A monovalent ion-selective cation current activated by noradrenaline in smooth muscle cells of rabbit ear artery

We have studied chloride influx and efflux in a highly purified preparation of type n cells freshly isolated from adult guinea-pig lung using ³⁶Cl^–. Chloride uptake was time-dependent, saturable (K_m<10 mM) and was inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS; K_i80 M). In the absence of external chloride (substituted by gluconate), ³⁶Cl^– uptake exhibited an overshoot above equilibrium. The rate of ³⁶Cl^– entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. ³⁶Cl^– efflux was stimulated by external bromide > bicarbonate chloride citrate; and was inhibited by proprionate > acetate > oxalate. Although the chloride channel blocker 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, ³⁶Cl^– influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anionexchange pathway for chloride transport in freshly isolated adult type II pneumocytes.     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Plasma membrane structure at the axon hillock, initial segment and cell body of frog dorsal root ganglion cells

Summary Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (300 m^–2) is much lower than that at the axon hillock (900 m^–2), proximal initial segment (1000 m^–2), or intermediate portion of the initial segment (800 m^–2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.     

Metabolic control of respiratory neuronal activity and the accompanying changes in breathing movements of the rabbit

Summary The property of the neuronal membrane to be permeable to metabolic modifiers of two regulatory enzymes has been utilized to manipulate the spike activity of inspiratory (I) and expiratory-inspiratory (EI) neurons of the bulbar respiratory centre. The neurons have been classified according to their response to lung distension or collapse (- or -type) and to hyperventilation (tonic firing denoted by +, cessation of activity by –). Using extracellular microelectrodes for single unit recording, the medulla oblongata was superfused with a metabolite-containing CSF. The various neuronal sub-types exhibited a differential activating or inhibitory response to one or several metabolic effectors. For example I ⁺ units were activated by 5 mM glucose-6-phosphate (G-6-P) and 3.5 mM 3-phosphoglycerate (3-PGA), which both inhibited I ⁺ neurons, while 5 mM AMP inhibited I ⁺ much more strongly than I ⁺ cells. The spike density of I ^– and I ^– neurons was increased in the presence of 2.5 mM fructose-6-phosphate and 3.5–5 mM AMP, but became reduced by G-6-P. In contrast, 3 mM fructose-1,6-diphosphate and 5 mM 3-PGA activated the I ^– but inhibited the I ^– neurons. The EI units were characteristically activated by 10 mM citrate, which inhibited all I-type neurons. Activations of the I and I neurons led to an accelerated respiratory rate and a higher tidal volume, while the opposite was true for EI neurons. Intravenous injection of metabolites could not duplicate the striking effects under local applications.Supported by the Deutsche Forschungsgemeinschaft, Grant Ch 25/1.     

β-Adrenoceptor-mediated depolarization of the resting membrane in guinea-pig papillary muscles: changes in intracellular Na+, K+ and Cl− activities

Effects of -adrenergc stimulation on the membrane potential and intracellular Na⁺, K⁺ and Cl^– activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM–1 M produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect (pD₂) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 M)-induced depolarization was not blocked by tetrodotoxin (10 M), nifedipine (10 M), Cs⁺ (5 mM), Ba²⁺ (0.3 mM), amiloride (1 mM) or ouabain (10 M), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl^–-channel blocker. Intracellular K⁺ activity increased, whereas intracellular Na⁺ activity slightly decreased during the transient depolarization. Intracellular Cl^– activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl^– movement, rather than inward Na⁺ movement, may be involved in the -adrenoceptor-mediated membrane depolarization.     

Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca2+-activated K+ channel in cultured cerebellar granule cells

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K⁺ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca²⁺-activated K⁺ channels (maxi K⁺ channels) using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (–) BAY K 8644, nitrendipine, nisoldipine and (–) nimodipine block 10–60% of macroscopic K⁺ currents. The most potent of these DHPs was nicardipine and the least potent, (–) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of maxi K⁺ channels showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca²⁺ and K⁺ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of maxi K⁺ channels having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K⁺ channels.     

