (GTPgammaS, 100 microM), a non-hydrolysable GTP analogue, mimicked the FMRFamide inhibition of the Ca2+ current and occluded any further FMRFamide-induced inhibition. Internal dialysis of these cells with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS, 100 microM) reduced the FMRFamide-induced inhibition of the peak Ca2+ current. The inhibitory effects of FMRFamide were abolished by pre-incubation of the cells with pertussis toxin (200 ng ml-1). The activation kinetics of ICa,L were not affected by FMRFamide application, nor by internal perfusion with GTPgammaS, and the FMRFamide-induced reduction in ICa,L was not relieved by large depolarising prepulses. These data indicate that FMRFamide can modulate ICa,L, but not ICa,T, in squid heart muscle cells, and that the underlying G protein pathway is dissimilar to that commonly associated with transmitter modulation of channel activity. The FMRFamide-modulated increase in ICa,L seen in the type II heart muscle cells was not mediated by a PTX-sensitive G protein pathway.

Accurate placement of the esophageal pH electrode for 24-hour pH monitoring using a combined pH/manometry probe

OBJECTIVE: Accurate placement of a pH electrode requires manometric localization of the lower esophageal sphincter (LES). Combined manometry/pH devices using water-perfused tubes attached to pH catheters and the use of an electronic “LES locator” have been reported. We investigated whether accurate placement of pH probes can be achieved using such a probe, and whether this may reduce the need for the performance of the usual stepwise pull-back manometry. METHODS: Thirty consecutive patients (15 men, 15 women; median age, 56 yr; interquartile range, 42–68 yr) referred for manometry and pH testing were included in the study. The localization of the LES was determined with standard esophageal manometry. After that, a second 3-mm pH electrode with an internal perfusion port was passed into the stomach. Using this catheter, a single stepwise pull-through manometry was performed and the LES position was noted. LES location, mean pressure, and length obtained with standard manometry were compared to data from the combined pH/manometry catheter. Additionally the time necessary to perform each of the procedures was noted and the patient’s discomfort caused by the catheter was evaluated using a standardized questionnaire. RESULTS: The LES location with the pH/manometry probe was proximal to that with standard manometry in 19 patients (63%), the same in nine patients (30%), and distal in two patients (7%). The differences were <2 cm in 29 of 30 (97%) patients. The LES location with the pH/manometry probe required a median of 6.5 min (interquartile range: 3.5–8.5 min) versus a median of 21.5 min (interquartile range: 14.5–26.5 min) for standard manometry ( p < 0.0001). In addition, LES evaluation using the combined pH/manometry probe provided accurate data on the resting pressure, as well as overall and intraabdominal length of the LES. All patients tolerated the combination probe better than the standard manometry probe ( p < 0.001). CONCLUSIONS: Placement of the esophageal electrode for 24-h esophageal pH monitoring using a combined pH/manometry probe is accurate. The technique is simple, time-saving, and convenient for the patients. Because it is possible to accurately evaluate the LES using this technique, it may even replace conventional manometry before pH probe placement.

Accurate placement of the esophageal pH electrode for 24-hour pH monitoring using a combined pH/manometry probe

