Showing papers in "Pflügers Archiv: European Journal of Physiology in 1988"

Rates of diffusional exchange between small cells and a measuring patch pipette.

Abstract: (1) Fluorescent compounds and specific ion currents (Na+, K+) were used to study the kinetics of the diffusional exchange between small cells and patch pipettes in the tight seal whole cell configuration of the patch clamp technique. (2) Changes in the intracellular concentration of the test substances following patch rupture could be fitted with single exponentials, provided the access resistanceR A of the pipette remained constant during diffusional equilibration. The diffusion time constants were linearly related to the access resistance. (3) When apparent diffusion rates were normalized with respect to access resistance they were found to be dependent on the cell size. However, the cell capacitance, which is proportional to the membrane area turned out not to be a precise measure of the cell size. (4) The experimental diffusion rates changed systematically with the aqueous diffusion coefficient and the inverse third root of the molecular weight. Linear interpolation with respect to these quantities provided estimates of diffusion time constants for the diffusion between patch pipettes and the cytoplasm for substances of interest.

Patch-clamp techniques for time-resolved capacitance measurements in single cells.

Abstract: Two methods are described for estimation of passive cell parameters such as membrane capacitance, membrane conductance and access resistance in tight-seal whole cell recording. Both methods are restricted in their application to cases where the cell under study can be approximated by a simple three-component network with linear properties over some voltage range. One method, referred to as the time domain technique, requires only standard electrophysiological equipment and a computer. Parameters are derived from an analysis of capacitive transients during square wave stimulation. It is readily adaptable to wide variations in experimental parameters. Particurlarly, it is equally applicable to the “slow whole-cell” configuration (access resistance in the range 100 MΩ to 1 GΩ) and to normal whole-cell measurements (access resistance typically 10 MΩ). The other method applies a sine wave command signal to the cell and employs a lock-in amplifier to analyse the resulting current signal. Two modes of operating the lock-in amplifier are described. One mode provides an output signal directly proportional to small changes in capacitance at maximum resolution (1–10 fF). The other mode, in conjunction with a digital computer, supplies estimates of all passive cell parameters, as does the time domain technique, but with a large amount of data reduction performed by the lock-in amplifier itself. Due to the special hardware, however, this method is not as flexible as the time domain technique.

Adenosine-5′-triphosphate-sensitive ion channels in neonatal rat cultured central neurones

Abstract: ATP-sensitive channels were observed in isolated inside-out membrane patches from rat cultured central neurones. Two types of ATP-sensitive K+ channels were present in cortical neurones, one which had its open-state probability increased, the other its open-state probability decreased by application of ATP to the cytoplasmic membrane surface. Another, ATP-sensitive channel differing in ion conductance from all previously reported ATP-sensitive channels was also seen in patches from cortical neurones. This channel was nonselective with respect to Na+, K+ and Cl- ions and ATP produced a "flickery" type of block. The non-hydrolysable analogue, AMPPNP, did not mimic ATP and prevented ATP action. Preliminary experiments indicate that similar, but not, identical ATP-sensitive channels exist in cerebellar neurones.

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes

Abstract: The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (I Ca) was recorded as peak inward current in response to test pulse to O mV. Extracellular application of OA (5–100μM) produced an increase ofI Ca. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline.I Ca was increased from 11.3±0.8μA cm−2 to 19.0±1.1μA cm−2 (n=4) by 5μM-OA in the presence of 1nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the β-adrenergic increase ofI Ca was markedly prolonged by OA. The β-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA onI Ca is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

“Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium

Abstract: We examined by a statistical approach the decrease of the Ca current (“run-down”) during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (ICa) occurs in three phases (T1–T3). T1 (38±19 min,n=135) and T3 (35±17 min,n=23) are characterized by a slow rate of decay ofICa [90±20 and 60±20 nA·cm−2·min−1, respectively]. T1 and T3 are separated by T2 (6±4 min,n=135) during which the current decays quickly [1200±230 nA·cm−2·min−1]. Between the onsets of T1 and T3,ICa decreases from 11±3 to 3.5±1 μA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofICa are globally shifted toward more negative potentials during the run-down process by 10–15 mV. (3)ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the originalICa. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down ofICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesICa survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallICa survival. (6) The results are consistent with the hypothesis that run-down ofICa can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.