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

Summary The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy.The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (1600 1100 m^–2, respectively), while relatively few IMPs (150 m^–2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (600–2300 m^–2) and, in most regions, E-faces displayed a relatively low IMP density (175 m^–2). Axons of > 0.4 m diameter exhibited significantly greater mean P-face IMP density than axons < 0.4 m diameter. Aggregations of E-face IMPs (350 m^–2) were occasionally observed along amyelinated axon membrane from md optic nerves.Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (1800 and 140 m^–2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (> 0.4 m) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (< 0.4 m) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (200 m^–2); however, focal aggregations (400 m^–2) of E-face particles are present.Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves. These results are consistent with persistence of action potential conduction in md axons, despite the absence of myelination in the optic nerves of the md mutant.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

A T cell/B cell/epithelial cell internet for mucosal inflammation and immunity

Conclusions Mucosal immune responses are strongly regulated by CD4⁺ T cells and their derived cytokines. In this regard, IFN-^–/– mice (i.e., which lack Th1 and have elevated Th2 cells) showed strong mucosal Th2-type responses together with S-IgA production, while IL-4^–/– (e.g., dominant Th1 and lack of Th2 cells) mice had impaired mucosal Th2 and IgA responses following oral delivery of TT and CT. However, when rSalmonella or radenovirus were used for antigen delivery, significant levels of mucosal IgA responses were induced in both IFN-^–/^– and IL-4^–/^– mice. The choice of the antigen delivery system which leads to optimal Th and B cell interactions are important for the induction of effective IgA responses, even in situations where the immune system is compromised. It is clear that Th2-type cytokines are important in mucosal IgA responses; however, other cytokine combinations can compensate for mucosal immunity in situations in which Th2 cell responses are absent. Mucosally induced tolerance may be one approach to prevent several systemic immune disorders; however, the mechanism of this phenomenon still needs to be elucidated. Our recent findings have suggested that IFN- may play an important role in induction of systemic unresponsiveness since oral tolerance was not induced in IFN-^–/^– mice.Our studies as well as those of others indicated that at least two phases of a triad of cell interactions are important for the mucosal immune system. First, it has been shown that epithelial cell-produced IL-7 and SCF and T cell-derived IL-2 are essential activation and growth signals for intestinal T cells. Second, our studies with TCR knockout mice have suggested that mucosal T cells also play a critical role in the regulation of mucosal IgA responses. Thus, a mucosal internet among T cells, T cells, and IgA B cells appear critical for mucosal homeostasis and for regulation of specific mucosal immune responses.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

On the mechanism of β-adrenergic regulation of the Ca channel in the guinea-pig heart

Dose-response relations for the increase in the amplitude of Ca current (I _Ca) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10^–9 M, for cAMP 3 M (pipette concentration to which 10^–5 M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 M (pipette concentration). The concentrations for the half-maximal effect were 3.7×10^–8 M (ISP), 5.0 M (cAMP) and 0.95 M (C subunit) and for the maximum effect 10^–6 M (ISP), 15–20 M (cAMP) and 3–4 M (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3–4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit onI _Ca were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects onI _Ca could be estimated and were compared with those obtained in broken cell preparations.A competitive inhibitor of phosphorylation, 5-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit onI _Ca. Cell dialysis with 3 mM adenosine-5-(-thio)-triphosphate, which produces a dephosphorylationresistant phosphorylation, markedly potentiated the effects of ISP and cAMP onI _Ca.The results support the hypothesis that phosphorylation of a protein within, or close to, the Ca channel by cAMP-dependent protein kinase is the mechanism of -adrenergic stimulation.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 38 (membranforschung), Projekt G, and H0579/6-2     

Muscarinic modulation of calcium dependent plateau potentials in rat neostriatal neurons

Intracellular recording from neostriatal neurons in rat brain slices revealed effects of the acetylcholine (ACh) agonist carbachol (Cch, 1–10 mol/l), of the anticholinesterase physiostigmine (10 mol/l) and of the muscarinic antagonist atropine (10 mol/l) on plateau potentials elicited in the presence of K-blockers were Cadependent, elicited in the presence of K-blockers were Cadependent, since they persisted in Na-free solution, were resistant to tetrodotoxin (TTX, 3 mol/l) and blocked by Cd (0.1–0.5 mmol/l). Cch reduced the duration of the plateau potentials and made them more susceptible to fatigue. These effects were antagonized by atropine (1–10 mol/l), but not by Ba (100–200 mol/l) or 4-aminopyridine (4-AP, 0.5 mmol/l). Physostigmine (10 mol/l) had the same atropine-sensitive effects as Cch on the plateau potential. Atropine (10 mol/l), by itself, prolonged the duration of the plateau potential. High concentrations (100 mol/l) of Cch did not further reduce the duration of the plateau potential, instead, the duration re-increased with prolonged exposure. The re-increase of the plateau-spike duration was later masked by bursting activity. The opposing effects of low and high concentrations of Cch on the plateau potential duration corresponded to effects of this drug on intrastriatally evoked EPSPs in that low concentrations of Cch reduced the EPSP amplitude, but high concentrations re-increased it after a transient decrease. It is concluded that the muscarinic effect of Ach in the neostriatum is to modulate Ca-influx and that this effect is exerted in a tonic manner. On leave from absence from: Clinica Neurologica II, Universita di Roma. Tor Vergata, Roma, Italy.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.     