Accurate placement of a pH electrode requires manometric localization of the lower esophageal sphincter (LES). Combined manometry/pH devices using water-perfused tubes attached to pH catheters and the use of an electronic "LES locator" have been reported. We investigated whether accurate placement of pH probes can be achieved using such a probe, and whether this may reduce the need for the performance of the usual stepwise pull-back manometry. Thirty consecutive patients (15 men, 15 women; median age, 56 yr; interquartile range, 42-68 yr) referred for manometry and pH testing were included in the study. The localization of the LES was determined with standard esophageal manometry. After that, a second 3-mm pH electrode with an internal perfusion port was passed into the stomach. Using this catheter, a single stepwise pull-through manometry was performed and the LES position was noted. LES location, mean pressure, and length obtained with standard manometry were compared to data from the combined pH/manometry catheter. Additionally the time necessary to perform each of the procedures was noted and the patient's discomfort caused by the catheter was evaluated using a standardized questionnaire. The LES location with the pH/manometry probe was proximal to that with standard manometry in 19 patients (63%), the same in nine patients (30%), and distal in two patients (7%). The differences were <2 cm in 29 of 30 (97%) patients. The LES location with the pH/manometry probe required a median of 6.5 min (interquartile range: 3.5-8.5 min) versus a median of 21.5 min (interquartile range: 14.5-26.5 min) for standard manometry (p < 0.0001). In addition, LES evaluation using the combined pH/manometry probe provided accurate data on the resting pressure, as well as overall and intraabdominal length of the LES. All patients tolerated the combination probe better than the standard manometry probe (p < 0.001). Placement of the esophageal electrode for 24-h esophageal pH monitoring using a combined pH/manometry probe is accurate. The technique is simple, time-saving, and convenient for the patients. Because it is possible to accurately evaluate the LES using this technique, it may even replace conventional manometry before pH probe placement.

Pharmacological Isolation of the Synaptic and Nonsynaptic Components of the GABA-Mediated Biphasic Response in Rat CA1 Hippocampal Pyramidal Cells

High-frequency stimulation (HFS) applied to stratum radiatum of a rat hippocampal slice in the presence of ionotropic glutamate receptor antagonists evokes a biphasic GABA(A) receptor-dependent response in CA1 pyramidal neurons, with a brief hyperpolarizing IPSP (hIPSP) followed by a long-lasting depolarization. We show now that it is possible to pharmacologically separate the hIPSP and late depolarization from one another. In neurons intracellularly perfused for 1-2 hr with F(-) as the major anion and no ATP, the hIPSP (and the corresponding current, hIPSC) evoked by HFS was blocked, whereas neither the late depolarization nor its underlying current was attenuated. In contrast, internal perfusion with a high concentration (5 mM) of the impermeant lidocaine derivative QX-314 selectively abolished the depolarizing component of the biphasic response and also strongly reduced depolarizations evoked by extracellular microinjection of K(+). Bath application of quinine (0. 2-0.5 mM) or quinidine (0.1 mM) resulted in a pronounced inhibition of the HFS-induced extracellular K(+) concentration ([K(+)](o)) transient but not of the bicarbonate-dependent alkaline shift in extracellular pH. The attenuation of the [K(+)](o) transient was closely paralleled by a suppression of the HFS-evoked depolarization but not of the hIPSP. Quini(di)ne did not affect depolarizations induced by exogenous K(+) either. These data provide direct pharmacological evidence for the view that the HFS-induced biphasic response of the pyramidal neuron is composed of mechanistically distinct components: a direct GABA(A) receptor-mediated phase, which is followed by a slow, nonsynaptic [K(+)](o)-mediated depolarization. The bicarbonate-dependent, activity-induced [K(+)](o) transient can be blocked by quini(di)ne, whereas its depolarizing action in the pyramidal neuron is inhibited by internal QX-314. The presence of fundamentally distinct components in GABA(A) receptor-mediated actions evoked by HFS calls for further investigations of their functional role(s) in standard experimental maneuvers, such as those used in studies of synaptic plasticity and induction of gamma oscillations.

Molecular and Functional Characterization of a Novel Mouse Transient Receptor Potential Protein Homologue TRP7: Ca2+-PERMEABLE CATION CHANNEL THAT IS CONSTITUTIVELY ACTIVATED AND ENHANCED BY STIMULATION OF G PROTEIN-COUPLED RECEPTOR

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca(2+) influx activated in response to stimulation of G(q) protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features, compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca(2+) release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca(2+) influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and for Ca(2+) release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca(2+) release. In fact, TRP7 expression did not affect capacitative Ca(2+) entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn(2+) influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca(2+) at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid revealed an essential role of intracellular Ca(2+) in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced cation currents in various native systems.

