Rhythmic Ca2 Research Articles

Circadian rhythms in Ca2+ release from ryanodine-sensitive Ca2+ stores in suprachiasmatic nucleus neurons

Circadian rhythms in Ca2+ release from ryanodine-sensitive Ca2+ stores in suprachiasmatic nucleus neurons

Rhythmic Ryanodine Receptor Ca 2+ Releases During Diastolic Depolarization of Sinoatrial Pacemaker Cells Do Not Require Membrane Depolarization

Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.

Spontaneous waves in the ventricular zone of developing mammalian retina.

Spontaneous rhythmic waves in the developing mammalian retina are thought to propagate among differentiated neurons in the inner retina (IR) and play an important role in activity-dependent visual development. Here we report a new form of rhythmic Ca(2+) wave in the ventricular zone (VZ) of the developing rabbit retina. Ca(2+) imaging from two-photon optical sections near the ventricular surface of the whole-mount retina showed rhythmic Ca(2+) transients propagating laterally as waves. The VZ waves had a distinctively slow Ca(2+) dynamics (lasting approximately 20 s) but shared a similar frequency and propagation speed with the IR waves. Simultaneous Ca(2+) imaging in VZ and multi-electrode array recording in the ganglion cell layer (GCL) revealed close spatiotemporal correlation between spontaneous VZ and IR waves, suggesting a common source of initiation and/or regulation of the two waves. Pharmacological studies further showed that all drugs that blocked IR waves also blocked VZ waves. However, the muscarinic antagonist atropine selectively blocked VZ but not IR waves at this developmental stage, indicating that IR waves were not dependent on VZ waves, but VZ waves likely relied on the initiation of IR waves. Eliciting IR waves with puffs of nicotinic or non-N-methyl-d-aspartate agonists in GCL produced atropine-sensitive waves in the VZ, demonstrating a unique, retrograde signaling pathway from IR to VZ. Thus differentiated neurons in the IR use spontaneous, rhythmic waves to send both forward signals to the central visual targets and retrograde messages to the developing cells in the VZ.

Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia.

Calsequestrin is a high-capacity Ca-binding protein expressed inside the sarcoplasmic reticulum (SR), an intracellular Ca release and storage organelle in muscle. Mutations in the cardiac calsequestrin gene (CSQ2) have been linked to arrhythmias and sudden death. We have used Ca-imaging and patch-clamp methods in combination with adenoviral gene transfer strategies to explore the function of CSQ2 in adult rat heart cells. By increasing or decreasing CSQ2 levels, we showed that CSQ2 not only determines the Ca storage capacity of the SR but also positively controls the amount of Ca released from this organelle during excitation-contraction coupling. CSQ2 controls Ca release by prolonging the duration of Ca fluxes through the SR Ca-release sites. In addition, the dynamics of functional restitution of Ca-release sites after Ca discharge were prolonged when CSQ2 levels were elevated and accelerated in the presence of lowered CSQ2 protein levels. Furthermore, profound disturbances in rhythmic Ca transients in myocytes undergoing periodic electrical stimulation were observed when CSQ2 levels were reduced. We conclude that CSQ2 is a key determinant of the functional size and stability of SR Ca stores in cardiac muscle. CSQ2 appears to exert its effects by influencing the local luminal Ca concentration-dependent gating of the Ca-release channels and by acting as both a reservoir and a sink for Ca in SR. The abnormal restitution of Ca-release channels in the presence of reduced CSQ2 levels provides a plausible explanation for ventricular arrhythmia associated with mutations of CSQ2.

Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos.

Through the injection of f-aequorin and the use of a photon imaging microscope, we have previously reported that a rhythmic series of intercellular Ca2+ waves circumnavigate zebrafish embryos over a 10 h period during gastrulation and axial segmentation. These waves first appear at about 65% epiboly and continue to arise every 5-10 min up to at least the 16-somite stage. In response to our publication, it was suggested that the waves may be an artefact caused by dechorionation of the embryos and would not be observed during the development of intact embryos (i.e. those with chorions). Here we demonstrate (again initially by aequorin imaging) that the rhythmic intercellular Ca2+ waves that traverse the blastoderm margin can also be observed in embryos that have an intact chorion. In addition, the appearance time, propagation pathway, velocity, duration and Ca2+ rise of the waves, as well as the interwave interval and the timing of wave onset, are approximately the same in both dechorionated embryos and those with an intact chorion. Furthermore, by loading intact embryos with Ca(2+)-green dextran at the single-cell stage and then using scanning confocal microscopy to obtain high-resolution images, we confirm the presence of circumferential Ca2+ waves and show that they pass through a population of deep cells located at the blastoderm margin. The confirmation of these pan-embryonic Ca2+ waves in zebrafish further corroborates our earlier suggestion that such waves might play a fundamental role in normal embryonic patterning during the gastrula period.

