Calcium-dependent immediate feedback control of inositol 1,4,5-triphosphate-induced Ca2+ release.

The temporal and spatial distribution of increases in intracellular Ca2+ concentration is an important factor in cellular signal transduction. Inositol 1,4,5-trisphosphate (InsP3) plays a key part in agonist-induced Ca2+ release, which can take place abruptly and in a confined space by a mechanism that is not fully understood. Here we analyse the kinetics of InsP3-induced Ca2+ release following flash photolysis of caged InsP3 or caged Ca2+, and demonstrate that Ca(2+)-dependent immediate feedback control is an important determinant of the time course of Ca2+ release. The positive feedback mechanism is also important for the 'loading dependence' of InsP3-induced Ca2+ release. Furthermore, our results support the operation of positive cooperativity in channel opening and feedback control augments the steep InsP3 concentration-Ca2+ release relation. These inherent properties of InsP3-induced Ca2+ release are expected to give rise to temporally abrupt and/or spatially confined Ca2+ release within the cell.     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.     

Multiple transport modes of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.     

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.     

The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons.

The dendrites of many types of neurons contain voltage-dependent Na+ and Ca2+ conductances that generate action potentials (see ref. 1 for review). The function of these spikes is not well understood, but the Ca2+ entry stimulated by spikes probably affects Ca(2+)-dependent processes in dendrites. These include synaptic plasticity, cytotoxicity and exocytosis. Several lines of evidence suggest that dendritic spikes occur within subregions of the dendrites. To study the mechanism that govern the spread of spikes in the dendrites of hippocampal pyramidal cells, we imaged Ca2+ entry with Fura-2 (ref. 9) and Na+ entry with a newly developed Na(+)-sensitive dye. Our results indicate that Ca2+ entry into dendrites is triggered by Na+ spikes that actively invade the dendrites. The restricted spatial distribution of Ca2+ entry seems to depend on the spread of Na+ spikes in the dendrites, rather than on a limited distribution of Ca2+ channels. In addition, we have observed an activity-dependent process that modulates the invasion of spikes into the dendrites and progressively restricts Ca2+ entry to more proximal dendritic regions.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells.

Low concentrations of inositol 1,4,5-trisphosphate (InsP3) evoke a very rapid mobilization of intracellular Ca2+ stores in many cell types, which can be followed by a further, much slower efflux. Two explanations have been suggested for this biphasic release. The first proposes that the Ca2+ stores vary in their sensitivity to InsP3, and each store releases either its entire contents or nothing (all-or-none release); the second proposes instead that the stores are uniformly sensitive to the effects of InsP3, but that they can release only a fraction of their Ca2+ before their sensitivity is somehow attenuated (steady-state release). Experiments using purified InsP3 receptor molecules reconstituted into lipid vesicles have shown heterogeneity of the receptors in their response to InsP3 under conditions in which the total Ca2+ level at both sides of the receptor is held constant. We now report that in permeabilized A7r5 smooth-muscle cells incubated in Ca(2+)-free medium, the amount of 45Ca2+ remaining in the stores after the rapid transient phase of release is independent of their initial Ca2+ levels, indicating that partially depleted stores are less sensitive to InsP3. Moreover, if the stores are reloaded with 40Ca2+ after the first stimulus, reapplication of the same low concentration of InsP3 will release further 45Ca2+. This recovery of InsP3 sensitivity is almost complete. Under these conditions, Ca2+ release must thus occur by a steady-state mechanism, in which the decreasing Ca2+ content of the stores slows down further release.     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.

Calcium has an important role in regulating epithelial cell ion transport and is itself transported by tissues involved in the maintenance of extracellular Ca2+ homeostasis. Although the mechanism of Ca2+ entry in electrically excitable cells is well-documented little is known about it in epithelial cells. Calcium absorption in polarized epithelial cells is a two-step process in which Ca2+ enters cells across apical plasma membranes and is extruded across basolateral membranes. Efflux may be mediated by an energy-dependent Ca2(+)-ATPase or by Na+/Ca2+ exchange. We examined Ca2+ influx in single cultured cells from distal renal tubules sensitive to parathyroid hormone by measuring intracellular Ca2+. Our results demonstrate that parathyroid hormone activates dihydropyridine-sensitive channels responsible for Ca2+ entry. We also show that microtubule-dependent exocytosis stimulated by parathyroid hormone may be necessary for the insertion or activation of Ca2+ channels in these cells. Once inserted or activated, dihydropyridine-sensitive channels mediate Ca2+ entry into these Ca2(+)-transporting epithelial cells. Our results support the view that agonist-induced exocytosis may represent a general paradigm for modulation of transport in epithelial cells by delivery and incorporation of transport proteins to plasma membranes or by delivery to plasma membranes of factors regulating these proteins.     

Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation

Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.     

Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells

Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

Vanadate inhibits uncoupled Ca efflux but not Na--Ca exchange in squid axons.

Nerve cells can maintain a very low intracellular calcium concentration ([Ca2+]i) against large Ca2+ electrochemical gradients (see ref. 1 for review). The properties of the calcium efflux from these cells depend on [Ca2+]i (ref. 2), and within the physiological range, most Ca efflux depends on ATP (which stimulates with high affinity) and is insensitive to Na1, Na0 and Ca0 (uncoupled Ca efflux). When the [Ca2+]i is well above the physiological range, Ca efflux becomes only partially dependent on ATP (acting now with low affinity), is inhibited by Nai and is stimulated by Na0 and Ca0 (Na--Ca exchange). Orthovanadate, a powerful inhibitor of the (Na+ + K+)ATPase and the Na pump, also inhibits the Ca-stimulated ATPase activity, which is the enzymatic basis for the uncoupled Ca pump, in human red cells. The experiments reported here show that in squid axons the ATP-dependent uncoupled Ca efflux can be fully and reversibly inhibited by vanadate, whereas concentrations of vanadate 10 times higher have no effect on the Na--Ca exchange. This is another indication that the uncoupled Ca efflux represents an ATP-driven Ca pump, and supports the suggestion that the uncoupled Ca efflux and Na--Ca exchange are mediated by different mechanisms.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Paraneoplastic myasthenic syndrome IgG inhibits 45Ca2+ flux in a human small cell carcinoma line

Certain cancers exert unexplained remote effects on the nervous system. Small cell carcinoma (SCC) of the lung, a tumour capable of spike electrogenesis and which is of possible neural crest origin, is present in approximately 70% of patients with the Lambert-Eaton myasthenic syndrome (LEMS), a disorder characterized by fatigable muscle weakness. Patients with this syndrome have a defect in the (Ca2+-dependent) quantal release of acetylcholine from motor nerve terminals evoked by a nerve impulse or by high K+ (ref.5), and a decreased number of presynaptic active zone particles. The physiological and morphological features of the syndrome can be transferred to mice by the patients' IgG, consistent with an autoantibody interfering with the function of voltage-dependent Ca2+ channels. Here we demonstrate that K+-induced 45Ca2+ flux in a cultured human SCC line is significantly reduced by LEMS IgG, suggesting that in SCC-LEMS an autoantibody to tumour Ca2+-channel determinants is triggered; its cross-reaction with similar determinants at the motor nerve terminal could lead to the remote neurological syndrome.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials

Electrical activity in non-neuronal cells can be induced by altering the membrane potential and eliciting action potentials. For example, hormones, nutrients and neurotransmitters act on excitable endocrine cells. In an attempt to correlate such electrical activity with regulation of cell activation, we report here direct measurements of cytosolic free Ca2+ changes coincident with action potentials. This was achieved by the powerful and novel combination of two complex techniques, the patch clamp and microfluorimetry using fura 2 methodology. Changes in intracellular calcium concentration were monitored in single cells of the pituitary line GH3B6. We show that a single action potential leads to a marked transient increase in cytosolic free calcium. The size of these short-lived maxima is sufficient to evoke secretory activity. The striking kinetic features of these transients enabled us to identify oscillations in intracellular calcium concentration in unperturbed cells resulting from spontaneous action potentials, and hence provide an explanation for basal secretory activity. Somatostatin, an inhibitor of pituitary function, abolishes the spontaneous spiking of free cytosolic Ca2+ which may explain its inhibitory effect on basal prolactin secretion. Our data therefore demonstrate that electrical activity can stimulate Ca2+-dependent functions in excitable non-neuronal cells.