Dynamic Video Imaging Research Articles

Contribution of Motion Information to Maternal Face Discrimination in Infancy

The contribution of motion and feature invariant information in infants' discrimination of maternal versus female stranger faces was assessed. Using an infant controlled habituation–dishabituation procedure, 4‐ and 8‐month‐old infants (N = 62) were tested for their ability to discriminate between their mother and a female stranger in 4 different conditions varying whether motion or feature information about the faces was available. The faces were presented in a still or dynamic video image with either a positive or a negative contrast. In each condition, infants habituated to a stranger's face and then viewed, in 3 pairs of alternating novelty test trials, either a new stranger or their mother's face. Results show that motion information contributes to the 8‐month‐old infants', but not the 4‐month‐old infants' discrimination of maternal faces. These results are interpreted in relation to recent findings and models in the adult literature suggesting that there is an enhanced contribution of dynamic information in face recognition when the face is familiar. Our data confirm that from the outset, there is a complex interplay of feature and motion information in the discrimination of the mother's face when the viewing condition is not optimal.

The extracellular calcium-sensing receptor increases the number of calcium steps and action currents in pituitary melanotrope cells

Secretion of α-melanophore-stimulating hormone (α-MSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland of the clawed frog Xenopus laevis is regulated by various inhibitory, stimulatory and autocrine factors. The neuropeptide sauvagine stimulates α-MSH secretion by changing the pattern of intracellular Ca 2+ oscillations and the electrical properties of the cell membrane. In the present study we investigated whether another secreto-stimulator, the extracellular Ca 2+-sensing receptor (CaR), also affects the Ca 2+ oscillatory pattern and electrical membrane properties. Using high-speed dynamic video-imaging we show that activation of the CaR with the specific agonist l-phenylalanine ( l-Phe) changes the Ca 2+ oscillatory pattern by increasing the number of Ca 2+ steps, which are the “building blocks” of the oscillations. Moreover, using patch-clamp electrophysiology it is demonstrated that l-Phe affects membrane properties by increasing frequency and duration of action currents. Compared to sauvagine, the CaR has different effects on the action current parameters, suggesting that multiple mechanisms regulate the electrical properties of the melanotrope cell membrane and, thereby, the Ca 2+ oscillation-dependent level of α-MSH secretion.

Evaluation of Shoulder Integrity in Space: First Report of Musculoskeletal US on the International Space Station

Investigative procedures were approved by Henry Ford Human Investigation Committee and NASA Johnson Space Center Committee for Protection of Human Subjects. Informed consent was obtained. Authors evaluated ability of nonphysician crewmember to obtain diagnostic-quality musculoskeletal ultrasonographic (US) data of the shoulder by following a just-in-time training algorithm and using real-time remote guidance aboard the International Space Station (ISS). ISS Expedition-9 crewmembers attended a 2.5-hour didactic and hands-on US training session 4 months before launch. Aboard the ISS, they completed a 1-hour computer-based Onboard Proficiency Enhancement program 7 days before examination. Crewmembers did not receive specific training in shoulder anatomy or shoulder US techniques. Evaluation of astronaut shoulder integrity was done by using a Human Research Facility US system. Crew used special positioning techniques for subject and operator to facilitate US in microgravity environment. Common anatomic reference points aided initial probe placement. Real-time US video of shoulder was transmitted to remote experienced sonologists in Telescience Center at Johnson Space Center. Probe manipulation and equipment adjustments were guided with verbal commands from remote sonologists to astronaut operators to complete rotator cuff evaluation. Comprehensive US of crewmember's shoulder included transverse and longitudinal images of biceps and supraspinatus tendons and articular cartilage surface. Total examination time required to guide astronaut operator to acquire necessary images was approximately 15 minutes. Multiple arm and probe positions were used to acquire dynamic video images that were of excellent quality to allow evaluation of shoulder integrity. Postsession download and analysis of high-fidelity US images collected onboard demonstrated additional anatomic detail that could be used to exclude subtle injury. Musculoskeletal US can be performed in space by minimally trained operators by using remote guidance. This technique can be used to evaluate shoulder integrity in symptomatic crewmembers after strenuous extravehicular activities or to monitor microgravity-associated changes in musculoskeletal anatomy. Just-in-time training, combined with remote experienced physician guidance, may provide a useful approach to complex medical tasks performed by nonexperienced personnel in a variety of remote settings, including current and future space programs.

Inhibition of Mitochondrial Na+-Ca2+ Exchange Restores Agonist-induced ATP Production and Ca2+ Handling in Human Complex I Deficiency

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.