Simultaneous imaging of Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity in mouse ileum

Electrical rhythmicity in smooth muscle cells is essential for the movement of the gastrointestinal tract. Interstitial cells of Cajal (ICC) lie adjacent to smooth muscle layers and are implicated as the pacemaker cells. However, the pace making mechanism remains unclear. To study the intercellular interaction during electrical rhythm generation, we visualized changes in intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle cells and myenteric ICC within segments of mouse ileum loaded with a fluorescent Ca2+ indicator, fluo-3. We observed rhythmic [Ca2+]i changes in longitudinal smooth muscle cells travelling rapidly through the smooth muscle cell layer. Between the rhythmic Ca2+ transients, we found brief Ca2+ transients localized to small areas within smooth muscle cells. The amplitude but not the periodicity of rhythmic [Ca2+]i transients in both cell types was partially inhibited by nicardipine, an L-type Ca2+ channel antagonist, suggesting that the rhythmic [Ca2+]i transients reflect membrane potential depolarizations corresponding to both slow waves and triggered Ca2+ spikes. Longitudinal smooth muscle cells and myenteric ICC showed synchronous spontaneous [Ca2+]i transients in eight out of 21 ileac preparations analysed. In the remaining preparations, the synchrony between ICC and smooth muscle cells was absent, although the rhythmicity of the smooth muscle cells was not disturbed. These results suggest that myenteric ICC may play multiple roles including pace making for physiological bowel movement.

Ca2+entry into primary cultured pig coronary smooth muscle cells after previous store depletion by repetitive P2Y purinoceptor stimulation

Store-operated Ca2+entry, stimulated by depletion of intracellular Ca2+pools, has not been fully elucidated in vascular smooth muscle cells of pig coronary arteries. Therefore, [Ca2+]iwas measured in cultured cells derived from extramural pig coronary arteries using the Fura-2/AM fluorometry. Divalent cation entry was visualized with the Fura-2 Mn2+-quenching technique. Ca2+stores were depleted either by repetitive stimulation of P2Y purinoceptors with ATP (10μmol/L), or by the sarcoendoplasmic Ca2+-ATPase inhibitor 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ; 1μmol/L) in Ca2+-free medium (EGTA 1mmol/L). Addition of Ca2+(1mmol/L) induced refilling of ATP-sensitive Ca2+stores and an increase in [Ca2+]iin the presence of BHQ. Both could be significantly diminished by Ni2+(5 and 1mmol/L), La3+(10μmol/L), Gd3+(10μmol/L), and Mg2+(5.1mmol/L). In contrast to the BHQ-mediated rise in [Ca2+]i, refilling of ATP-depleted stores was affected by neither flufenamate (0.1mmol/L), nor by nitrendipine, nifedipine, and nisoldipine (each 1μmol/L). The data suggest that after store depletion in pig coronary smooth muscle cells ATP and BHQ may converge on a common, Ni2+−, La3+−, Gd3+−, and Mg2+− sensitive Ca2+entry pathway, i.e. on a store-operated Ca2+entry. An additional contribution of the Na+/Ca2+exchanger cannot be excluded. Flufenamate-sensitive non-selective cation channels and dihydropyridine-sensitive L-type Ca2+channels are not involved in refilling of Ca2+stores after previous depletion by repetitive P2Y purinoceptor stimulation. The store-operated Ca2+entry in-between repetitive purinoceptor stimulation, i.e. in the absence of the agonist, may be responsible for the maintenance of agonist-induced rhythmic Ca2+responses.