Extracellular calcium regulates responses of both alpha3- and alpha7-containing nicotinic receptors on chick ciliary ganglion neurons.

Neuronal nicotinic receptors are generally both permeable to calcium and potentiated by it. We have examined acute calcium regulation of both native alpha7-containing and the less abundant alpha3-containing nicotinic receptors on chick ciliary ganglion neurons. Most of the receptors are concentrated on somatic spines tightly overlaid in situ by a large presynaptic calyx. Whole cell patch-clamp recording from dissociated neurons using perforated patch-clamp techniques indicates that the rapidly desensitizing nicotinic response of alpha7-containing receptors achieves maximum amplitude in 2 mM calcium; both lower and higher concentrations of calcium are less effective. Barium and strontium but not magnesium can substitute for calcium in potentiating the response. Neither calcium current through the receptors nor calcium action at intracellular sites is necessary. These latter conclusions are supported by current-voltage analysis of the nicotine-induced response, ion substitution experiments, and internal perfusion of the cells with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) via a conventional patch pipette. Varying the agonist concentration indicates that some of the calcium-dependent enhancement may involve a shift in the dose-response curve for agonist binding, but much of the effect is also likely to involve increased receptor responsiveness. Blockade of alpha7-containing receptors with alpha-bungarotoxin showed that the heteromeric alpha3-containing nicotinic receptors also undergo calcium-dependent potentiation. Calcium did not have a major effect on the desensitization rate of either receptor class but did have a selective effect on the rise time of alpha7-containing receptors. Analysis of stably transfected cells expressing an alpha7 gene construct showed that the calcium potentiation observed for native receptors did not require neuron-specific modifications or components and that it could be seen with the natural agonist acetylcholine. Receptor dependence on extracellular calcium may provide a regulatory mechanism for constraining synaptic signaling, avoiding local depletion of external calcium, and limiting calcium buildup in postsynaptic compartments.

Differential control of three after-hyperpolarizations in rat hippocampal neurones by intracellular calcium buffering.

1. The whole-cell recording technique, combined with internal perfusion, was used to study the effects of intracellular Ca2+ buffering on fast, medium and slow after-hyperpolarizations (fAHP, mAHP and sAHP) in hippocampal CA1 pyramidal neurones in rat brain slices at room temperature. 2. The action potentials and the fAHP were unaffected by 100 microM to 3 mM concentrations of the internally applied fast Ca2+ chelator BAPTA. At higher (10-15 mM) concentrations, BAPTA inhibited the fAHP and prolonged the decay of the action potential, suggesting that the corresponding large-conductance Ca2+-activated K+ channels are located close to the sites of Ca2+ entry during an action potential. Addition of Ca2+ to the BAPTA-containing solution (at a ratio of 4.5 [Ca2+] : 10 [BAPTA]) to maintain the control level of [Ca2+]i did not prevent the effects of high concentrations of BAPTA. 3. The mAHP, activated by a train of action potentials, was inhibited by internally applied BAPTA within the range of concentrations used (100 microM to 15 mM), and this effect could not be reversed or prevented by addition of Ca2+ to the BAPTA-containing solution. The inhibition of the mAHP by BAPTA could also be observed after blockade of the hyperpolarization-activated IQ type mixed Na+-K+ current (also known as Ih) component of the mAHP by bath-applied 3-5 mM Cs+, suggesting that the inhibition of the mAHP by BAPTA is due to inhibition of the depolarization-activated IM (muscarinic) type K+ current. 4. The sAHP, activated by a train of action potentials, was potentiated by 100-300 microM internally applied BAPTA, both with and without added Ca2+. At 1-2 mM or higher concentrations, the potentiation of the sAHP by BAPTA without added Ca2+ was transient and was followed by a fast decrease. With added Ca2+, however, BAPTA caused a persistent potentiation of the sAHP with more than a 10-fold increase in duration for periods exceeding 1 h even at concentrations of the buffer as high as 10-15 mM. Earlier reports showing a blockade of the sAHP by BAPTA, based on experiments without added Ca2+, were apparently due to a sharp reduction in intracellular free [Ca2+] and to a high intracellular concentration of the free buffer. 5. Internally applied BAPTA caused a prolongation of the spike discharge during an 800 ms-long depolarizing current step. At 100-300 microM BAPTA, but not at 1-2 mM or higher concentrations, this effect could be reversed by addition of Ca2+. The effects of BAPTA on the spike discharge occurred in parallel with the changes in the sAHP time course, which was more prolonged at higher concentrations of the buffer. 6. The concentration-dependent differential control of the three types of AHP in hippocampal neurones by BAPTA is related to modulation of intracellular Ca2+ diffusion by a fast acting mobile Ca2+ buffer.