The microscopic visualisation of the sonocrystallisation of ice using a novel ultrasonic cold stage

This work reports dynamic video images of the influence of ultrasonic cavitation on the sonocrystallisation of ice at a microscopic level. This has been achieved through the construction of a unique ultrasonic system for an optical microscope. The system consists of (1) an ultrasonic cold stage, (2) a temperature control system, and (3) a microscope and imaging setup. This allows the temperature of a sample to be systematically controlled while it is subjected to simultaneous excitation with alternating pressures in the ultrasonic frequency range. Both the amplitude of excitation and the frequency can be varied. Experiments on ice crystals in pure water and sucrose solutions were conducted. Three distinct phenomena were observed. Firstly, there is a tendency for cavitation bubbles to form at the grain boundaries between ice crystals. Secondly, there is a progressive melting of ice by cavitation bubbles which appear to eat their way into the ice phase. Thirdly, the dendritic ice structures may fragment under the influence of ultrasound, thus increasing the number of nuclei which may subsequently grow (secondary nucleation). These observations form the basis of a significantly enhanced understanding and exploitation of the sonocrystallisation of ice.

Computer Simulated 3D Virtual Reality for Dynamical Modeling and Calculations of Carbon-Based Composite Materials

AbstractThe principal goal of the present paper is to demonstrate an application of modern software engineering tools for modeling virtual reality and molecular dynamics of novel nanocomposites. The main technical components of presented system are 1) software and nanoengineering tools for modeling of virtual reality, molecular dynamic and 3D video images of novel diamonds- containing nanocomposites and 2) Problem tracking system to be used during modeling of virtual reality. For a realistic simulation of the stability behavior of the reinforced material, the nonlinear intramolecular inter-actions between neighboring atoms have to be taken into account. A comparison shows the buckling sensitivity of different geometries. In order to reduce computational costs, it is necessary to develop suited homogenization techniques, so that shell elements can be applied.

THE EFFECT OF HeNe LASER (633 nm) RADIATION ON INTRACELLULAR CA2+ CONCENTRATION IN FIBROBLASTS

The increasing use of phototherapy (for example in wound healing) has led to a great interest in the mechanism of light-biosystem interaction. Therefore, recent observations on the enhancement of the proliferation of irradiated skin cells (fibroblasts and keratinocytes) by visible light are important to understand phototherapy at the cellular level. In the present communication we show that at the doses appropriate for enhanced fibroblast proliferation, HeNe irradiation produces transiently increasing calcium concentration in the target cells. Fibroblasts were irradiated with 1, 3 and 5 J/cm2HeNe (633 nm) laser and intracellular Ca2+ concentration ([Ca2+ ]i) in single cells was analyzed by Fluo-3/AM using a dynamic video imaging system with frame-grabbing soft ware. Such observations can explain photobiostimulation because of the well-known vital role played by calcium in important biological processes such as proliferation and exocytosis.

The Cysteinyl-Leukotriene D4 Induces Cytosolic Ca2+ Elevation and Contraction of the Human Detrusor Muscle

The role of intracellular Ca2+ in the activation of human detrusor smooth muscle cells (SMCs) is pivotal. Recently we showed that the mast cell derived pro-inflammatory mediator leukotriene D(4) (LTD(4)) induces increase in intracellular free Ca2+ ([Ca2+](i)) in human detrusor myocytes. In the current study we examined the mechanisms underlying LTD(4) induced increase in [Ca2+](i) and tested whether LTD(4) induces muscle contraction by measuring force development in human detrusor tissue. Cultures of human detrusor SMCs were obtained from patients with benign bladder diseases undergoing cystoscopy. [Ca2+](i) was measured in fura-2 loaded SMCs using micro-spectrofluorometry and dynamic video imaging. Contractile force was monitored with an especially built mini-myograph. Spontaneous oscillations in [Ca2+](i) and force were observed. In the absence of calcium these oscillations were absent. LTD(4) caused a concentration dependent increase in [Ca2+](i) and isometric force. Calcium was released exclusively from intracellular stores. Increases in [Ca2+](i) and force were inhibited in dose dependent fashion by the LTD(4) receptor antagonists montelukast and zafirlukast. Likewise, LTC(4) and LTE(4) induced an increase in [Ca2+](i) and contractile force in the rank order LTD(4) >LTC(4) >LTE(4). Inhibition of Ca2+ induced Ca2+ release (CICR) with thapsigargin and ryanodine suggested the presence of a functional CICR in SMCs. To our knowledge this study demonstrates for the first time that the cysteinyl-leukotriene LTD(4) induces contraction in human detrusor SMCs. LTD(4) induced force and increased [Ca2+](i) were entirely dependent on Ca2+ release from intracellular stores. The action of LTD(4) on force development and increased [Ca2+](i) appeared to be specific, mediated by the binding and activation of specific LTD(4) receptors on SMCs. Also, to our knowledge this report is the first to show that human detrusor SMCs are sensitive to ryanodine, consistent with the hypothesis that a CICR is present and functional in these cells. The presence and role of endogenous cysteinyl leukotrienes for normal contractile functioning of the human detrusor during inflammation remains to be elucidated.

Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca 2+-dependent production of NO, without modifying the [Ca 2+] i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca 2+] i levels. The data demonstrate that Ca 2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.

Fluorescence Imaging of Changes in Intracellular Chloride in Living Brain Slices

In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl− tracer methods, Cl−-selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noinvasive technique that uses the fluorescent Cl− ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl− by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl−-sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl− concentrations can be calibrated from the Stern–Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl−, Cl− flux through the γ-aminobutyric acid (GABA)-gated Cl− channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl− quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl− can also be studied under various conditions such as oxygen/glucose deprivation (“in vitro ischemia”) and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl− flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl− and an increase in cell volume.

Inhibition of Intracellular Ca2+ Mobilisation by Low Antiproliferative Concentrations of Thapsigargin in Human Vascular Smooth-Muscle Cells

Low nanomolar concentrations of thapsigargin, a modulator of intracellular Ca2+ ([Ca2+]i) pools, inhibit vascular smooth-muscle cell (VSMC) proliferation. Because the mechanisms underlying this effect have not been defined, the effect of antiproliferative concentrations of thapsigargin on [Ca2+]i in fura-2-loaded VSMCs was studied by using dynamic video imaging of [Ca2+]i. After seeding on coverslips, human VSMCs were incubated for 1-48 h with thapsigargin before loading with fura-2 or during imaging. Mobilisation of [Ca2+]i was stimulated with 1 microM ionomycin in Ca2+-free medium and the increase in [Ca2+]i detected by using Ca2+ imaging techniques. Continuous exposure of cells to low concentrations of thapsigargin (which failed measurably to increase in [Ca2+]i) reduced the ionomycin response in a time- and dose-dependent manner (100% inhibition at 10 nM thapsigargin after 1 h exposure). After exposure of cells to 10 nM thapsigargin for 1 h followed by washing and further incubation for < or = 72 h, there was a time-dependent recovery of the ionomycin response. Because the concentrations of thapsigargin and exposure times are identical to those that inhibit replication in VSMCs, it is proposed that depletion of [Ca2+]i pools mediates the inhibitory effect of thapsigargin on VSMC proliferation.

Image Light Amplifier (ILA) Technology for Large-Screen Projection

The paper describes the configuration and performance of Image Light Amplifier (ILA <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">®</sup> ) projectors. The ILA projector combines the advantages of a CRT-type projector with the high brightness capability of a xenon arc lamp projection light source. The projector has no defined pixel structure such as that found on liquid crystal display (LCD) and digital mirror device (DMD) projectors. Thus, dynamic video images with continuous edges and a highly realistic image quality are projected. The ILA projector will directly accept all standard video and computer formats from PAL/NTSC at 15.625/15.750 kHz horizontal scan frequencies to work station formats at 90 kHz. The enabling technology of the projector is the Hughes-JVC ILA modulator, which accepts a low-intensity image from a cathode ray tube (CRT) and replicates the image on a high-intensity white xenon arc light beam. The ILA uses solid-state thin film and liquid crystal technology to create a compact, rugged, image light modulator. The ILA projector incorporates three ILA modulators for the RGB channels. Projectors with up to 12,000 lumens output, a contrast ratio of 1000:1, and a resolution of 2800 × 1500 TV lines are available. Two series of models, with one or three projection lenses, are available. Besides single-projector applications, such as electronic cinema, ILA projectors also are used in installations requiring high levels of raster shaping and the one-and two-dimensional seamless blending of multiple projector images.

How Do Monkeys Look at Faces?