Mechanisms of respiratory rhythm generation in vitro. I. Pacemaker neurons and networks in the pre-Bötzinger complex (pre-BötC)

We have proposed that the inspiratory rhythm-generating kernel in the pre-BotC consists of a heterogeneous network of glutamatergic neurons with voltage-dependent pacemaker-like bursting properties. This model is based on our optical imaging [1], electrophysiological [2], and mathematical modeling [3] studies of inspiratory (I) neurons within the pre-BotC of rhythmically active in vitro transverse slice preparations from neonatal rats. For electrophysiological and imaging studies, we have developed novel methods to optically identify I neurons with Ca2+-sensitive dyes combined with IR-DIC imaging for whole-cell current camp (CC) and voltage clamp (VC) analyses of neuronal membrane conductance and synaptic mechanisms. In computational modeling studies we have developed models of single pacemaker neurons and synaptically-coupled networks of these neurons (50–500 cells) with heterogeneous distributions of cellular/network parameters. Model predictions have been tested experimentally from single-cell electrophysiological measurements and from macroscopic recordings of neuron population activity within the pre-BotC [2]. Electrophysiological and optical measurements demonstrate a sub-population of pre-BotC I neurons that exhibit voltage-dependent rhythmic bursting under CC after blockade of non-NMDA gluta-matergic synaptic transmission or after blocking synaptic transmission with Ca2+ channel blockers. The intrinsic bursting frequency of these pacemaker-type cells was a monotonic function of baseline membrane potential, spanning a frequency range of over an order of magnitude (~0.05–1 Hz); the voltage-dependence and frequency range varied for different cells indicating heterogeneity. Under VC with synaptic transmission intact, these pacemaker neurons exhibited glutamatergic synaptic drive currents. Optical imaging and cross-correlation of rhythmic Ca2+ activities of multiple cells demonstrate that the glutamatergic synaptic interactions synchronize bursting within the heterogeneous population, but with a temporal dispersion in neuronal spiking including pre-I spiking patterns [2,3]. Measurements of pre-BotC population activity show network frequency control by changing pre-BotC excitability, like predictions from our network models [3]. We have obtained evidence that a persistent Na+ current (INaP) is the main subthreshold-activating cationic conductance underlying the voltage-dependent pacemaker bursting. We measured Na+ currents in bursting pacemaker and non-bursting pre-BotC I cells. In all cells tested under VC, voltage ramp commands at rates of <100 mV/s inactivated the fast Na+ current and yielded N-shaped IV curves with a negative slope region between -60 and -35 mV. TTX (1 μM) blocked this inward current. The slowest ramp speed tested (3.3 mV/s) failed to fully inactivate the negative slope generating current. The current is therefore a TTX-sensitive INaP. The peak amplitude of INaP ranged from -50 to -100 pA at Em = -20 mV (peak conductance = ~1–2 nS). Boltzmann plots gave half-maximal activation voltage of ~-40 mV and a slope factor of ~5, very similar to values used for INaP in our pacemaker neuron models [3]. In non-pacemaker Icells, the ratio of INaP/Ileak was insufficient to support intrinsic bursting as predicted by our models. Further tests of the role of INaP have been performed using the dynamic clamp to artificially add INaP to non-pacemaker neurons. INaP transformed these neurons into rhythmic bursters with voltage-dependent behavior quantitatively similar to our models and experimental observations. These results generally support our conceptual and computational models for the rhythm-generation kernel as a heterogeneous pacemaker network.

Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria

Pacemaker cells, known as interstitial cells of Cajal (ICC), generate electrical rhythmicity in the gastrointestinal tract. Pacemaker currents in ICC result from the activation of a voltage-independent, non-selective cation conductance, but the timing mechanism responsible for periodic activation of the pacemaker current is unknown. Previous studies suggest that pacemaking in ICC is dependent upon metabolic activity 1y1yand1 Ca2+ release from intracellular stores. We tested the hypothesis that mitochondrial Ca2+ handling may underlie the dependence of gastrointestinal pacemaking on oxidative metabolism. Pacemaker currents occurred spontaneously in cultured ICC and were associated with mitochondrial Ca2+ transients. Inhibition of the electrochemical gradient across the inner mitochondrial membrane blocked Ca2+ uptake and pacemaker currents in cultured ICC and blocked slow wave activity in intact gastrointestinal muscles from mouse, dog and guinea-pig. Pacemaker currents and rhythmic mitochondrial Ca2+ uptake in ICC were also blocked by inhibitors of IP3-dependent release of Ca2+ from the endoplasmic reticulum and by inhibitors of endoplasmic reticulum Ca2+ reuptake. Our data suggest that integrated Ca2+ handling by endoplasmic reticulum and mitochondria is a prerequisite of electrical pacemaking in the gastrointestinal tract.

