Showing papers in "Pflügers Archiv: European Journal of Physiology in 1989"

A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.

Abstract: (1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.

Characterization of two kinds of high-voltage-activated Ca-channel currents in chick sensory neurons

Abstract: High-voltage-activated (HVA) Ca-channel currents in chick sensory neurons were characterized by dihydropyridine compounds (DHPs) and ω-conotoxin GVIA (ωCTX) using patch-clamp methods. In single-channel recordings, two HVA-currents were identified by their single-channel conductances, 13 pS and 25 pS in 110 mM BaCl2. DHPs selectively affected the large-conductance channel. ωCTX (5 μM), on the other hand, irreversibly eliminated only the small-conductance channel, while the large-conductance channel was either unaffected or only transiently blocked. In whole-cell recordings the macroscopic HVA-current was completely and irreversibly blocked by ωCTX but insensitive to DHPs in 60% of the cells. This current presumably was carried by the 13 pS channel. In the remaining cells, a part of the HVA-current (10%, SD=11%) was either unaffected or transiently blocked by ωCTX and was sensitive to DHPs. This current presumably was carried by the 25 pS channel. Inactivation of both macroscopic current component was incomplete during a 150 ms long depolarization. Our data suggest that the HVA-currents in chick sensory neurons are carried by two distinct Ca-channels that are differentially affected by ωCTX and DHPs.

Variations of membrane cholesterol alter the kinetics of Ca2(+)-dependent K+ channels and membrane fluidity in vascular smooth muscle cells.

Abstract: The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca2+-dependent K+ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (Po). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in Po. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca2+-dependent K+ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca2+-dependent K+ channels kinetics in different preparations.

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.

Abstract: The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PDbl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PDbl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na+2Cl-K+ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PDbl from -55 +/- 5 mV to -79 +/- 4 mV (n = 7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PDbl from -48 +/- 3 mV to -66 +/- 5 mV (n = 4). A Cl- concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24 +/- 4 mV (n = 4) depolarization of PDbl. This depolarization of PDbl was absent when furosemide was present during the Cl- concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp

Abstract: With a new method called “Action Potential-Clamp” (APC) we studied in single SA nodal cells the contribution of both transient and long lasting calcium currents (L-type and T-type) to the action potential. Action potentials were recorded by a computer and a representative cycle was subsequently repetitively replayed to the same cell under voltage clamp. Blockade of the L-type calcium current (D600) or T-type calcium current (nickel) revealed the quantitative and time related contributions of these currents to the action potential, since the blocked current is compensated by the clamp amplifier.

Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes

Abstract: The effect of intracellular perfusion with cyclic GMP (cGMP) on Ca current (ICa) was investigated in Cs-loaded isolated cells from guinea pig ventricle using the whole-cell patch-clamp technique and a perfused patch pipette. cGMP (5 μM) strongly reduced ICa which had been elevated by intracellular perfusion with 50 μM of either cyclic AMP (cAMP) or its hydrolysis-resistant analog 8-Bromo-cAMP. In addition, cGMP prevented the stimulation of ICa by IBMX, a phosphodiesterase inhibitor. The membrane permeant cGMP analog 8-Bromo-cGMP (100 μM), when applied outside the cell, also antagonized the stimulatory effect of IBMX on ICa. It is concluded that cGMP inhibits ICa in guinea pig ventricular cells by a mechanism different from the activation of a cGMP-stimulated phosphodiesterase recently found in frog ventricular cells.

Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents

Abstract: A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10–16 μm with 5–25 GΩ seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15–40 min with peak current densities of 120 to 350 μA/cm2. Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by 20 μA/cm2.

Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke.