Nonselective cation channels in endothelial cells derived from human umbilical vein.

(i) We have used a combined patch-clamp and fura-2 fluorescence technique to characterize a nonselective cation channel (NSC) in Ea. hy926 (EA) cells, an endothelial cell line derived from human umbilical vein. (ii) Stimulation with ATP, histamine and bradykinin activated slowly and with a long delay after application of the agonist, a nonselective cation current (INSC) which is time- and voltage-independent. The permeability sequence for cations was PNa > PCs >> PNMDG, PCa. In the absence of external Ca2+ and at rather high concentrations, La3+ and Gd3+ blocked INSC. (iii) Single channel analysis revealed that ATP activates in the cell-attached configuration a nonselective cation channel with a conductance of approximately 24 pS and a permeation sequence identical to that of the macroscopic current. The channel activity disappeared after membrane excision. (iv) Activation of NSC required physiological intracellular Ca2+ levels (100 nm or higher). All agonists failed to activate NSC if cytosolic Ca2+ ([Ca2+]i) was lowered by 10 mm BAPTA. Clamping internal Ca2+ at 1 microm sometimes (8 out of 17 cells) spontaneously activated INSC in the absence of any additional stimulus. (v) Application of 2,5-di-tert-butylhydroquinone and internal perfusion of inositol 1,4,5-trisphosphate also activated INSC. The phospholipase C inhibitor, U-73122 inhibited INSC and the sustained Ca2+ plateau during agonist stimulation whereas the inactive analogue, U-73343 had no effect. (vi) These results indicate NSC may act as a Ca2+ entry pathway in endothelium. [Ca2+]i and inositol 1,4,5-trisphosphate play a role in the activation cascade of NSC, and possibly also store depletion.

Permeability of a capsaicin derivative, [14C]DA-5018 to blood-brain barrier corrected with HPLC method.

In the present work, the transport mechanism of a capsaicin derivative, DA-5018, through blood-brain barrier (BBB) has been investigated to evaluate the feasibility of potential drug development. The result of pharmacokinetic parameters obtained from the intravenous injection of plasma volume marker, [3H]RSA and [14C]DA-5018, indicated that both AUC, area under the plasma concentration curve and VD, volume of distribution in brain of [3H]RSA agreed with those reported (1620 +/- 10 percentage injected dose minute per milliliter (%IDmin/ml) and 12.0 +/- 0.1 microliters/g, respectively). Elimination half-life and AUC of [14C]DA-5018 is corrected by the HPLC analysis, 19.6 +/- 1.2 min and 7.69 +/- 0.85% IDmin/ml, respectively. The metabolic rate of [14C] DA-5018 was very rapid. The blood-brain barrier permeability surface area (PS) product of [14C]DA-5018 was calculated to be 0.24 +/- 0.05 microliter/min/g. The result of internal carotid artery perfusion and capillary depletion suggested that [14C]DA-5018 pass through BBB with the time increasingly. Investigation of transport mechanism of [14C]DA-5018 using agonist and antagonist suggested that vanilloid (capsaicin) receptor did not exist in the BBB, and nutrient carrier system in the BBB has no effect on the transport of DA-5018. In conclusion, despite the fact that penetration of DA-5018 through BBB is significant, the intact drug found in the brain tissue is small because of a rapid metabolism. Therefore, for the central analgesic effect of DA-5018, the method to increase the metabolic stability in plasma and the brain permeability should be considered.

ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation.

1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

Modulation of the sarcolemmal L-type current by alteration in SR Ca2+ release.

Modulation of the L-type current by sarcoplasmic reticulum (SR) Ca2+ release has been examined in patch-clamped mouse myotubes. Inhibition of SR Ca2+ release by inclusion of ryanodine in the internal solution shifted the half-activating voltage (V0.5) of the L-type current from 1.1 +/- 2.1 to -7.7 +/- 1.7 mV. Ruthenium red in the internal solution shifted V0.5 from 5.4 +/- 1.9 to -3.2 +/- 4.1 mV. Chelation of myoplasmic Ca2+ with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid perfusion shifted V0.5 from 4.4 +/- 1.7 to -3.5 +/- 3.3 mV and increased the peak current. Extracellular caffeine (1 mM), which should enhance SR Ca2+ release, significantly decreased the peak Ca2+ current. In low (0.1 mM) internal EGTA, myotube contraction was abolished by internal perfusion with ryanodine or ruthenium red, whereas addition of caffeine to the extracellular solution lowered the contractile threshold, indicating that these modulators of SR Ca2+ release had the expected effects on contraction. Therefore, SR Ca2+ release appears to modulate the sarcolemmal L-type current, suggesting a retrograde communication from the SR to the sarcolemmal L-type channels in excitation-contraction coupling.

A voltage clamp method with internal perfusion for quasi-intact axon of the crayfish

A voltage-clamp system with internal perfusion for quasi-intact crayfish giant axons was developed using a partition method where three pools were formed by two partitions. The membrane potential was extracellularly recorded across the one side partition whose resistance was highly increased using a water-absorbing material, Sephadex G25 mixed with vaseline and liquid paraffin so that no short circuiting correction became necessary. Axon membranes were voltage-clamped at the center by feeding current through the other side pool. For the internal perfusion with the axoplasm left intact, the membranes of the lateral pools were perforated with a 0.1% of saponin-treatment for 1.5-2min, through which the axon was intracellularly“perfused”with a reasonable diffusion rate. This system was well bearable to long-term experiments under both extracellularly and intracellularly controlled conditions of the cell.

Charge Immobilization Caused by Modification of Internal Cysteines in Squid Na Channels

We studied the effects of modification of native cysteines present in squid giant axon Na channels with methanethiosulfonates. We find that intracellular, but not extracellular, perfusion of axons with positively charged [(2-trimethylammonium)-ethyl]methanethiosulfonate (MTSET), or 3(triethylammonium)propyl]methanethiosulfonate (MTS-PTrEA) irreversibly reduces sodium ionic ( I Na) and gating ( I g) currents. The rate of modification of Na channels was dependent on the concentration of the modifying agent and the transmembrane voltage. Hyperpolarized membrane potentials (e.g., −110 mV) protected the channels from modification by MTS-PTrEA. In addition to reducing the amplitudes of I Na and I g, MTS-PTrEA also altered their kinetics such that the remaining I Na did not appear to inactivate, whereas I g was made sharper and declined to baseline more quickly. The shape and amplitude of I g after modification of channels with MTS-PTrEA appeared to be “charge-immobilized,” as if the modified channels were inactivated. MTS-PTrEA did not affect I Na or I g when inactivation was removed by internal perfusion of the axon with pronase. In addition, we find that the steady-state inactivation curve of modified Na channels is made much shallower and is markedly shifted to hyperpolarized potentials. The rates of activation, deactivation, or open-state inactivation were not altered in MTS-PTrEA-modified channels. The uncharged sulfhydryl reagent methymethanethiosulfonate (MMTS) did not affect either I Na or I g, but prevented the irreversible effects of MTS-PTrEA or MTSET on Na channels. It is proposed that the positively charged methanethiosulfonates MTS-PTrEA and MTSET modify a native internal cysteine(s) in squid Na channels, and by doing so promote inactivation from closed states, resulting in charge immobilization and reduction of I Na.