Patch-Clamp-Induced Perturbations of [Ca2+]i Activity in Somatotropes

Somatotropes and GC cells, a GH-producing cell line, exhibit [Ca²⁺]_i oscillations that result from rhythmic Ca²⁺ action potentials. Determination of this operating mode required simultaneous recording of both parameters by fura-2 imaging and patch-clamp techniques. In order to test whether patch recording induces artificial alteration of the [Ca²⁺]_i oscillatory pattern, we recorded separately or simultaneously [Ca²⁺]_i and membrane potential. In the absence of any other stimulation, seal formation in patch-clamp recording evoked by itself a 2.5- to 4-fold persistent increase in basal [Ca²⁺]_i, speeded up their frequency (from 0.03–0.17 to 0.4 Hz) and changed their pattern to a tonic mode. Patch-induced [Ca²⁺]_i increase was reproduced by mechanical contact between the pipette and the membrane. It was reduced by nifedipine, a blocker of L-type Ca²⁺ channels, as well as by removal of external Na⁺. It was fully blocked by external Ca²⁺ removal or gadolinium. All patch-clamp-induced perturbations were reversed by membrane hyperpolarization. We propose that patch-clamp recording evokes Ca²⁺ entry through L-type Ca²⁺ channels either directly, or indirectly via membrane depolarization. This shows that patch recordings in endocrine cells showing mechanosensitivity have to be interpreted with caution, and explains why long-lasting patch recordings are so difficult to obtain.

Role of Voltage-Dependent Na + Channels for Rhythmic Ca 2+ Signalling in Glucose-Stimulated Mouse Pancreatic β-Cells

The putative role of voltage-dependent Na + channels for glucose induction of rhythmic Ca 2+ signalling was studied in mouse pancreatic β-cells with the use of the Ca 2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca 2+ concentration ([Ca 2+] i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na + channel blocker tetrodotoxin. During exposure to 1–10 μM veratridine, which facilitates the opening of voltage-dependent Na + channels, the slow oscillations were replaced by repetitive and pronounced [Ca 2+] i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca 2+] i transients were critically dependent on the influx of Ca 2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca 2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca 2+] i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na + channels is a useful approach for amplifying Ca 2+ signals for insulin release.

Oxytocin-induced Ca2+ responses in human myometrial cells.

Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of </= 6 mHz in cells was from the inositol 1,4,5-trisphosphate-sensitive pool, accounting for about 60% of the frequencies. Most of the remaining frequencies were attributable to extracellular Ca2+, which presumably comes from plasma membrane channels other than L-type channels. When oscillation frequencies exceeded 6 mHz, a significant contribution from a ryanodine-sensitive Ca2+ pool was detected. Eight-bromo-cAMP suppressed both the initial Ca2+ spike and the long-term oscillations. Prostaglandin E1 and E2 caused a significant increase in the frequency of oxytocin-induced Ca2+ oscillations. FFT analysis may be of considerable value for study of the mechanisms of rhythmic Ca2+ transients and their function in myometrial cells, as well as the mechanisms by which uterotonins and tocolytic agents impact myometrial Ca2+ regulation.

Rhythmic CA 2+ activity of rat pituitary somatotropes: A comparison between normal and tumoral cells

Much physiological evidence is available to show that acetylcholine (ACh) hyperpolarizes the outer hair cells (OHCs) of guinea pig cochlea and induces Ca2+-activated K+ currents. In this study, using the nystatin perforated patch-clamp technique, we investigated the effects of various K+ channel blockers on the ACh-induced currents (IACh) in dissociated OHCs of guinea pig cochlea. The IACh were inhibited by apamin in a concentration-dependent manner. The half-maximal inhibitory concentration for apamin on the ACh-induced response was 1.59×10−9 M. Charybdotoxin and iberiotoxin had no inhibitory effect on the IACh. The inhibitory potency of the various K+ channel blockers on the IACh was as follows: apamin>>quinine≈quinidine≈d-tubocurarine>tetraethylammonium chloride>4-aminopyridine≈Ba2+>Cs2+. It is proposed that the Ca2+-activated K+ channels of mammalian cochlear OHCs should be classified as small conductance Ca2+-activated K+ (SK) channels according to their pharmacological properties.

