Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs.

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs

Basal and receptor-regulated changes in cytoplasmic calcium concentration ([Ca2+]i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1. Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal [Ca2+]i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers. Such random fluctuations in [Ca2+]i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion. The physiological agonist GnRH induced high amplitude [Ca2+]i oscillations; when a threshold [Ca2+]i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients. The time required to reach the threshold [Ca2+]i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration. The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature. At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations. The presence of spontaneous fluctuations in basal [Ca2+]i did not significantly change the patterns of agonist-induced [Ca2+]i responses. Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase. Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in [Ca2+]i. In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response. In about 10% of the cells, however, high thapsigargin concentrations induced coarse [Ca2+]i oscillations; subsequent stimulation of such cells with GnRH was ineffective. The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion. The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.     

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.     

Delphinidin induces antiproliferation and apoptosis of endometrial cells by regulating cytosolic calcium levels and mitochondrial membrane potential depolarization

Endometriosis is a benign gynecological disease of women of reproductive ages, wherein endometrial cells grow ectopically, decreasing their quality of life due to chronic pelvic pain and severe dysmenorrhea. Although surgery and hormone therapies are gold standards for treating endometriosis, side effects are common and the recurrence rate is nearly 50%. Recent studies are exploring phytochemicals as pharmacological adjuvants for treating endometriosis. Delphinidin is an anthocyanin with anti-inflammatory, antioxidative, and anticancerous properties. In this study, delphinidin showed antiproliferative and apoptotic effects on human endometrial cells. Additionally, treatment with delphinidin decreased the mitochondrial membrane potential and increased cytosolic calcium levels in VK2/E6E7 and End1/E6E7 cells. Delphinidin decreased the phosphorylation of proliferative signaling molecules, including ERK1/2, AKT, P70S6K, and S6, while increasing the phosphorylation of P38 MAPK and P90RSK. These results imply that delphinidin is a novel therapeutic agent for treating and managing endometriosis, and has fewer side effects.     

Activin A increases cytosolic free calcium concentration in rat pituitary somatotropes.

The effect of activin A on the cytosolic free calcium concentration ([Ca2+]i) in normal rat pituitary cells was examined using a calcium sensitive fluorescent dye, indo 1 AM, and a digital imaging fluorescent microscope system. The cells showing an increase in [Ca2+]i in response to activin A were then characterized by comparison with cells responding to growth hormone releasing hormone (GRH), thyrotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), and gonadotropin releasing hormone (GnRH) in monolayer cultures of normal rat pituitary cells. Activin A increased [Ca2+]i in some cells in a mixed population of normal rat pituitary cells. The cells that responded to activin A also responded to GRH. Most of these cells were not affected by other tropic hormones (CRH, TRH, and GnRH), but a few cells responded to both GRH and TRH. None of the activin A-responding cells responded to CRH or GnRH, and none of the CRH- or GnRH-responding cells responded to activin A. In a preparation of somatotropes purified 80-90% by fluorescence-activated cell sorting, activin A increased [Ca2+]i in 30% of the cells that shows a [Ca2+]i-response to GRH. These findings suggest direct involvement of somatotropes in activin A-induced biological events in the rat pituitary gland.     

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals

Cytosolic Ca²⁺ buffers bind to a large fraction of Ca²⁺ as it enters a cell, shaping Ca²⁺ signals both spatially and temporally. In this way, cytosolic Ca²⁺ buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca²⁺ entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca²⁺ buffers in controlling pituitary Ca²⁺ signaling is poorly understood. Here, cytosolic Ca²⁺ buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca²⁺ indicator fluo-8 revealed stepwise increases in free Ca²⁺ after a series of brief depolarizing pulses in rapid succession. These Ca²⁺ increments grew larger as free Ca²⁺ rose to saturate the cytosolic buffers and reduce the availability of Ca²⁺ binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a K_d of 1.5–4.7 µM, and approximately half contained an additional higher-affinity buffer with a K_d of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca²⁺ buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca²⁺ signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.     

Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.     

Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes.

Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media. The first sign of meiotic resumption is germinal vesicle breakdown (GVB). Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3. The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h. Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s. The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3. Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations. In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin. Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB. The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation. However, the spontaneous oscillations do not appear to trigger GVB. Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.     

Transient but not oscillating component of the calcium mobilizing response to gonadotropin-releasing hormone depends on calcium influx in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation.     

Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs.

Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.     

Maitotoxin induces a calcium-dependent membrane depolarization in GH4C1 pituitary cells via activation of type L voltage-dependent calcium channels.

Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.     