Positive chronotropic effects of endothelin, a novel endothelium-derived vasoconstrictor peptide.

Abstract: Endothelin (ET) is a novel 21-amino-acid peptide isolated from the culture supernatant of porcine aortic endothelial cells, which has a potent vasoconstricting effect. It has been suggested that ET increases Ca2+ influx via a voltage-dependent Ca2+ channel. The purpose of the present study is to investigate the mode of actions of ET on the cardiac automaticity. ET increased the sinoatrial nodal rate in a dose-dependent manner on the isolated right atria of guinea pigs. The positive chronotropic effect of ET was very potent and the ED50 value was 0.4 nM. These results suggest that ET is one of the most potent cardio-acceleratory substances.

Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction

Abstract: As in many smooth muscle tissue preparations, single smooth muscle cells freshly dissociated from the stomach of the toadBufo marinus contract when stretched. Stretch-activated channels have been identified in these cells using patch-clamp techniques. In both cell-attached and excised inside-out patches, the probability of the channel being open (P o) increases when the membrane is stretched by applying negative pressure to the extracellular surface through the patch pipette. The increase inP o is mainly due to a decrease in closed time durations, but an increase in open time duration is also seen. The open-channel current-voltage relationship shows inward rectification and is not appreciably altered when K+ is substituted for Na+ as the charge-carrying cation in Ca2+-free (2 mM EGTA) pipette solutions bathing the extracellular surface of the patch. The inclusion of physiological concentrations of Ca2+ (1.8 mM) in pipette solutions (containing high concentrations of Na+ and low K+) significantly decreases the slope conductance as well as the unitary amplitude. The channel also conducts Ca2+, since inward currents were observed using pipette solutions in which Ca2+ ions were the only inorganic cations. When simulating normal physiological conditions, we find that substantial ionic current is conducted into the cell when the channel is open. These characteristics coupled with the high density of the stretch-activated channels point to a key role for them in the initiation of stretch-induced contraction.

Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport.

Abstract: The aim of the present study was to investigate by what transport mechanism does HCO3− cross the luminal membrane of pancreatic duct cells, and how do the cells respond to stimulation with dibytyryl cyclic AMP (db-cAMP). For this purpose a newly developed preparation of isolated and perfused intra-and interlobular ducts of rat pancreas was used. Responses of the epithelium to inhibitors and agonists were monitored by electrophysiological techniques. Addition of HCO3−/CO2 to the bath side of nonstimulated ducts depolarized the PD across the basolateral membrane (PDbl) by about 9mV, as also observed in a previous study [21]. This HCO3− effect was abolished by Cl− channel blockers or SITS infused into the lumen of the duct: i. e. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 10−5 M) hyperpolarized PDbl by 8.2±1.6 mV (n=13); 3′,5-dichlorodiphenylamine-2-carboxylic acid (DCl-DPC, 10−5 M) hyperpolarized PDbl by 10.3±1.7 mV (n=10); and SITS hyperpolarized PDbl by 7.8±0.9 mV (n=4). Stimulation of the ducts with dbcAMP in the presence of bath HCO3−/CO2 resulted in depolarization of PDbl, the ductal lumen became more negative and the fractional resistance of the luminal membrane decreased. Together with forskolin (10−6 M), db-cAMP (10−4 M) caused a fast depolarization of PDbl by 33.8±2.5 mV (n=6). When db-cAMP (5×10−4 M) was given alone in the presence of bath HCO3−/CO2, PDbl depolarized by 25.3±4.2 mV (n=10). In the absence of exogenous HCO3−, db-cAMP also depolarized PDbl by 24.7±3.0 mV (n=10). The present data suggest that in the luminal membrane of pancreatic duct cells there is a Cl− conductance in parallel with a Cl−/HCO3− antiport. Dibutyryl cyclic AMP increases the Cl− conductance of the luminal membrane. Taking together our present results, and the recent data obtained for the basolateral membrane [21], a tentative model for pancreatic HCO3− transport is proposed.