Calcium oscillations and calcium waves coordinate rhythmic contractile activity within the stellate cell layer of medaka fish embryos

AbstractDuring gastrulation and body axis formation in medaka (Oryzias latipes) fish embryos, rhythmic contraction waves sweep across a tissue sheet known as the periderm, which covers both the developing embryo and its associated yolk cell. Cope et al. ([1990] J. Exp. Zool., 254:270–275) have found that these contractions are generated by a layer of stellate‐shaped cells which is closely apposed to the inner surface of the periderm. In this paper, we show that rhythmic contractions in the stellate cell layer are correlated with elevations of intracellular [Ca2+]. Medaka periderms were first explanted and secured to microscope coverslips using a modified plasma clot culture technique. The explants were then loaded with the calcium indicator dye Fluo‐3AM and observed with time‐lapse epifluorescence confocal microscopy. Rises of intracellular [Ca2+] were observed to propagate from cell to cell in the stellate cell layer, in the form of intercellular waves. After a latency of a few seconds, a coordinated contraction within the stellate cell layer ensued. Rhythmic Ca spikes were also observed within individual stellate cells. In many cases, rises of intracellular [Ca2+] preceded cellular contraction in these individual cells by several seconds. The Ca oscillations and Ca waves that occur in medaka stellate cells represent one of the earliest expressions of Ca‐based excitability and contractility within the blastoderm of post‐fertilization vertebrate embryos. © 1995 Wiley‐Liss, Inc.

Active calcium transport in the skin of the frog Rana pipiens: kinetics and seasonal rhythms.

The frog Rana pipiens takes up Ca2+ against an electrochemical gradient from dilute external solutions that are similar to natural freshwater environments. The influx is dependent upon external [Ca2+] and is saturable. Kinetic analysis yielded a Km of 0.625 mmol l-1 and a Jmax of 38 nmol cm-2h-1. These kinetic variables suggest that both the affinity and capacity are smaller than those for Na+ and Cl- transport in the skin of the same species. They are also smaller than those for Ca2+ transport in fish gill. A significant portion (20-25%) of the Ca2+ entering a frog remains in Ca(2+)-rich layers of the skin, with ventral skin containing about three times as much Ca2+ as dorsal skin. There are seasonal rhythms in Ca2+ exchange: although Ca2+ influx does not vary significantly over the year, efflux is minimal in July, while net flux, which is negative most of the year, appears to be positive in July. Since these fluxes do not include dietary calcium, one cannot conclude that feeding frogs are in negative Ca2+ balance.

Bradykinin and muscarine induce Ca 2+-dependent oscillations of membrane potential in rat glioma cells indicating a rhythmic Ca 2+ release from internal stores: Thapsigargin and 2,5-Di( tert-butyl)-1, 4-benzohydroquinone deplete InsP 3-sensitive Ca 2+ stores in glioma and in neuroblastoma-glioma hybrid cells

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 n M) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-γ-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 n M to 2 μ M, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca 2+, the substances thapsigargin, 2,5-di( tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHQ, which are known to block the Ca 2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca 2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca 2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca 2+ response to bradykinin which is known to be due to release of Ca 2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca 2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP 3-sensitive Ca 2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to “quantal” Ca 2+ release from stores with different inositol 1,4,5-trisphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca 2+ seems to be due to a lack of replenishment of the intracellular stores with Ca 2+, which comes from the extracellular compartment.

Oscillations: a key event in transformed renal epithelial cells.

Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form "foci" in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.

Calcium homeostasis in growth hormone (GH)-secreting adenoma cells: effect of GH-releasing factor.