Efficacy of the two-microelectrode voltage clamp technique in crayfish muscle

Crayfish muscle fibres of different dimensions were voltage clamped and white noise current was injected into the fibres at various distances from the voltage clamp current electrode. The clamp current was measured and power spectral densities were calculated. This method revealed the efficacy of the voltage clamp in these fibres. In large fibres (l=1.8–2.0 mm; =100–180m) a space clamp was achieved only for a band width f=40Hz. At a distance of 100m from the clamp electrodes f was 250–500Hz. In fibres of medium size (l=1.0–1.3mm; =60–120m) f was about 80Hz and about 800 Hz at a distance of 100m. In experiments with very small muscle fibres (l=400–600m; =30–50m) f was more than 500Hz. The improvement of the space clamp for the smaller muscle fibres resulted mainly from the reduced total membrane capacity,c _m, of these fibres. The limitations of the space clamp could be derived from the impedance properties of the fibres. The band width of the space clamp correlated with the band width for which the square of the absolute impedance, |Z _p|², of the muscle fibre could be described by a simple RC-model. This correlation was demonstrated in a model circuit.Power density spectra of membrane current fluctuations were measured also. To optimize the resolution of these measurements the contribution of instrumental noise was minimized. The effects of instrumental noise are discussed.This investigation was supported by the Deutsche Forschungs-gemeinschaft     

Glucose dependent K+-channels in pancreaticβ-cells are regulated by intracellular ATP

The resting conductance of cultured-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (>20 G per cell or>100 k·cm²), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K⁺ conductance. In cell-attached patches single K⁺-channels were observed in the absence of glucose. Adition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K⁺]₀=145 mM, T=21° C) and kinetics (at –70 mV: _open=2.2 ms and _closed=0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the-cell by increasing the cytoplasmic concentration of ATP.     

An investigation of intrinsic buffering power in rat vascular smooth muscle cells

Intrinsic buffering power ( _i) has been measured in vascular strips and single cells from rat mesenteric artery. Intracellular pH (pH_i) regulation was inhibited to prevent overestimation of _i due to acid extrusion or entry via regulatory processes. At resting values of pH_i (7.0–7.2), a mean value of 41±4 mM/pH unit for _i was found. _i increased approximately fivefold from 30 to 150 mM/pH unit over the pH_i range 7.5–6.5. The mean data relating _i to pH_i could be described by relating _i to buffer concentrations and pK _a. This gave a value of 310 mM for buffer concentration and a pK _a of 6.0. As changes in pH_i are known to have marked effects on vascular tone then the increase in _i as pH_i falls may be considered as a means of attenuating pH_i decreases, before pH regulation restores pH_i to resting levels.     

Bursting electrical activity in pancreatic β-cells: evidence that the channel underlying the burst is sensitive to Ca2+ influx through L-type Ca2+ channels

In glucose-stimulated pancreatic -cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca²⁺ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2–12.8 mM Ca²⁺, 1 M Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the presence of 100 M tolbutamide. The [Ca²⁺]_i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca²⁺ and Bay-K-8644 evoked [Ca²⁺]_i oscillations overshot the [Ca²⁺]_i recorded in tolbutamide. Nifedipine (10–20 M) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca²⁺ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 M) caused membrane hyperpolarization in the presence of 7.7 mM Ca²⁺, which was not abolished by nifedipine. Charybdotoxin (CTX, 40–80 nM), TEA (2 mM) and quinine (200 M) did not suppress the high-Ca²⁺-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca²⁺]_i rises mediated by Ca²⁺ influx through L-type Ca²⁺ channels, (2) both the ATP-dependent K⁺ channel and the CTX and TEA-sensitive Ca²⁺-dependent K⁺ channel are highly unlikely to provide the pacemaker current underlying the burst. We propose that the burst is mediated by a distinct Ca²⁺-dependent K⁺ channel and/or by [Ca²⁺]_idependent slow processes of inactivation of Ca²⁺ currents.     