Abstract: We have measured the effect of phosphate (Pi) on the tension and maximum shortening velocity of permeable rabbit psoas fibers. Work in a number of laboratories has established that addition of phosphate (0-25 mM) to active muscle fibers at physiological MgATP concentrations decreases isometric tension with little effect on the maximum shortening velocity. Here we extend these results to a wider range of Pi concentrations and to low MgATP concentrations. Low levels of Pi (approx. 150 microM-200 microM) were obtained by using sucrose phosphorylase and sucrose to reduce contaminating Pi in the solutions used to activate the fiber, and high levels (52-73 mM) were obtained by replacing acetate with Pi as the principal anion. In an activating solution containing either 50 microM or 4 mM MgATP, pH 6.2 or 7.0, isometric tension declines linearly with the logarithm of Pi concentration. Although the isometric tension decreases with increasing concentrations of H+ or MgATP, the slope of relative isometric tension as a function of log[Pi] is the same at the two values of pH and [MgATP]. At pH 7 and 4 mM MgATP, the velocity of contraction increased slightly as Pi increased from 0.2 to 52 mM. At 50 microM MgATP the velocity decreased slightly as Pi increased from 0.2 to 10 mM with a substantial decrease as Pi increased from 10 to 52 mM. These results are discussed in terms of models of cross-bridge energetics. The observation that force declines linearly with the logarithm of [Pi] is compatible with models in which a major force producing state occurs subsequent to Pi release.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle.

Abstract: The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca2+]i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca2+]i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K+ (21 mM), both LC20 phosphorylation and [Ca2+]i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca2+]i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha 1-mediated activation by 10(-5) M phenylephrine whereas a higher force was developed at a shorter time compared to K+. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10(-6) M) induced a slow contraction of similar magnitude to that induced by K+ without significantly changing either [Ca2+]i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca2+]i in all three types of activation. The initial levels of [Ca2+]i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca2+]i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca2+]i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.

Uric acid as radical scavenger and antioxidant in the heart.

Abstract: Uric acid (UA) is released from the heart of many species, including man, and its site of formation has been shown to be the microvascular endothelium. Since UA reacts with oxygen radicals in vitro, experiments were conducted on guinea pig hearts perfused with Krebs-Henseleit buffer (KHB) to evaluate whether the formation of UA could afford protection from damage by radicals and oxidants. The following results were obtained: (1) Upon addition of the hydroxyl radical scavenger DMSO to the perfusate, the coronary rate of release of endogenous uric acid was increased relative to the precursor purines. (2) UA was degraded during passage through the coronary system and also in KHB in vitro after addition of substances generating hydroxyl radicals or hypochlorite. Superoxide (O 2 − ) radicals did not seem to react directly with UA, though UA concentration-dependently quenched the chemiluminescence generated from luminol in the presence of O 2 − and OH radicals. (3) Coronary dilatation by acetylcholine (Ach) and sub-μM concentrations of adenosine, induced by both via endothelial mechanisms, was attenuated after prolonged inhibition of endothelial UA formation by allopurinol. Furthermore, the effect of Ach but not of adenosine proved acutely sensitive to methylene blue and O 2 − , substances known to inactivate EDRF. This finding suggests involvement of EDRF in Ach-mediated, but not in adenosine-induced dilatation of the intact coronary system. Exogenously applied UA prevented the impairment of vascular responses to Ach and adenosine caused by allopurinol, and to Ach upon generation of O 2 − .(4) Hearts performed more pressure-volume work and exhibited greater functional stability when perfused with KHB supplemented with UA in a physiological concentration. It is concluded that uric acid can actually serve as a physiologic radical scavenger and antioxidant, maintaining functional responsiveness of the coronary system and of the myocardium.

Modulation of Ca-channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons.

Abstract: Inhibitory modulation of the high-voltage-activated (HVA) Ca-channel current by 2-chloroadenosine (2CA) was studied in chick sensory neurons using the whole-cell clamp method. 2CA reduced the ωCTX-sensitive HVA-current (Aosaki and Kasai 1989) in a dose-dependent manner with aK d of 0.8 μM. The inhibition by 2CA was also voltage-dependent, being maximal at hyperpolarized potentials, and completely removed at potentials more positive than 30 mV. This voltage-dependence of 2CA action was also evident as a progressive increase in Ca-channel current magnitude during a depolarization which could be described by a single exponential function and which became faster at larger depolarizations. The concentration of 2CA affected the steady-state reduction in Ca-channel current, but did not alter the time-course of current increase during depolarization. The voltage-dependent effect of 2CA was mimicked by intracellular application of GTP-γS, but not by phorbol ester, arachidonic acid or nitroprusside. These results are consistent with model in which 2CA activates a G-protein, which then unmasks an additional activation gate on the Ca-channel.

