Rapid Kinetic Studies of the Light Emitting Protein Aequorin

SHIMUMURA et al.^1,2 isolated from luminescent jelly fish (Aequora forskalea) a protein (aequorin) which on addition of Ca²⁺ emits light. In contrast to other bioluminescent compounds, aequorin luminescence requires neither oxygen nor FMNH₂, ATP nor long chain aldehydes, but only Ca²⁺. Recently, other bioluminescent proteins reacting only with Ca²⁺ have been isolated and termed Ca²⁺-activated photoproteins^3,4. Since the intensity of the emitted light varies with the Ca²⁺ concentration, aequorin and related proteins can serve as useful tools for detecting small changes in Ca²⁺ concentration in biological systems^5,6. Little is known, however, about the mechanism of this reaction. Since light emission from aequorin occurs within a few milliseconds of rapid mixing with Ca²⁺, kinetic studies are possible only with rapid mixing instruments, stopped and continuous flow. Hastings et al. studied the kinetics of aequorin using stopped flow and double stopped flow apparatuses⁷. Because both have a dead time of about 2 ms, which is in the same range as the rise time of the light emission from aequorin, they were unable to establish a Ca²⁺ dependence for the rate of rise of the light emission. Ca²⁺ dependence of this rate might be very important for delineation of the reaction mechanism.     

Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.

Parthenogenetic activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. Parthenogenetic treatments, including the nonelectrolyte urea, hypertonic sea water, and ionophore A23187, all acted to release Ca²⁺ from intracellular stores. Ionophore and urea solutions release Ca²⁺ from the same intracellular store as normal fertilization. This intracellular store can be reloaded after 40 min and discharged again. Hypertonic medium appears to release Ca²⁺ from a different intracellular store. Treatment with the weak base NH₄Cl did not release intracellular Ca²⁺ but did result in a momentary Ca²⁺ influx if Ca²⁺ was present in the external solution. Ca²⁺ influx was not required for ammonia activation.     

Evidence that myosin light chain phosphorylation regulates contraction in the body wall muscles of the sea cucumber

The Ca²⁺ activation mechanism of the longitudinal body wall muscles of Parastichopus californicus (sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca²⁺-activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca²⁺ (10^-5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca²⁺ insensitive tension. In contrast, pretreatment with low Ca²⁺ (10^-8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca²⁺-sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca²⁺-activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP-dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca²⁺-activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal body wall muscle.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin.2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca²⁺ for aequorin and at least 3Ca²⁺ for obelin.3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units.4. Other monovalent cations, such as Na⁺ are able to compete with Ca²⁺ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions.5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca²⁺ solutions or of calcium/EGTA buffers.     

Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons

To gain a better understanding of Ca²⁺-induced Ca²⁺ release in central neurons, we have studied the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca²⁺]_i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca²⁺]_i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca²⁺] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ responses to caffeine were observed in Ca²⁺-containing and nominally Ca²⁺-free external solutions, suggesting that caffeine was inducing Ca²⁺ release from intracellular stores. Ca²⁺ responses to caffeine were potentiated by inducing a tonic Ca²⁺ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca²⁺ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca²⁺ release was use- and concentration-dependent; the median effective concentration EC₅₀ for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca²⁺ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca²⁺ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.     

A Comparison between Quin-2 and Aequorin as Indicators of Cytoplasmic Calcium Levels in Higher Plant Cell Protoplasts

Assessment of the regulation of plant metabolism by the calcium ion requires a knowledge of its intracellular levels and dynamics. Technical problems have prevented direct measurement of the concentration of intracellular Ca²⁺ in plant cells in all but a few cases. In this study we show that electropermeabilized protoplasts of Daucus carota and Hordeum vulgare took up the Ca²⁺ indicating fluorescent dye methoxyquinoline(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Quin-2) and the Ca²⁺ indicating photoprotein, aequorin. These protoplasts subsequently recovered their plasma membrane integrity. However, up to 10% of intracellularly trapped Quin-2 was associated with a protoplast vacuolar fraction. Also, Quin-2 loading reduced total ATP levels by approximately 60% and inhibited subsequent protoplast division whereas aequorin loading reduced ATP content by only 20% and did not prevent division. Therefore, the basal cytoplasmic Ca²⁺ level measured with aequorin (less than 200 nanomolar) may more reliably reflect that found in vivo in the unperturbed protoplast than that measured with Quin-2 (120-360 nanomolar). However, measurements made with aequorin were found to be inaccurate at Ca²⁺ levels below 200 nanomolar, Quin-2 proving complementary in indicating these low Ca²⁺ concentrations. Cytosolic Ca²⁺ was observed to increase on treatment with azide and silver ions.     