Tetrodotoxin exerts a large frequency-dependent depression of the maximum rate of rise of action potentials in guinea pig ventricular myocytes

The action potential configuration in guinea pig ventricular myocytes was unaffected by low concentrations (0.3–1 M) of tetrodotoxin (TTX); high concentrations (10–30 M) depressed both the overshoot (5–10 mV) and duration (5–10%). Although the control was unaffected by stimulation rate (0.1–5 Hz), the depression of by TTX was greatly potentiated at rates above 1 Hz: on dose-response curves, 50% control occurred at 4.3 M (5 Hz) versus 22 M ( 1 Hz). The frequency dependent component of the depression reported here is much larger than the extra block of Na channels observed by others in voltage clamp studies on Purkinje strands. This is not a discrepancy; rather it is a consequence of a non-linear relation between and available Na conductance.     

Modulation of sarcoplasmic reticulum Ca2+-release channels by caffeine,Ca2+, and Mg2+ in skinned myocardial fibers of fetal and adult rats

Ryanodine causes depression of the caffeine-induced tension transient (ryanodine depression) in skinned muscle fibers, because it blocks the sarcoplasmic reticulum (SR) Ca²⁺-release channels [Su, J. Y. (1988) Pflügers Arch 411:132–136, 371–377; (1992) Pflügers Arch 421:1–6]. This study was performed to examine the sensitivity of SR Ca²⁺-release channels to ryanodine in fetal compared to adult myocardium and to investigate the influence of Ca²⁺, caffeine, and Mg²⁺ on ryanodine depression in skinned fibers. Ryanodine (0.3 nM–1 M) caused a dose-dependent depression in skinned myocardial fibers of the rat, and the fetal fibers (IC₅₀74 nM) were 26-fold less sensitive than those of the adult (IC₅₀2.9 nM). The depression induced by 0.1 M or 1 M ryanodine was a function of [caffeine], or [Ca²⁺] (pCa<6.0), which was potentiated by caffeine, and an inverse function of [Mg²⁺]. At pCa>8.0 plus 25 mM caffeine, a 20% ryanodine depression was observed in both the fetal and adult fibers, indicating independence from Ca²⁺. Ryanodine depression in skinned fibers of the fetus was less affected than that seen in the adult by pCa_i, [caffeine]_i, or 25 mM caffeine plus pCa_i or plus pMg_i (IC₅₀pCa 4.5 versus 5.1; caffeine 12.7 mM versus 2 mM; pCa 6.7 versus 7.3; and pMg 3.9 versus 3.3 respectively). The results show that the SR Ca²⁺-release channel in both fetal and adult myocardium is modulated by Ca²⁺, caffeine, and Mg²⁺. It is concluded that less ryanodine depression seen in the skinned fibers of the fetus, indicating a relatively insensitive SR Ca²⁺-release channel, could contribute to the resistance of intact myocardium to ryanodine.     

Chloride channels in cultured human skeletal muscle are regulated by G proteins

The regulation of Cl^– channels in human myoballs by G proteins was studied using whole-cell and inside-out patch recordings. After perfusion of the cell with 0.1 mM GTP[S], the specific Cl^– conductance, G _Cl, at standard resting potential (–85 mV) was increased from 5.9 S/cm² to 103 S/cm², and the kinetics upon stepping the potential to positive values was changed from an activating current with very slow inactivation to a fast inactivating current with no potential-dependent activation. These effects were not affected by the simultaneous blockade of several signal cascades involving G proteins. Addition of the protein kinase blockers PKI (25 M), H8 (10M), or of the phospholipase-A₂-blocking agent quinacrine (10 M), had not much influence on these GTP[S] effects. Buffering of the intracellular Ca²⁺ concentration (0.1 M) or addition of the Ca²⁺/calmodulin antagonist trifluoperazine (50 M) was also without effect. Pre-incubation of the cells with pertussis toxin or with cholera toxin did not change G _Cl. In excised inside-out patches voltage-clamped at –85 mV, application of GTP[S] influenced the intermediate Cl^– channel, the Cl^– channel type having the highest density in these cells, by increasing the number of transitions in a half-conductance state. The probability of the channel being in one of the two conducting states rose from 0.015 to 0.67, and the kinetics of the single-channel currents was changed so that, on average, it was similar to the whole-cell current kinetics seen after application of GTP[S]. It is concluded that a G protein is directly interacting with these channels.