Changes in velocity of shortening, power output and relaxation rate during fatigue of rat medial gastrocnemius muscle

Abstract: The force-velocity characteristics of rat medial gastrocnemius muscle have been determined by measuring the force sustained during constant velocity releases of the muscle stimulated in situ at an ambient temperature of 26°C. The velocity of unloaded shortening was determined using the “slack” test and rate of relaxation from the half time of force loss at the end of stimulation. Measurements were first made on fresh muscles using short contractions and then during a series which consisted of a 15 s contraction (fatigued muscle), followed by 15 min recovery and a 1 s contraction (recovered muscle). After a 5 min recovery period the sequence was repeated. Comparison was made between the fatigued and recovered state in each preparation in order to allow for any change in the preparation during the course of the experiment. After 15 s contraction the fatigued muscles showed a marked reduction in all parameters measured. In fatigued muscles the isometric force fell to 48±15% (mean±SD) and there was a decrease in maximum velocity of shortening to 66%. These changes in the force-velocity relationship were accompanied by slowing of relaxation so that the half time of relaxation nearly doubled. The consequence of these changes was that the maximum power output was reduced by a much greater extent that was the isometric force (75% vs. 52%). It is suggested that the changes in force-velocity characteristics reflect a reduction in cross-bridge cycling in fatigued muscle.

Sensitization of articular afferents to mechanical stimuli by bradykinin.

Abstract: In 18 cats anaesthetized with alpha-chloralose, we recorded from thin myelinated and unmyelinated articular afferents of the medial articular nerve of the knee joint. Bradykinin was injected intra-arterially close to the knee, alone and in combination with prostaglandin E2 (PGE2), and changes of the responses of single afferents to movements of the knee were monitored. Bradykinin changed the mechanosensitivity in 20 of 28 afferents inducing movement sensitivity in initially unresponsive units, lowering the threshold for movements in high-threshold afferents and/ or enhancing pre-existing responses to innocuous and/or noxious joint movements in low and high threshold units. Also the application of PGE2 and bradykinin within a short interval sensitized the majority of these afferents, and in about 50% of the afferents the effect of the combination was superior to those induced by the single substances. We conclude that the inflammatory mediator bradykinin is able to sensitize articular afferents for movement stimuli and that PGE2 may enhance this effect. It is suggested that in arthritis inflammatory mediators act synergistically in the initiation and stabilization of the increased mechanosensitivity of slowly conducting articular afferents.

Voltage- and time-dependent chloride currents in chick skeletal muscle cells grown in tissue culture.

Abstract: Membrane chloride currents in chick skeletal muscle cells grown in tissue culture were studied by use of the whole cell variation of the patch electrode voltage clamp technique. Small diameter myoballs were obtained by adding colchicine to the growth media. To isolate the currents through the chloride channels, the currents through the sodium, calcium and potassium channels were minimized. With symmetrical chloride concentrations bathing the membrane, inward currents were activated by depolarizations above −45 mV. Above 0 mV, the currents became outward. The reversal potential for the currents shifted with the chloride concentration gradient in a manner consistent with the Nernst relation, indicating that the currents were predominantly carried by chloride ions. The instantaneous current-voltage relation obtained from tail current data was linear. The relationship between conductance and membrane potential was sigmoid. The conductance activated above −45 mV, increased steeply between −45 and −10 mV and saturated above +20 mV. Over the range of potentials where the conductance was just beginning to activate, the conductance increasede-fold for a 7 mV depolarization. The currents activated with an exponential time course and did not decline during step depolarizations. Tail currents declined slowly as the sum of two exponential components. The currents were reversibly suppressed by 100 μM SITS and were irreversibly suppressed by 10 μM DIDS.

Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture.

Abstract: Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of −70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to −70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 μM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.

Potassium channel openers act through an activation of ATP-sensitive K+ channels in guinea-pig cardiac myocytes