Activation of heterologously expressed Drosophila TRPL channels: Ca 2+ is not required and InsP 3 is not sufficient

Light-sensitive channels encoded by the Drosophila transient receptor potential-like gene ( trpl) are activated in situ by an unknown mechanism requiring activation of G q and phospholipase C(PLC). Recent studies have variously concluded that heterologously expressed TRPL channels are activated by direct G q-protein interaction, InsP 3 or Ca 2+. In an attempt to resolve this confusion we have explored the mechanism of activation of TRPL channels coexpressed with a PLC-specific muscarinic receptor in a Drosophila cell line (S2 cells). Simultaneous whole-cell recordings and ratiometric .Indo-1 Ca 2+ measurements indicated that agonist (CCh)-induced activation of TRPL channels was not always associated with a rise in Ca 2+. Internal perfusion with BAPTA (10 mM) reduced, but did not block, the response to agonist. In most cases, releasing caged Ca 2+ facilitated the level of spontaneous channel activity, but similar concentrations (200–500 nM) could also inhibit TRPL activity. Releasing caged InsP 3 invariably released Ca 2+ from internal stores but had only a minor influence on TRPL activity and none at all when Ca 2+ release was buffered with BAPTA. Caged InsP 3 also failed to activate any light-sensitive channels in situ in Drosophila photoreceptors. Two phospholipase C inhibitors (U-73122 4 μM and bromo-phenacyl bromide 50 μM) reduced both spontaneous and agonist-induced TRPL activity in S2 cells. The results suggest that, as in situ, TRPL activation involves G-protein and PLC; that Ca 2+ can both facilitate and in some cases inhibit TRPL channels, but that neither Ca 2+ nor InsP 3 is the primary activator of the channel.

Na+-activated K+ channels in small dorsal root ganglion neurones of rat.

1. Whole-cell Na+-activated K+ (KNa) channel currents and single KNa channels were studied with the patch-clamp method in small (20-25 micrometer) dorsal root ganglion (DRG) neurones in slices of rat dorsal root ganglia. 2. The whole-cell KNa channel current was identified as an additional K+-selective leakage current which appeared after cell perfusion with internal solutions containing different [Na+]. The concentration for half-maximal activation of KNa channel current was 39 mM and the Hill coefficient was 3.5. At [Na+]i above 12 mM, KNa channel current dominated the unspecific leakage current. The ratio of maximum KNa channel current to unspecific leakage current was 45. 3. KNa channel current was not activated by internal Li+. It was suppressed by external 20 mM Cs+ but not by 10 mM tetraethylammonium. 4. Single KNa channels with a conductance of 142 pS in 155 mM external K+ (K+o)-85 mM internal K+ (K+i) solutions were observed at a high density of about 2 channels micrometer-2. 5. In two-electrode experiments, a direct correlation was seen between development of whole- cell KNa channel current and activation of single KNa channels during perfusion of the neurone with Na+-containing internal solution. 6. Under current-clamp conditions, KNa channels did not contribute to the action potential. However, internal perfusion of the neurone with Na+ shifted the resting potential towards the equilibrium potential for K+ (EK). Varying external [K+] indicated that in neurones perfused with Na+-containing internal solution the resting potential followed the EK values predicted by the Nernst equation over a broader voltage range than in neurones perfused with Na+-free solution. 7. It is concluded that the function of KNa channels has no links to firing behaviour but that the channels could be involved in setting or stabilizing the resting potential in small DRG neurones.

Rapid modulation of L-type calcium current by acutely applied oestrogens in isolated cardiac myocytes from human, guinea-pig and rat.