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization

Phosphorylation of the regulatory light chain of myosin by the Ca²⁺/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca²⁺/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca²⁺/calmodulin required for activation and hence increases the Ca²⁺ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca²⁺ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca²⁺ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca²⁺ influx and mitochondrial Ca²⁺ overload. A substantial change in intracellular Ca²⁺ homeostasis or remodeling of intracellular Ca²⁺ homeostasis may favor neuron damage in old neurons. For investigating Ca²⁺ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca²⁺ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca²⁺ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca²⁺ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca²⁺ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.     

Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca²⁺ concentration. To better understand this process, we measured the cytosolic Ca²⁺ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca²⁺ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca²⁺-sensitive dyes that possess different affinities for Ca²⁺ allowed the quantitative determination of the cytosolic Ca²⁺ concentration in the steady state. Determining the cytosolic Ca²⁺ concentration as a function of the adapting light intensity shows that the Ca²⁺ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca²⁺ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca²⁺ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca²⁺ concentration, we conclude that the Ca²⁺-buffering capacity is limited. The percentage of the Ca²⁺ influx that is buffered gradually decreases with increasing Ca²⁺ concentrations; at cytosolic Ca²⁺ concentration levels above 10 μM, buffering becomes minimal.     

Mechanisms Underlying Nitroglycerin-Induced Suppression of Phenylephrine- and Caffeine-Evoked Contractions of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a strain measurement technique, we studied the mechanisms of the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on contractions of smooth muscles of the main pulmonary artery of the rabbit induced by phenylephrine and caffeine in normal Krebs solution (NKS) or in nominally calcium-free solution (NCFS). Phenylephrine applications caused contractions consisting of an initial fast phasic low-amplitude component followed by a tonic higher-amplitude component. After caffeine-induced monophasic low-amplitude contraction, tension of the smooth muscle strip shifted below the conventional zero. Addition of NG to NKS resulted in a decrease in the smooth muscle tension below the conventional zero. Under the influence of NG, the initial phasic component of phenylephrine-induced contraction was partially suppressed, whereas the next tonic component was suppressed to a greater extent. At the same time, NG exerted nearly no influence on the amplitude of caffeine-induced contractions. Washing out by NKS of phenylephrine dissolved in NCFS resulted in initiation of a fast phasic high-amplitude contraction. Such a contraction did not develop either in the presence of NG or phenylephrine in NCFS or in the case of washing out of caffeine dissolved in NCFS. Our findings allow us to conclude that phenylephrine or caffeine added to the superfusate induce contractions of the smooth muscle cells (SMC) of the main pulmonary artery of the rabbit due to activation of Ca²⁺ release from the respective intracellular calcium stores. In addition, calcium ions entering SMC through the calcium channels of the plasma membrane are also involved in activation of the phenylephrine-induced contraction. The inhibitory effect of NG on the phenylephrine-induced contraction is related to the influence of NO on the release of Ca²⁺ from the inositol trisphosphate-sensitive intracellular calcium store and receptor-operated inflow of Ca²⁺ to SMC. Nitroglycerin did not significantly influence the caffeine-induced contraction and, therefore, Ca²⁺ release from the caffeine-sensitive store.     

Analysis of the Ca<Superscript>2+</Superscript> response of mycelial fungi to external effects by the recombinant aequorin method

Using the mutant strain Aspergillus awamori 66A, producing the recombinant Ca²⁺-dependent photosensitive protein aequorin, the dynamics of Ca²⁺ was studied for the first time in the cytosol of micromycetes exposed to stressful factors, such as an increase in extracellular Ca²⁺ to 50 mM, hypoosmotic shock, and mechanical shock. The cell response to stress proved to involve an increase in the Ca²⁺ concentration in the cytosol, which was determined from the amplitude of aequorin luminescence and the time of the amplitude enhancement and relaxation. The level of the Ca²⁺ response depended on the physiological stimulus. Inhibitory analysis with various agents that block Ca²⁺ channels and with agonists that specifically enhance the activity of the channels suggested that (1) the level of Ca²⁺ in the cytosol of micromycetes increases in response to stress because of the ion influx from both the growth medium and intracellular reservoirs and (2) potential-dependent transport systems play the major role in the Ca²⁺ influx into the cytosol of the micromycete cells.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 734–740.Original Russian Text Copyright © 2004 by Kozlova, Egorov, Kupriyanova-Ashina, Rid, El-Registan.     

Free-calcium and tension responses in single barnacle muscle fibres following the application of l-glutamate

The effect of the putative transmitter, l-glutamate, on free intracellular Ca²⁺, tension and membrane potential in single muscle fibres from the barnacle Balanus nubilus has been investigated. External application of l-glutamate (0.1–10 mM) resulted in a transient increase in free intracellular Ca²⁺, monitored by the Ca²⁺-activated protein aequorin. This increase in free intracellular Ca²⁺ was associated with membrane depolarization and force development, and was followed by a period of ‘desensitization’ in which the preparation was unresponsive to l-glutamate. This could be reversed by removing l-glutamate from the external saline. External application of a number of closely related compounds, including d-glutamate and l-aspartate, were ineffective for initiating the transient light response. The l-glutamate response was virtually abolished in Na-free (Li) medium and completely abolished in Ca-free (Na) medium. The responses to l-glutamate were not reduced in Mg-free medium. The fibre's response to 1 mM l-glutamate was also inhibited by D-600 (10 μM) or by La³⁺ (1 mM), suggesting that Ca was directly involved in the underlying ionic conductance changes brought about by this putative excitatory transmitter.     