Abstract: In a previous article (Escande et al. 1988a), we have shown that cromakalim (BRL 34915), a potassium channel opener (PCO), is a potent activator of ATP-sensitive K+ channels in cardiac cells. In the present article, the influence on K+ channels of two other potassium channel openers chemically unrelated to cromakalim, RP 49356 and pinacidil, has been investigated in patch-clamped isolated cardiac myocytes. In the whole-cell configuration, K+ currents were recorded in the presence of 50 μM TTX and 3 μM nitrendipine or 3 mM cobalt. Like cromakalim, RP 49356 or pinacidil activated a time-independent outward current at 33–35°C but not at 19–21°C, which showed little voltage-dependency in the potential range −60 to +60 mV. Its amplitude was a function of the agonist concentration, e.g. it was 2.1±0.4 nA at +60 mV with 30 μM RP 49356 and 4.3±0.8 nA with 300 μM. In control conditions, glibenclamide, a blocker of K+-ATP channels in pancreatic and heart cells, affected neither the inward rectifier,iK1, nor the delayed K+ current,iK. At 3 μM, glibenclamide fully prevented the effects of 300 μM RP 49356 or pinacidil. At lower concentrations, glibenclamide partially counteracted the activation by PCOs of a K+ current. In the cell-attached contiguration, externally applied RP 49356 or pinacidil caused opening of large channels which reversed around 0 mV in a high K+ external medium. In inside-out patches, both RP 49356 or pinacidil activated K+-ATP channels by increasing the time period for which the channels remained in the open state. It is concluded that, like cromakalim, RP 49356 and pinacidil are potent activators of K+-ATP channels in cardiac myocytes.

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

Abstract: The present study examines the properties of Cl−channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl− channels were larger at positive as compared to negative clamp voltages (V c): 74±2.6 (V c > 0) and 47±2.0 pS (V c < 0) for CF (n= 57) and 69±3.6 (V c > 0) and 45±2.3 pS (V c < 0) for N (n=35). The open probability (P o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl−=Br− =I− > NO 3 − ≫ gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10−7 mol/l to 10−5 mol/l. While Cl− channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl− channels was 76±3.2 pS for positive clamp voltages (V c) and 46±3.9 pS for negative V c (n=8). When the membrane patches were excised from CF cells Cl− currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl− channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). “Excision activation” of CF Cl− channels was observed at low (< 10−9 mol/l) or high (10−3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca+ activity (< 10−9–10−3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl− channels. These results suggest that Cl− channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic “inhibitor” leads to an activation of Cl− channels. The Cl− channels in excised patches of N and CF cells have identical properties.

Water, K+, H+, lactate and glucose fluxes during cell volume regulation in perfused rat liver.

Abstract: The present study has been performed to test for ion release from isolated perfused rat liver exposed to hypotonic perfusates. Replacement of 40 mmol/l NaCl in perfusate by 80 mmol/l raffinose leads to slight alkalinization and slight decrease of liver weight. Subsequent decrease of perfusate osmolarity by omission of raffinose results in an increase of liver weight and a parallel increase of effluent sodium, chloride and potassium activity pointing to net uptake of solute free water. While effluent chloride and sodium activities approach perfusate activities within less than 2 min, a second, 6 min lasting increase of effluent potassium activity is observed, pointing to potassium release by the liver. This transient increase of effluent potassium activity is paralleled by a decrease of liver weight. Throughout exposure to hypotonic perfusates, lactate, pyruvate and glucose release by the liver is significantly decreased and effluent pH is rendered alkaline. Readdition of 80 mmol/l raffinose leads to rapid decrease of liver weight and a parallel decrease of effluent sodium, chloride and potassium activities followed by a 10–20 min lasting decrease of effluent potassium activity, pointing to net uptake of potassium, which almost matches the net release observed before. The transient decrease of potassium activity is paralleled by an increase of liver weight, an increase of effluent glucose, lactate and pyruvate concentration and an acidification of the effluent. Similar decrease of effluent potassium activity, acidification of effluent and increase of effluent glucose, lactate and pyruvate concentration are observed, if perfusates are made hypertonic by addition of raffinose. In conclusion, both, volume regulatory decrease (VRD) and increase (VRI) can be elicited in liver and are in large part achieved by movements of potassium. Lactate and pyruvate production is decreased throughout exposure to hypotonic perfusates and enhanced following exposure to hypertonic perfusates.

Electrical properties of gap junction channels in guinea-pig ventricular cell pairs revealed by exposure to heptanol.