Gender-based differences in cardiovascular mortality may be due to a cardio-protective effect of oestrogens on the myocardium. However, mRNA expression of oestrogen receptors in myocardial tissue of the adult heart has yet to be demonstrated. Furthermore, a calcium antagonistic action of 17beta-oestradiol on myocardial tissue has been discussed. Therefore, two subjects were investigated in atrial myocytes of the human, and ventricular myocytes of guinea-pig and rat in this study. (1) Are oestrogen receptors expressed in adult myocardial cells? (2) Is there an influence of oestrogens on the L-type calcium current of cardiac myocytes? Expression of oestrogen receptors was investigated by reverse polymerase chain reaction. L-type calcium current was usually measured by the patch-clamp technique in whole-cell recording mode under selective recording conditions, i.e. overlapping currents were blocked. One series of experiments was performed in perforated patch configuration to avoid internal perfusion. 17beta-oestradiol inhibited L-type calcium current reversibly in all three species. At 10(-5) M, the inhibition was 15-20%. This inhibition was independent of the sex and the species. A full concentration response curve of 17beta-oestradiol on basal L-type current was recorded from female guinea-pig myocytes. The inhibition increased from 2% at 10(-7) M to about 30% at 10(-4) M 17beta-oestradiol. The values could be fitted by a sum of two sigmoidal functions with log EC50 values of -6.5 and -4.9 M and Hill slopes of 2.5 for both. The specificity of the 17beta-oestradiol action was tested by recording the L-type current in the presence of 17alpha-oestradiol and oestrone. 17alpha-oestradiol also inhibited the current, but with a maximal inhibition of only 17%. The concentration-response curve could be fitted by a single sigmoidal function (log EC50 -6-3 M; Hill slope 0.55). Oestrone did not influence the current at all. The decrease in L-type current after the application of 17beta-oestradiol via a rapid perfusion system developed with a time constant of 3-4 s, which was in the same range as that for the influence of isoprenaline. The isoprenaline-stimulated L-type current was much more susceptible to the inhibition by 17beta-oestradiol, i.e. in pre-stimulated cells (1) the inhibitory effect is significantly higher (e.g. at 10(-5) M, inhibition was 36.3% compared with 11.2% in untreated cells) and (2) an inhibitory effect can be seen with oestradiol concentrations as low as 10(-9) M. Although the concentrations needed to gain a calcium antagonistic influence on the basal current were much too high to explain a cardio-protective influence of oestrogens, the presence of oestrogen receptors in cardiac myocytes of all three species, together with the shift in concentration dependence following pre-stimulation by isoprenaline, suggest that myocytes are a potential target for oestrogen.

Thiamine and its derivatives inhibit delayed rectifier potassium channels of rat cultured cortical neurons

We examined the effects of thiamine and its derivatives on voltage-gated ion channels of neuronal cells isolated from fetal forebrain cortex and cultured for 6–14 days. Under the whole-cell voltage clamp, thiamine tetrahydrofurfuryl disulfide (TTFD), a membrane-permeable derivative of thiamine, inhibited the delayed rectifier K + current (I K) in a concentration-dependent manner (10 −4–10 −3 M). The I K-suppressing effect was also observed by internal perfusion with 1 mM thiamine, but not by the external application of thiamine, indicating the poor permeability of thiamine through the cell membrane. However, thiamine which was applied directly to the intracellular side of patch membranes in the inside-out configuration failed to decrease the open probability of the single I K channel. In contrast, thiamine diphosphate decreased both the open probability and the open-time of the channel without changing the single channel conductance. These results suggest that phosphorylated thiamine can function as an endogenous K + channel blocker in neuronal cells. TTFD, when applied extracellularly at a concentration of 1 mM, prolonged the action potential (AP) duration of neurons (172.8±6.6%) without changing the resting membrane potential or AP amplitude, while the same concentration of thiamine did not influence any parameters of the AP, implying that TTFD may cause the potentiation of neuronal AP through the inhibition of I K.