A new short-term toxicity assay using <Emphasis Type="Italic">Aspergillus awamori</Emphasis> with recombinant aequorin gene

Background  

Most currently available short-term toxicity assays are based on bacterial cells. Therefore there is a need for novel eukaryotic microbial bioassays that will be relevant to higher eukaryotes such as animals and plants. Ca²⁺ is a universal intracellular signalling molecule found in all organisms from prokaryotes to highly specialized animal cells. In fungi calcium has been demonstrated to be involved in control of many important processes. The recombinant aequorin gene from the jellyfish Aequorea victoria responsible for the expression of the Ca²⁺-sensitive aequorin photoprotein has been cloned in the filamentous fungus Aspergillus awamori. This has allowed real life monitoring of [Ca²⁺]_c changes in living fungal cells. When subjected to different physico-chemical stimuli fungal cells respond by transiently changing the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_c) and the pattern of these changes (Ca²⁺ signature) is specific to each particular stimulus. Therefore it was interesting to investigate whether different environmental toxicants would be able to affect the pattern of [Ca²⁺]_c changes in a reproducible and dose dependant manner.     

Staphylococcal leukotoxins trigger free intracellular Ca2+ rise in neurones,signalling through acidic stores and activation of store‐operated channels

Headache, muscle aches and chest pain of mild to medium intensity are among the most common clinical symptoms in moderate Staphylococcus aureus infections, with severe infections usually associated with worsening pain symptoms. These nociceptive responses of the body raise the question of how bacterial infection impinges on the nervous system. Does S. aureus, or its released virulence factors, act directly on neurones? To address this issue, we evaluated the potential effects on neurones of certain bi‐component leukotoxins, which are virulent factors released by the bacterium. The activity of four different leukotoxins was verified by measuring the release of glutamate from rat cerebellar granular neurones. The bi‐component γ‐haemolysin HlgC/HlgB was the most potent leukotoxin, initiating transient rises in intracellular Ca²⁺ concentration in cerebellar neurones and in primary sensory neurones from dorsal root ganglia, as probed with the Fura‐2 Ca²⁺ indicator dye. Using pharmacological antagonists of receptors and Ca²⁺ channels, the variations in intracellular Ca²⁺ concentration were found independent of the activation of voltage‐operatedCa²⁺ channels or glutamate receptors. Drugs targeting Sarco‐Endoplasmic Reticulum Ca²⁺‐ATPase (SERCA) or H⁺‐ATPase and antagonists of the store‐operated Ca²⁺ entry complex blunted, or significantly reduced, the leukotoxin‐induced elevation in intracellular Ca²⁺. Moreover, activation of the ADP‐ribosyl cyclase CD38 was also required to initiate the release of Ca²⁺ from acidic stores. These findings suggest that, prior to forming a pore at the plasma membrane, leukotoxin HlgC/HlgB triggers a multistep process which initiates the release of Ca²⁺ from lysosomes, modifies the steady‐state level of reticular Ca²⁺ stores and finally activates the Store‐Operated Calcium Entry complex.     

Aberrant Ca2+ oscillations in smooth muscle cells from overactive human bladders

Overactive bladder (OAB) syndrome is highly prevalent and costly, but its pathogenesis remains unclear; in particular, the origin of involuntary detrusor muscle activity. To identify the functional substrate for detrusor muscle overactivity, we examined intracellular Ca²⁺ oscillations in smooth muscle cells from pathologically overactive human bladders. Basal cytoplasmic Ca²⁺ concentration was elevated in smooth muscle cells from overactive bladders. Unprovoked, spontaneous rises of Ca²⁺ were also identified. These spontaneous Ca²⁺ oscillations were Ca²⁺-dependent, sensitive to L-type Ca²⁺ channel antagonist verapamil and also attenuated by blocking SR Ca²⁺ reuptake. The fraction of spontaneously active cells was higher in cells from overactive bladders and the magnitude of spontaneous Ca²⁺ oscillations also greater. Spontaneous action potentials or depolarising oscillations were also observed, associated with Ca²⁺ rise; with a higher percentage of cells from idiopathic OAB, but not in neurogenic OAB. Low concentrations of NiCl₂ attenuated both spontaneous electrical and Ca²⁺ activation. This study provides the first evidence that spontaneous, autonomous cellular activity—Ca²⁺ and membrane potential oscillations, originates from detrusor smooth muscle in human bladders, mediated by extracellular Ca²⁺ influx and intracellular release. Such cellular activity underlies spontaneous muscle contraction and defective Ca²⁺ activation contributes to up-regulated contractile activity in overactive bladders.