Abstract: Cell pairs were isolated from adult guinea pig ventricles to study the electrical properties of gap junction channels. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Heptanol decreased the intracellular current, In, in a dose-dependent fashion. Before complete uncoupling, In showed fluctuations suggesting the operation of gated channels. In the presence of 3 mM heptanol, In showed quantal steps arising from spontaneous opening and closing of single channels. The IV-relationship of the channels was linear (range: ±95 mV). Analysis of current records revealed the following singlechannel conductances, γn: Mean value = 37 pS; median value = 33 pS. γn was insensitive to the non-junctional membrane potential (range: −90 to +10 mV). 3 mM ATP4− in the pipette solution had no effects on γn, 6 mM ATP4− produced a small decrease, and 6 mM ATP+0.1 mM cAMP− an increase in γn. Channel transitions from closed to open state were variable (range of apparent time constants: 2.5–32 ms; mean: 11 ms).

The effectiveness of different isomers of octanol as blockers of harmaline-induced tremor.

Abstract: Intracellular recording in the guinea-pig brainstem slice has demonstrated that high molecular weight alcohols block the low threshold calcium channel (LTCC) in the inferior olive (IO). These alcohols thus provide a tool for understanding the function of the pacemaking cellular networks of the olivo-cerebellar system, since the LTCC has been implicated in the oscillatory behavior of these neurons. Aspects of normal and pathological tremor are also believed to be mediated by these circuits, and thus development of effective ways of blocking the LTCC in vivo may eventually lead to novel treatments for essential tremor. The present experiments evaluated the effectiveness of the isomers of octanol in decreasing harmaline-induced tremor in vivo in the rat. Harmaline was used in this study because its tremorgenic action is mediated at the level of IO; octanol was found to be a potent antagonist of harmaline-induced tremor. Significant differences between the isomers further suggested conformational differences. This, taken in conjunction with the lack of effect of octanol in both IO lesioned rats and oxotremorine-induced tremor, implied that the action of the alcohol may be mediated at a specific binding site. These findings thus support the conclusions that the antagonism of harmaline-induced tremor by octanol occurs in the IO, and, in view of the previously reported in vitro data, that octanol may be an effective blocker of the LTCC in vivo.

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

Abstract: The relative importance of intracellular and extracellular Ca2+ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca2+ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC50 of 4×10−10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca2+. However, in the absence of external Ca2+, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with45Ca, bradykinin increased the45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in45Ca efflux induced by bradykinin of 1.3×10−9 M. The involvement of an intracellular Ca2+ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca2+,Mg2+-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca2+ concentrations showed at least two compartments involved in the uptake of Ca2+. The45Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the45Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP3). The IP3-induced release was not affected by apyrase or exogenous GTP. The EC50 for the release of Ca2+ by IP3 was 1.0 μM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca2+ influx and the release of intracellular Ca2+. The release of calcium from one of the high affinity intracellular Ca2+ stores is mediated by the intracellular second messenger, IP3.

Single channel currents of homo- and heterologous gap junctions between cardiac fibroblasts and myocytes

Abstract: Recently, the use of the double whole-cell patch-clamp technique enabled conductance measurements of single gap junctional channels. Different values have been measured in pairs of rat lacrimal cells (6), murine acinar cells and chinese hamster ovary cells (9), embryonic chick heart- (10) and neonatal rat heart myocytes (7). We here present evidence that the conductance of gap junction channels between two different cell types originating from the same tissue, neonatal rat heart, is different. In mixed cultures of cardiac fibroblasts and myocytes, gap junction channels between fibroblasts have a single channel conductance of only 22 pS, while those between myocytes have a conductance of 43 pS. Fibroblasts can be electrically coupled to myocytes through channels having an intermediate conductance of 29 pS, a value which matches very well with the theoretically expected conductance of a gap junction channel composed of a fibroblast- and a myoblast connexon (hemichannel). These data provide direct evidence on the single channel level that in heterologous gap junction channels the composing connexons retain their cell-specific properties.

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas. Inhibition by 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) and activation by stilbene disulfonates.

Abstract: Nonselective Ca2+-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca2+ on the cytosolic side, the mean openstate probabilityPo of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3′,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 μmol/l of NPPB or DPC decreasedPo from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 μmol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedPo, but in contrast to the abov mentioned substances, it induced fast flickering. Ba2+ (70 mmol/l) and tetraethylammonium (TEA+; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and 4,4′-dinitro-2,2′-stilbenedisulfonate (DNDS). 10 μmol/l SITS applied to the cytosolic side increasedPo from 0.5 to 0.7 and with 100 μmol/l SITS the channels remained nearly permanently in its open state (Po≅1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca2+ concentration to 0.1 μmol/l, addition of 100 μmol/l of SITS had no effect. Similarly, reducing bath Ca2+ concentration from 1.3 mmol/l in presence of 100 μmol/l SITS (channels are maximally activated) to 0.1 μmol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca2+. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca2+ ions.