Agonist-induced cytosolic calcium oscillations originate from a specific locus in single hepatocytes

Digital imaging fluorescence microscopy of fura-2-loaded hepatocytes in primary culture has been used to examine the changes of cytosolic free Ca2+ ([Ca2+]i) in response to receptor activation by alpha 1-adrenergic agonists and vasopressin at the subcellular level. Agonist-induced Ca2+ oscillations did not occur synchronously within the cell but originated from a specific region adjacent to the cell membrane and then propagated throughout the rest of the cell, with each oscillation within a series originating from the same locus. Furthermore, hormones acting through different receptors produced Ca2+ waves with similar rates of progress (20-25 microns.s-1) which originated from the same subcellular locus. For a given cell, the rate of progress and amplitude of the Ca2+ waves were independent of applied agonist concentration and were unaffected by depletion of extracellular Ca2+. The kinetics of Ca2+ increase at different points within the cell indicated that the Ca2+ waves were not driven by diffusion but were characteristic of a self-propagating mechanism. Significantly, when cells were treated with A1F-4 to directly activate the G-protein which couples receptor occupancy to [Ca2+]i mobilization, the origin and kinetics of the Ca2+ waves were identical to those observed with hormonal stimulation. It is proposed that the spatial organization of the intracellular Ca2+ release mechanisms may have significance in the regulation of the asymmetric metabolic functions of hepatocytes and other functionally polarized cells.     

Regulation of L-type calcium channels in pituitary GH(4)C(1) cells by depolarization.

The neurosecretory anterior pituitary GH(4)C(1) cells exhibit the high voltage-activated dihydropyridine-sensitive L-type and the low voltage-activated T-type calcium currents. The activity of L-type calcium channels is tightly coupled to secretion of prolactin and other hormones in these cells. Depolarization induced by elevated extracellular K(+) reduces the dihydropyridine (+)-[(3)H]PN200-110 binding site density and (45)Ca(2+) uptake in these cells (). This study presents a functional analysis by electrophysiological techniques of short term regulation of L-type Ca(2+) channels in GH(4)C(1) cells by membrane depolarization. Depolarization of GH(4)C(1) cells by 50 mm K(+) rapidly reduced the barium currents through L-type calcium channels by approximately 70% and shifted the voltage dependence of activation by 10 mV to more depolarized potentials. Down-regulation depended on the strength of the depolarizing stimuli and was reversible. The currents recovered to near control levels on repolarization. Down-regulation of the calcium channel currents was calcium-dependent but may not have been due to excessive accumulation of intracellular calcium. Membrane depolarization by voltage clamping and by veratridine also produced a down-regulation of calcium channel currents. The down-regulation of the currents had an autocrine component. This study reveals a calcium-dependent down-regulation of the L-type calcium channel currents by depolarization.     

Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.     

Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes

Changes of cytosolic free Ca2+ [( Ca2+]i) in response to receptor activation were studied at the single cell level by using digital imaging fluorescence microscopy of fura-2-loaded primary cultured hepatocytes. In response to phenylephrine and vasopressin, individual hepatocytes displayed dose-dependent oscillations of [Ca2+]i similar to those observed in aequorin-injected hepatocytes by Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 329, 719-721). With increasing agonist concentration, the frequency of oscillations increased and the latent period decreased. For a given cell, peak [Ca2+]i was independent of applied agonist concentration. However, there was considerable variation from cell to cell in the absolute value of peak [Ca2+]i. There was also marked intercellular heterogeneity in the latency, frequency, and overall pattern of the Ca2+ responses. Such asynchronous responses can be explained in part by the apparent differential agonist sensitivity of individual cells for latency and frequency. At high doses, phenylephrine maintained an oscillatory pattern, whereas vasopressin produced a complex mixture of spiking and sustained [Ca2+]i responses. Vasopressin and phenylephrine also displayed differently shaped [Ca2+]i oscillations at submaximal doses, due primarily to a slower rate of decay with vasopressin. Despite the large cell-cell variation in the patterns of [Ca2+]i oscillations, successive readditions of the same agonist elicited identical cell-specific patterns of oscillation. In the absence of extracellular Ca2+ the frequency but not the magnitude of [Ca2+]i oscillations was decreased. Buffering of [Ca2+]i by increasing the fura-2 load of single hepatocytes also decreased the frequency of oscillations without affecting the peak Ca2+ level. These data provide further support for the importance of frequency modulation in agonist-induced Ca2+ responses and suggest that Ca2+ itself plays an important role in regulating the frequency of [Ca2+]i oscillations. Furthermore, the data demonstrate a broad heterogeneity in hepatocyte [Ca2+]i oscillations which may underlie the nonoscillatory responses of cell populations.     

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.