Paralysis of skeletal muscle by butanedione monoxime, a chemical phosphatase

Abstract: The chemical phosphatase butanedione monoxime (BDM) reversibly inhibited twitches and tetanic contractions in bundles of rat soleus fibres in a dose-dependent manner (2–20 mM) but had no effect on the amplitude or time course of action potentials. In addition, BDM reversibly reduced the amplitude of potassium contractures demonstrating a depressant effect on contraction not mediated by action potentials. BDM had no effect on asymmetric charge movement but depressed calcium currents across the surface membrane in voltage-clamped fibres. The most significant effect of BDM on excitation-contraction coupling was a reduction in the amplitude of the calcium transient associated with contraction in aequorin-injected fibres. While these experiments do not eliminate the possibility of a direct effect of BDM on contractile filaments, reduction of calcium release from the sarcoplasmic reticulum, at least at low concentrations of BDM (below 2 mM), would seem to be the main mechanism for the inhibition of contractions in rat skeletal muscle.

Probing a Ca2+-activated K+ channel with quaternary ammonium ions.

Abstract: A series of quaternary amonium (QA) ions were used to probe the gross architecture of the ion conduction pathway in a Ca2+-activated K+ channel from rat muscle membrane The channels were inserted into planar phospholipid membranes and the single channel currents were measured in the presence of the different QA ions Internally applied monovalent QA ions (eg tetramethylammonium and analogues) induced a voltage-dependent blockade with a unique effective valence of the block equal to 030, and blocking potency increases as the compound is made more hydrophobic Blockade is relieved by increasing the K+ concentration of the internal or external side of the channel The effective valence of block is independent of K+ concentration These results suggest that, from the internal side, all monovalent QA ions interact with a site located in the channel conduction system Divalent QA ions of the type n-alkylbis-α,β-trimethylammonium (bisQn) applied internally also block the channel in a voltage dependent fashion For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 041 to 027, it remains constant for bisQ5 to bisQ6 and increases up to 054 for bisQ10 This dependence of block with chain length implies that 27% of the voltage drop within the channel occurs over a distance of ≈ 1 nm Externally applied monovalent QA ions also block the channel The site is specific for tetraethylammonium; increasing or decreasing the side chains in one methylene group decrease potency by about 400-fold It is concluded that the Ca2+-activated K+ channel has wide mouths located at each end and that they are different in molecular nature

Force-length relation of in-vivo human rectus femoris muscles

Abstract: In this paper the force-length relation of intact, in vivo, human rectus femoris musles were determined experimentally and compared to a corresponding theoretical force-length relation based on the cross-bridge theory. The experimental force-length relation has a much smaller peak force and a much wider range of muscle fiber lengths where non-zero forces were observed than the theoretical relation. Possible reasons for the differences between the two forcelength relations are discussed.

Cardiovascular and respiratory responses to submaximal exercise training in the thoroughbred horse

Abstract: Cardiovascular and respiratory responses to submaximal exercise training were investigated in 6 thoroughbred racehorses. Oxygen uptake, heart rate (HR) and arteriovenous oxygen content difference were measured during incremental treadmill exercise tests, before and after 7 weeks of treadmill training. Cardiac output during exercise was calculated by the direct Fick technique. Maximal oxygen uptake (\(\dot VO_{2\max } \)) was increased by 23% after training, from 129.7 ml/kg/min to 160.0 ml/kg/min. The treadmill speed at which \(\dot VO_{2\max } \) was attained increased by 19%. The increased aerobic power after training was associated with an increase in maximal cardiac output and stroke volume, a decrease in arteriovenous oxygen difference and no change in HR. There was no change in pulmonary ventilation during exercise at \(\dot VO_{2\max } \). Mean mixed venous oxygen content (\(C\bar vO_2 \)) at \(\dot VO_{2\max } \) before training was 2.8±1.0 ml/100 ml blood (mean ±SE). After training the value was 8.6±1.4 ml/100 ml blood. It is concluded that the increase in \(\dot VO_{2\max } \) after training in the horse is dependant on augmented blood flow, and is not dependent on either increased arterial oxygen content or arteriovenous oxygen content difference. Cardiac capacity to pump blood is therefore of primary importance as a determinant of increases in \(\dot VO_{2\max } \) due to training in the horse.