A novel method for absolute calibration of intracellular pH indicators.

Abstract: In this paper we present methods to measure intracellular pH (pHi) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pHi that follows the addition of a weak acid or weak base to the extracellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pKa is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pHi. The null method only requires the first two assumptions. We have made estimates of pHi in four different cell types and compared the results obtained with these methods with those obtained from other methods of pHi calibration.

Direct modulation of voltage-dependent calcium channels by muscarinic activation of a pertussis toxin-sensitive G-protein in hippocampal neurons

Abstract: Acetylcholine (Ach) reversibly reduces the high voltage-activated (HVA) calcium (Ca) current in hippocampal neurons. Pretreatment of the cells with pertussis toxin (PTX) abolishes the Ach effect, suggesting that PTX-sensitive GTP-binding regulatory proteins (G-proteins) are involved in the signal transduction mechanism that links Ach receptor activation to inhibition of Ca channel activity. This effect is mimicked by intracellular application of the nonhydrolyzable GTP analog GTPγS. Intracellular application of purified G-proteins restored the response to Ach in PTX-treated cells. Furthermore, Ach inhibits the Ca current independently of the presence of cyclic AMP and of the protein kinase C inhibitor H-7 and neither does the Ach effect on the Ca current seem to be correlated to a transient increase in intracellular Ca. Our results suggest that activation of the α-subunit of the PTX-sensitive G-protein could directly modulate the HVA Ca channel without involving second messenger systems.

A method for making solution changes in the sub-millisecond range at the tip of a patch pipette

Abstract: A method is described here for making multiple fast external solution changes at the tip of a patch pipette. The time for the change, 0.2 ms, has been established by measuring changes in liquid junction potential at the tip of an open patch pipette. This technique of producing an abrupt change in solution allows agonist/receptor reactions to be studied under non-equilibrium conditions. We have applied this technique to the nicotinic receptors in outside-out patches from skeletal muscle cell line C2 (Jaffe and Saxel 1979) and from bovine adrenal chromaffin cells. The application of step changes in acetylcholine concentration produces current traces with a characteristic shape, which may be compared with the predictions of established models for the activation and desensitisation of the nicotinic receptor. The results of making single steps and also short pulses in acetylcholine concentration are demonstrated. The direct comparison of two different cholinergic agonists is demonstrated.

Effects of (―) baclofen on inhibitory neurons in the guinea pig hippocampal slice

Abstract: Intracellular recordings were made from electrophysiologically identified inhibitory neurons in the dentate hilus. (−)Baclofen (0.1–0.5 μmol/l), applied by the bath, strongly hyperpolarized inhibitory neurons, reduced their input resistance and induced outward currents under voltage clamp at holding potential of −60 mV in cells recorded with KCl-filled electrodes. Increasing the (−)baclofen concentration (up to 1 μmol/l) did not increase the amplitude of the outward current, but increased its duration. (−)Baclofen depressed Cl-dependent IPSPs evoked by perforant path stimulation in inhibitory neurones, granule cells and CA3 neurons. In the case of inhibitory neurons and CA3 neurons, depression of IPSPs, membrane hyperpolarization and increase in membrane conductance concurred. All effects were blocked by BaCl2 (1 mmol/l) in the superfusate. In the case of granule cells, depression of IPSPs by (−)balcofen out-lasted an only small membrane hyperpolarization, conductance increase or outward current. High concentrations (up to 10 μmol/l) of (−)baclofen depressed evoked IPSPs of granule cells for an extended period of time, but the other effects remained small and transient. IPSPs elicited in granule cells by microdrop application of glutamate to the dentate hilus were also blocked by (−)baclofen, but spontaneous IPSPs were only reduced in amplitude. We suggest that the blockade of GABAA receptor-mediated IPSPs of hippocampal neurons by the GABAB receptor agonist (−)baclofen can be explained by a K-dependent hyperpolarization of inhibitory neurons.

