Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Actin cytoskeletons regulate the stretch-induced increase of Ca2+ current in human gastric myocytes

Using the whole-cell and single channel recording techniques, the influence of actin cytoskeletons on L-type Ca2+ current was investigated in human gastric smooth muscle cells. In isotonic condition, an actin depolymerizer cytochalasin D (Cyt-D) markedly decreased the whole-cell current (I(Ba)) without changing steady-state voltage dependency and single channel conductance. Intracellular dialysis of phalloidin, an actin polymerizer, significantly increased the I(Ba). Hypotonic stretch (222 mOsm/L) of the myocytes increased the I(Ba), and Cyt-D significantly inhibited the I(Ba) increase by the stretch. Phalloidin was without effect on the I(Ba) increase by the stretch. Phalloidin antagonized the Cyt-D inhibition of the stretch-induced I(Ba) increase. Neither heterotrimeric G protein modifiers (GTPgammaS and GDPbetaS) nor rho GTPase inhibitor (C3 exoenzyme) influenced the stretch-induced responses. These results reveal that the integrity of the actin cytoskeleton is an important factor which determines the activity of L-type Ca2+ channels and a response to stretch.     

Sodium and Calcium Inward Currents in Freshly Dissociated Smooth Myocytes of Rat Uterus

Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both I_Na and I_Ca that are vastly different from those in tissue-cultured material. I_Na is abolished by Na⁺-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na⁺ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. I_Na reactivates with a τ of 20 ms. I_Ca is abolished by Ca²⁺-free medium, Co²⁺ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca²⁺, Ba²⁺, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca²⁺ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. I_Ca reactivates with τ''s of 27 and 374 ms. I_Na and I_Ca are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak I_Na/I_Ca changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of I_Na, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with I_Na vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of I_Na, and increased densities of I_Na and I_Ca as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Ca channels and skeletal muscle diseases

When observed under a microscope, skeletal muscle exhibits striations due to the highly organized arrangement of muscle proteins that interact with one another to induce muscle contraction. Muscle contraction requires transient increases in intracellular ‘Ca²⁺’ concentration. In this review, Ca²⁺ channels contributing to the functional integrity of intracellular Ca²⁺-release and extracellular Ca²⁺-entry during skeletal muscle contraction are reviewed in terms of their properties, newly emerging ancillary proteins to them, and their abnormalities related to human skeletal muscle diseases. Finally, the aim of this review is to show the big picture of the correlation among Ca²⁺ channels that participate in the Ca²⁺ homeostasis in skeletal muscle.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.     

Single-channel gating and regulation of human L-type calcium channels in cardiomyocytes of transgenic mice

Overexpression of human cardiac L-type Ca(2+) channel pores (hCa(v)1.2) in mice causes heart failure. Earlier studies showed Ca(v)1.2-mRNA increase by 2.8-fold, but whole-cell current density enhancement by 相似文献   

Properties of the apamin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigtaenia coli

With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca²⁺-dependent K⁺ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I _SK(Ca)). This component did not show the temporal inactivation;I _SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI _SK(Ca) is very sensitive to changes in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ): a decrease in [Ca²⁺] _i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca²⁺ channels of L-type was not an obligatory condition for activation ofI _SK(Ca). The current-voltage relationship forI _SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.     

Two populations of pancreatic islet alpha-cells displaying distinct Ca2+ channel properties

In low or absence of glucose, alpha-cells generate rhythmic action potentials and secrete glucagon. alpha-Cell T-type Ca(2+) channels are believed to be pacemaker channels, which are expected to open near the resting membrane potential (around -60 mV) to initiate a small depolarization. A previous publication, however, showed that alpha-cell T-type Ca(2+) channels have an activation threshold of -40 mV, which does not appear to fulfill their role as pacemakers. In this work, we investigated the Ca(2+) channel characteristics in alpha-cells of mouse-insulin-promoter green-fluorescent-protein (MIP-GFP) mouse. The beta-cells of MIP-GFP were conveniently distinguished as green cells, while immunostaining indicated that the majority of non-green cells were alpha-cells. We found that majority of alpha-cells possessed T-type Ca(2+) channels having an activation threshold of -40 mV; these cells also had high-voltage-activated (HVA) Ca(2+) channels (activation threshold of -20 mV). A novel finding here is that a minority of alpha-cells had T-type Ca(2+) channels with an activation threshold of -60 mV. This minor population of alpha-cells was, surprisingly, devoid of HVA Ca(2+) channels. We suggest that this alpha-cell subpopulation may act as pacemaker cells in low or absence of glucose.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

线粒体和细胞内钙自稳平衡

线粒体对胞浆钙信号调节作用的研究已经历较长时间.近年,随着研究方法和技术的不断改进,发现在绝大多数生理条件下,线粒体都能参与胞内钙通信过程.线粒体可感受其周围钙微区的存在从而摄取钙,又可以通过钠-钙交换和大分子孔道将钙释放出来,因此可以调节胞浆钙信号的时空特性,影响相关的细胞功能.但是,由于技术上的局限性,目前的研究仍然存在模糊不清和自相矛盾之处,有待于进一步研究.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Calcium and inositol trisphosphate receptors

Work from the authors' laboratory is supported by the Wellcome Trust, and the Medical, and Agricultural and Food Research Councils. CWT is a Lister Institute Research Fellow.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.