Human GH-secreting tumors are heterogenous regarding their basal secretory activity and response to GH-releasing factor (GRF). We have investigated whether such different secretory properties could be accounted for by alterations of intracellular mechanisms occurring at the calcium level. Basal free intracellular calcium concentrations ([Ca2+]i) and Ca2+ responses to GRF were studied in single cells cultured from fragments of five GH-secreting pituitary adenomas. We used the microspectrofluorimetric method and indo-1 as the fluorescent probe. The cell populations cultured from the tumors of patients A and C showed increased hormone secretion in response to GRF in vitro, whereas cultures from patients B, D, and E were unresponsive to the peptide. Basal [Ca2+]i measured in the five cell populations ranged from 82 +/- 18 to 118 +/- 27 nM. A 10-sec application of 10 nM GRF induced an increase in [Ca2+]i in 60% and 54% of A and C cells, respectively. In the nonresponsive cell populations, the number of calcium responses to GRF was lower, 26% (B cells), 5% (D cells), and 10% (E cells). Two principal responses types were observed: 1) an initial increase in [Ca2+]i, followed by a sustained plateau phase lasting for more than 200 sec; and 2) a monophasic peak of increased [Ca2+]i lasting approximately 1 min before returning to baseline levels. GRF responses were totally suppressed in the absence of Ca2+ ions in the external medium. Sixteen to 30% of the cells cultured from four of the five tumors showed spontaneous fluctuations of [Ca2+]i. These spontaneous Ca2+ transients were suppressed in Ca(2+)-free medium. The number of cells exhibiting such Ca2+ transients decreased with time in culture. Basal hormone secretion was higher in cultures from patient D, in which no spontaneous Ca2+ transients were observed in any of the 72 studied cells, and in cultures from patients E, in which only 16% of cells were spontaneously active. We conclude that 1) in human responsive somatotrophs, the involvement of Ca2+ in GRF stimulus-secretion coupling mechanisms is apparently similar to that described in somatotrophs of other species; 2) the lack of a secretory response to GRF observed in some tumors may result from impairment of Ca2+ responsiveness in either cell recruitment or response amplitude and/or duration; and 3) spontaneous rhythmic Ca2+ activity is apparently dissociated from basal hormone secretion in some of these tumor cells.

Ultraviolet light activates blocking actions of dantrolene on intracellular Ca2+ release in bullfrog sympathetic neurones.

Effects of dantrolene, a blocker of intracellular Ca2+ release, on the oscillation of the intracellular Ca2+ ([Ca2+]i) induced by caffeine were studied in bullfrog sympathetic ganglion cells, using a Fura-2 fluorescence technique. Dantrolene blocked the Ca2+ oscillation only in the cell illuminated by ultraviolet light (335-385 nm). Likewise, the blocking effects on rhythmic Ca(2+)-dependent hyperpolarizations, representing Ca2+ oscillations via activation of Ca(2+)-dependent K+ channel, occurred only under the illumination with ultraviolet light (335-385 nm), but not with visible light (404-417 nm). This wavelength dependence differs from the absorbance spectrum of dantrolene. On the other hand, dantrolene preirradiated with ultraviolet light under dark condition or ultraviolet light itself did not affect the [Ca2+]i oscillation. The blocking action was not prevented by the pretreatment of the cells with reducing agents. These results indicate that illumination of the Ca2+ release channel or dantrolene itself with ultraviolet light (possibly the former) is necessary for the drug to exert its blocking effect. Furthermore, dantrolene was found to decrease Fura-2 fluorescence and to increase cell autofluorescence, leading sometimes to a false decrease in the basal [Ca2+]i.

Ultraviolet light activates blocking actions of dantrolene on intracellular Ca 2+ release in bullfrog sympathetic neurones

Effects of dantrolene, a blocker of intracellular Ca2+ release, on the oscillation of the intracellular Ca2+ ([Ca2+]i) induced by caffeine were studied in bullfrog sympathetic ganglion cells, using a Fura-2 fluorescence technique. Dantrolene blocked the Ca2+ oscillation only in the cell illuminated by ultraviolet light (335-385 nm). Likewise, the blocking effects on rhythmic Ca(2+)-dependent hyperpolarizations, representing Ca2+ oscillations via activation of Ca(2+)-dependent K+ channel, occurred only under the illumination with ultraviolet light (335-385 nm), but not with visible light (404-417 nm). This wavelength dependence differs from the absorbance spectrum of dantrolene. On the other hand, dantrolene preirradiated with ultraviolet light under dark condition or ultraviolet light itself did not affect the [Ca2+]i oscillation. The blocking action was not prevented by the pretreatment of the cells with reducing agents. These results indicate that illumination of the Ca2+ release channel or dantrolene itself with ultraviolet light (possibly the former) is necessary for the drug to exert its blocking effect. Furthermore, dantrolene was found to decrease Fura-2 fluorescence and to increase cell autofluorescence, leading sometimes to a false decrease in the basal [Ca2+]i.