Calcium current inactivation in insulin-secreting cells is mediated by calcium influx and membrane depolarization

Abstract: Inactivation of voltage-dependent calcium currents was studied in single, dissociated insulin-secreting HIT cells voltage-clamped by the whole-cell patch-clamp method at room temperature. Na and K currents were suppressed by tetrodotoxin, tetraethylammonium, ATP, 4-aminopyridine and Cs. Ca currents activated in less than 10 ms by depolarizations beyond −50 mV from a holding potential of −100 mV and were identified, as in previous studies, by their sensitivity to divalent cation blockade and permeability to Ba as a charge carrier. Sustained depolarization revealed two kinetically distinct phases of inactivation: a rapid phase inactivated approximately 50% of the current in less than 100 ms while the remaining current was inactivated over the next 10–20 s. Rapid inactivation appeared to be due to Ca2+ influx since it was slowed markedly when Ba2+ was used as the current carrier, while the degree of inactivation mercased and decreased with increasing depolarization in direct parallel with the U-shaped current-voltage relationship for inward Ca current. Slow inactivation appeared to be voltage-dependent since current could be inactivated (by ≈20%) by 10 s long depolarizations to potentials below the threshold for activating Ca current, slow time constants of inactivation were voltage-dependent and slow inactivation persisted when Ca was replaced with Ba. Ca currents with low activation thresholds (in the −50 to −30 mV range) appeared to be preferentially inactivated by the rapid Ca-dependent mechanism. Recovery of slowly inactivated Ca current was very slow and currents inactivated by larger depolarizations required longer recovery time than those elicited by smaller depolarizations. Rapid and slow inactivation mechanisms may be important in understanding the fast spiking and slow plateau depolarizations seen in pancreatic B-cells exposed to stimulatory levels of glucose.

Effect of photoperiod and acclimation temperature on nonshivering thermogenesis and GDP-binding of brown fat mitochondria in the Djungarian hamster Phodopus s. sungorus.

Abstract: Acclimation to short photoperiod at 23° C constantT a causedP. sungorus to improve their NST capacity from 752 to 1,082 mW. Chronic cold exposure in short photoperiod further enhanced the NST capacity, reaching a maximum level of 1,573 mW at −5° C acclimation temperature. Improvements in NST capacity were always accompanied by an increase in brown fat mitochondrial mass and GDP-binding of brown fat mitochondria, in proportion with the cold load applied during temperature acclimation (23°, 15°, 5°, −5° C). Brown fat mitochondrial protein increased from 7.41 mg (23° CT a, long photoperiod) through 21.6 mg (23° CT a, short photoperiod) and 81.6 mg (−5° CT a, short photoperiod). This ∼10-fold increase was accompanied by a ∼35-fold increase in GDP-binding (2.0, 7.3 and 71.6 nmol GDP bound, respectively), demonstrating that the increase in capacity for uncoupled respiration in brown fat is of primary significance for thermogenic acclimation to cold as well as to short photoperiod.

Arachidonic acid closes gap junction channels in rat lacrimal glands.

Abstract: The effects of arachidonic acid (AA) on gap junction conductance of rat lacrimal glands have been studied with the double patch-clamp technique. Extracellular application of 50–100 μM AA for a few minutes induced a closure of gap junction channels. This effect was mimicked by linoleic acid and by other non-degradable fatty acids (myristic and lauric), and was not blocked by inhibitors of AA metabolism. This suggests that the active molecule was the fatty acid itself, and not one of its oxidative derivatives. Inhibitors of AA metabolism caused a certain degree of uncoupling by themselves, probably due to the accumulation of AA. This effect was reduced in the presence of 10 μM 4-bromophenacylbromide, an inhibitor of phospholipase A2. The effect of AA did not seem to be mediated by an increase of intracellular Ca concentration, nor by a change in the activity of guanylate or adenylate cyclases, nor by activation of protein kinase C. Therefore it could be attributed to a direct effect of AA on gap junctions. Analysis of single gap junction channel currents showed that AA-induced closure of these channels resulted from a change in the number of open channels, and not from a reduction of their conductance. Finally, acetylcholine-induced closure of gap junction channels was not primarily mediated by an increase in AA concentration, although such an increase may well follow the activation of muscarinic receptors and play a role in the acetylcholine effect.