A novel cardiac potassium channel that is active and conductive at depolarized potentials.

Abstract: We report the existence of a novel potassium channel revealed in single-channel recordings from guinea-pig ventricular heart cells. The channel, observed in approximately 10% of patches, demonstrates a 14 pS conductance at physiological potassium concentrations, does not rectify over the voltage range of the action potential, and is quite selective for K ions. The channel activates with depolarization, but does not require intracellular Ca2+ ions to open. Open channel probability increases rapidly (less than 10 ms) to a plateau in response to depolarizing voltage steps, and demonstrates no detectable inactivation (greater than 600 ms). These features clearly distinguish this channel from other known K channels in cardiac muscle. Because of its high activity at plateau potentials, we propose the name iKp.

Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.

Abstract: Tibialis anterior (TA) muscle of mouse, rat, guinea pig, and rabbit was indirectly stimulated for 10 h/day at 10 Hz up to 28 days. Changes in the activity levels of hexokinase (HK), phosphofructokinase (PFK) glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), creatine kinase (CK), citrate synthase (CS), malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HADH), and β-hydroxybutyrate dehydrogenase (HBDH) were compared. Although the direction of changes in the enzyme activity pattern was in accordance with previous findings on rabbit TA, the magnitude of the responses varied markedly between themammals under study. Mouse TA was almost unaffected. A major effect of chronic stimulation in rat, guinea pig and rabbit was an increase in enzyme activities of aerobic-oxidative metabolism. According to intrinsic differences of the muscles under study, the increases varied among the species and appeared to be inversely related to the basal levels of these enzymes in the unstimulated muscles. Conversely, glycolytic enzyme activities (PFK, GAPDH, LDH) markedly decreased in rat, guinea pig, and rabbit, and were only slightly reduced in mouse. Changes in HK and HBDH activities displayed the largest variations in the induced change between species. These results indicate species-specific patterns of metabolic adaptation to increased contractile activity.

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane.

Abstract: In order to study the mechanism of pancreatic HCO3- transport, a perfused preparation of isolated intra- and interlobular ducts (i.d. 20-40 microns) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PDte) in ducts bathed in HCO3(-)-free and HCO3(-)-containing solution was -0.8 and -2.6 mV, respectively. The equivalent short circuit current (Isc) under similar conditions was 26 and 50 microA . cm-2. The specific transepithelial resistance (Rte) was 88 omega cm2. In control solutions the PD across the basolateral membrane (PDbl) was -63 +/- 1 mV (n = 314). Ouabain (3 mmol/l) depolarized PDbl by 4.8 +/- 1.1 mV (n = 6) within less than 10 s. When the bath K+ concentration was increased from 5 to 20 mmol/l, PDbl depolarized by 15.9 +/- 0.9 mV (n = 50). The same K+ concentration step had no effect on PDbl if the ducts were exposed to Ba2+, a K+ channel blocker. Application of Ba2+ (1 mmol/l) alone depolarized PDbl by 26.4 +/- 1.4 mV (n = 19), while another K+ channel blocker TEA+ (50 mmol/l) depolarized PDbl only by 7.7 +/- 2.0 mV (n = 9). Addition of amiloride (1 mmol/l) to the bath caused 3-4 mV depolarization of PDbl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PDbl. An increase in the bath HCO3- concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PDbl by 8.5 +/- 1.0 mV (n = 149). It was investigated whether the effect of HCO3- was due to a Na+-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K+ conductance caused by lowered intracellular pH. Experiments showed that the HCO3- effect was present even when the bath Na+ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO3- effect remained unchanged after Ba2+ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na+ + K+)-ATPase, a Ba2+ sensitive K+ conductance and an amiloride sensitive Na+/H+ antiport. The HCO3- effect on PDbl is most likely due to rheogenic anion exit across the luminal membrane.

Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology.

Abstract: Fluorescent indicator molecules, such as fura-2, are useful probes for studying the concentrations of ions in single cells. A key feature of these fluorescent dyes is the shift in their excitation spectra upon binding the ion, thus making alternate excitation from two wavelengths desirable. In this report we describe an inexpensive system for making simultancous electrophysiological and dual excitation fluorescence measurements using equipment much of which is available in a typical biophysical laboratory. In order to synchronize the fluorescence signal with the appropriate excitation wavelength we devised a simple computer program which uses the output of photodiodes placed in the excitation beam to determine which wavelength is illuminating the cell. We also describe the use of a liquid light guide to transmit excitation illumination from the light source to the epifluorescence port of the microscope in order to isolate a perfusion chamber from light, electrical noise and vibration. A sensitive light detection system was assembled using a photomultiplier tube and discriminator that took advantage of sampling single unit events obtained with photon counting rather than the analog of annode current. However, rather than employing a sophisticated and expensive photon counting system, a filter was used to integrate the signal so that an analog output could be presented to a multichannel analog to digital converter commonly used for electrophysiological recordings. This apparatus was sensitive enough to allow the determination of the intracellular free Ca2+ concentration, [Ca2+]i, using fura-2 in the following situations: (a) in single processes of dorsal root ganglion (DRG) neurons grown in primary culture, (b) the release of Ca2+ from intracellular stores in single neurons, (c) the influx of Ca2+ through channels activated by excitatory amino acids and (d) it was also possible to measure [Ca2+]i transients while simultaneously recording Ca2+ currents at precisely controlled membrane potentials in DRG neurons. The instrumentation described here makes use of a number of innovations which investigators developing similar systems may find useful.

Three types of calcium channels in the membrane of mouse sensory neurons.

Abstract: Three types of electrically-operated calcium channels have been identified in the membrane of cultured dorsal root ganglion neurons from mouse embryos using patch clamp technique. Low-threshold inactivating (LTI) channels with the lowest unitary conductance (5.7 pS with 60 mM Sr2+ or 7.2 pS with 60 mM Ba2+) preserved their activity for a long time on excised membrane patches and were insensitive to dihydropyridine Ca channel agonist Bay K8644. Corresponding whole-cell current could be decreased by 40% with 25 μM D-600. High-threshold inactivating (HTI) channels had somewhat higher unitary conductance (7 pS and 11.4 pS for Sr2+ or Ba2+ at 60 mM concentration) and were much more dependent upon intracellular metabolic support. D-600 inhibited the corresponding HTI whole-cell current by about 10%. High-threshold non-inactivating (HTN) channels were the most conducting ones (9 pS and 18.4 pS for 60 mM Sr2+ or 60 mM Ba2+) and their functioning was strongly metabolic dependent. Whole-cell HTN current could be slightly enhanced by 10 μM Bay K8644 due to some prolongation of channel mean open time. Effect of Bay K8644 was much less pronounced than that reported for cardiac cells. HTN whole-cell current could be almost completely blocked by 25 μM D-600. The described three types of calcium channels revealed different potential dependence and absolute values of mean channel open time.

External ATP triggers a biphasic activation process of a calcium-dependent K^+ channel in cultured bovine aortic endothelial cells

Abstract: We have used the patch-clamp method in order to investigate the single-channel events underlying the effect of external ATP on the potassium permeability of bovine aortic endothelial cells (BAE). The results obtained from cell-attached and inside-out experiments led first to conclude that BAE cells possess an inward rectifying potassium channel activated by internal calcium at micromolar concentrations. The channel conductance for inward currents was estimated at 40 pS in symmetrical 200 mM KCl and the open-channel probability was found to be voltage insensitive within the membrane voltage range −50 to −100 mV. Based on results obtained in the cell-attached configuration, it could next be established that external ATP and ADP at micromolar concentrations could trigger, via the stimulation of P2 purinergic receptors, a time variable activation process of the observed calcium-dependent potassium channel. This activation process was found to occur in a biphasic manner with an initial phase independent of the presence of calcium in the cell bathing medium. The second phase which could be blocked by calcium channel blockers such as Co2+ or La3+ required, however, the presence of external calcium and could be abolished by depolarizing the cells using high K+ external solutions. Another important aspect related to this phenomenon was the observation that removing ATP from the external medium during the second phase led to a complete abolition of the associated calcium-dependent potassium channel activation process. It is suggested from these results that the action of ATP on the potassium permeability of BAE cells is related to a second messenger mediated release of calcium from internal calcium stores coupled to an ATP-dependent calcium influx abolished at depolarizing voltages.

Single Ca channel currents in mouse pancreatic B-cells.

Abstract: Barium currents flowing through single Ca2+ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca2+ channels was observed. In 110 mM Ba2+, the single channel conductance was 24pS (at negative membrane potentials) and the current amplitude at 0 mV was -0.7 pA. Channel openings were activated by depolarisations more positive than -30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd2+. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca2+ channels in other tissues. It is suggested that this Ca2+ channel participates in the generation of the B-cell action potential and mediates the increase in Ca2+ influx required for insulin secretion.

Fura-2 detected myoplasmic calcium and its correlation with contracture force in skeletal muscle from normal and malignant hyperthermia susceptible pigs.

Abstract: Fura-2 was used to estimate myoplasmic [Ca2+] in intact intercostal muscle fibers from normal and malignant hyperthermia susceptible (MHS) pigs. Small muscle bundles (20–50 fibers) were loaded with the membrane-permeant form of the dye. Resting myoplasmic [Ca2+] were not significantly different in normal and MHS muscles. Halothane produced increases in myoplasmic Ca2+ with associated contractures in MHS muscles, but not in normal muscles. These halothane effects were reversible. Caffeine produced increases in myoplasmic Ca2+ and contractures in both MHS and normal muscles. The threshold concentrations were lower in the MHS muscles. The correlations between myoplasmic [Ca2+] and force in MHS and normal muscles were similar.

A low cost high intensity flash device for photolysis experiments

Abstract: Novel techniques of flash photolysis experiments require high intensity light sources in the near UV. We describe here a simple and inexpensive flash device which may compete with bulky and expensive laser systems if the experiments do not necessitate very short light pulses. Using a particular optical arrangement and stored electrical energy, a variation of the parameters voltage and capacitance led to a difference in light output by a factor of more than two. The system is used to relax both skeletal and smooth muscle fibres in the rigor state by releasing up to 2 mM ATP from 12.5 mM caged-ATP.

A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes.

Abstract: The decrease (‘run-down’) of the L-type Ca2+-current during long-lasting recordings with the whole-cell patch-clamp technique was examined in guinea pig ventricular myocytes. We have tested whether proteolysis is involved in the decay of the Ca2+-current by intracellular application of several concentrations of the Ca2+-dependent proteases calpain I and II, as well as their endogenous inhibitor calpastatin. The major finding was that calpain I and calpain II accelerated the Ca2+-current run-down in a concentration dependent manner, whereas calpastatin retarded it. These observations indicate that a proteolytic degradation of Ca2+-channels might be the reason for the run-down phenomenon.

ATP4- and ATP.Mg inhibit the ATP-sensitive K+ channel of rat ventricular myocytes.

Abstract: K+ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes ATP-sensitive K+ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP4- Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATPMg Dose-response relationships for ATP-sensitive K+ channel inhibition evoked by ATP had a Hill coefficient of 2 and Ki of 17 and 30 microM for ATP in the presence and absence of magnesium respectively This was the obverse of the expected results if ATP4- were to be the sole form of ATP to effect channel closure ATP-sensitive K+ channel inhibition evoked by ATP gamma S, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium It is concluded that the ATP-sensitive K+ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cation-bound forms of ATP

Na+ currents in cultured mouse pancreatic B-cells.

Abstract: Pancreatic B-cells, kept in culture for 1–4 days, were studied in the whole-cell, cell-attached and outside-out modes of the patch clamp technique. B-cells were identified by the appearance of electrical activity in the cell-attached mode when the bath glucose was raised from 3 to 20 mM. In whole-cell, 80% of these cells showed a transient inward Na+ current, when depolarizing pulses were preceded by holding potentials, or prepulses to potentials more negative than −80 mV. The midpoint (Eh) of the inactivation curve (h∞) was at −109 mV in 2.6 mM Ca2+, 1.2 mM Mg2+ and −120 mV in 0.2 mM Ca2+, 3.6 mM Mg2+. In 2.6 mM Ca2+, inactivation was fully removed atE −60 mV and were largest at around −10 mV (120 mM Na+). The kinetic parameters of activation (tp) and inactivation (τ)h were similar to those of other mammalian Na+ channels. Unitary currents with an amplitude of approximately 1 pA at −30 mV (140 mM Na+) with a similar voltage-dependence and time-course of mean current were recorded from outside-out patches. The results show that B-cells have a voltage-dependent Na+ current which, owing to the voltage-dependence of inactivation, is unlikely to play a major role in glucose-induced electrical activity.

Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells.

Abstract: Using the patch clamp technique we show that exposure of opossum kidney cells to hypotonic shock evokes an outward rectifying potassium current. The corresponding single channel slope conductance approaches 15 pS at depolarizing voltages. The K current also becomes activated after addition of the ionophore A23187 to an isotonic bath medium containing Ca2+. We therefore conclude that the K selective channels are modulated by an elevation of cytoplasmic Ca2+. Evidence is presented that release of Ca2+ from internal stores is involved.

Differential effects of ADH on sodium, chloride, potassium, calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron.

Abstract: The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (JNa+, JCl-, JK+, JCa2+, JMg2+) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PDte) and transepithelial resistance (Rte) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO3- containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO3- free Ringer solutions. In cTAL segments, AVP (10(-10) mol.l-1) significantly increased JMg2+ and JCa2+ from 0.39 +/- 0.08 to 0.58 +/- 0.10 and from 0.86 +/- 0.13 to 1.19 +/- 0.15 pmol.min-1 mm-1 respectively. Neither JNa+ nor JCl-, (JNa+: 213 +/- 30 versus 221 +/- 28 pmol.min-1 mm-1, JCl-: 206 +/- 30 versus 220 +/- 23 pmol.min-1 mm-1) nor PDte (13.4 +/- 1.3 mV versus 14.1 +/- 1.9 mV) or Rte (24.6 +/- 6.5 omega cm2 versus 22.6 +/- 6.4 omega cm2) were significantly changed by AVP. No significant effect of AVP on net K+ transport was observed. In mTAL segments, Mg2+ and Ca2+ net transports were close to zero and AVP (10(-10) mol.l-1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone, JNa+ increased from 107 +/- 33 to 148 +/- 30 and JCl- from 121 +/- 33 to 165 +/- 32 pmol.min-1 mm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptors along the rat nephron: [125I] insulin binding in microdissected glomeruli and tubules.

Abstract: Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10−18 mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [125I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [125I] insulin binding sites may correspond to physiological insulin receptors.

Efficient K+ buffering by mammalian retinal glial cells is due to cooperation of specialized ion channels.

Abstract: Radial glial (Muller) cells were isolated from rabbit retinae by papaine and mechanical dissociation. Regional membrane properties of these cells were studied by using the patch-clamp technique. In the course of our experiments, we found three distinct types of large K+ conducting channels. The vitread process membrane was dominated by high conductance inwardly rectifying (HCR) channels which carried, in the open state, inward currents along a conductance of about 105 pS (symmetrical solutions with 140 mM K+) but almost no outward currents. In the membrane of the soma and the proximal distal process, we found low conductance inwardly rectifying (LCR) channels which had an open state-conductance of about 60 pS and showed rather weak rectification. The endfoot membrane, on the other hand, was found to contain non-rectifying very high conductance (VHC) channels with an open state-conductance of about 360 pS (same solutions). These results suggest that mammalian Muller cells express regional membrane specializations which are optimized to carry spatial buffering currents of excess K+ ions.

Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones.

Abstract: The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P3-ester derivatives of guanosine 5'-0(3-thio)triphosphate (caged GTP-gamma-S) and 5'-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-gamma-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-gamma-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5-10 min, and a single pulse of 300-350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30-120 s following photochemical release of approximately 20 microM GTP-gamma-S. The peak calcium channel current was reduced by about 70% with a slow time course [t1/2 1.5 +/- 0.2 min (mean +/- SEM); n = 5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP.(ABSTRACT TRUNCATED AT 250 WORDS)