Facial displays are an important form of social communication in nonhuman primates. Clues to the information conveyed by faces are the temporal and spatial characteristics of ocular viewing patterns to facial images. The present study compares viewing patterns of four rhesus monkeys (Macaca mulatta) to a set of 1- and 3-sec video segments of conspecific facial displays, which included open-mouth threat, lip-smack, yawn, fear-grimace, and neutral profile. Both static and dynamic video images were used. Static human faces displaying open-mouth threat, smile, and neutral gestures were also presented. Eye position was recorded with a surgically implanted eye-coil. The relative perceptual salience of the eyes, the midface, and the mouth across different expressive gestures was determined by analyzing the number of eye movements associated with each feature during static and dynamic presentations. The results indicate that motion does not significantly affect the viewing patterns to expressive facial displays, and when given a choice, monkeys spend a relatively large amount of time inspecting the face, especially the eyes, as opposed to areas surrounding the face. The expressive nature of the facial display also affected viewing patterns in that threatening and fear-related displays evoked a pattern of viewing that differed from that recorded during the presentation of submissive-related facial displays. From these results we conclude that (1) the most important determinant of the visual inspection patterns of faces is the constellation of physiognomic features and their configuration, but not facial motion, (2) the eyes are generally the most salient facial feature, and (3) the agonistic or affiliative dimension of an expressive facial display can be delineated on the basis of viewing patterns.

Ca2+ entry in gonadotrophs and αT3–1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+]i in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca(2+)-containing medium, GnRH caused a biphasic increase in [Ca2+]i whereas in Ca(2+)-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca(2+)-free medium was reduced by > 95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca(2+)-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca(2+)-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+]i in Ca(2+)-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca(2+)-containing medium because addition of ionomycin and Ca(2+)-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+]i rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca(2+)-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca(2+)-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+]i seen on return to Ca(2+)-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca(2+)-containing medium. Return of GnRH-treated cells to Ca(2+)-containing medium caused an increase in [Ca2+]i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca(2+)-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca(2+)-free medium did not enhance LH release on return to Ca(2+)-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells.

This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.

Pituitary ATP receptors: characterization and functional localization to gonadotropes.

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

Pituitary ATP receptors: characterization and functional localization to gonadotropes

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

Changes in intracellular calcium concentration in bovine oocytes following penetration by spermatozoa

The pattern of changes in intracellular calcium concentration ([Ca2+]i) in bovine oocytes after penetration by spermatozoa was determined. Dynamic video imaging, using Fura-2 as a probe for intracellular free calcium, showed that activation of oocytes by spermatozoa induced multiple transient increases in [Ca2+]i with a spike interval of 24.2 +/- 7.3 min, and that the early transient increases were propagated throughout the oocytes in the form of a wave. Calcium transients at fertilization are involved in the induction of cortical granule exocytosis and the resultant block to polyspermy. The hypothesis that the inhibition of Ca2+ release from inositol trisphosphate-sensitive stores would inhibit exocytosis and increase polyspermy was tested by injecting oocytes before fertilization with heparin, a potent inhibitor of inositol trisphosphate-activated Ca2+ release. There was no significant difference after fertilization in either [Ca2+]i spikes or in polyspermy rates between control and experimental groups injected with low molecular mass heparin up to a final cytoplasmic concentration of 400 mumol l-1. We conclude that inositol trisphosphate-independent Ca2+ stores may be mobilized during the fertilization of bovine oocytes.

Des‐Arg9‐bradykinin‐induced increases in intracellular calcium ion concentration in single bovine tracheal smooth muscle cells

1. Dynamic video imaging was used to measure the des-Arg9-bradykinin-induced changes in the intracellular free calcium ion concentration ([Ca2+]i) of single bovine tracheal smooth muscle (BTSM) cells. 2. In the presence of extracellular calcium ions, des-Arg9-bradykinin (1 nM-10 microM) produced a concentration-dependent increase in the [Ca2+]i over basal levels yielding an EC50 value of 316 nM. The percentage of cells responding to each concentration of des-Arg9-bradykinin also increased in a concentration-dependent manner (from 9% to 100%). 3. The bradykinin B2 receptor antagonist, D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 microM), was without effect on the calcium response of the cells when added 2 min prior to des-Arg9-bradykinin (100 nM). However, the B1 receptor antagonist, des-Arg9Leu8-bradykinin (10 microM), completely abolished the des-Arg9-bradykinin-induced response. 4. Under calcium-free conditions, des-Arg9-bradykinin induced an increase in [Ca2+]i at concentrations of 1 microM and 10 microM. The response to 10 microM des-Arg9-bradykinin was reduced by preincubation of either D-Arg[Hyp3, Thi5,8,D-Phe7]-bradykinin (10 microM) or des-Arg9Leu8-bradykinin (10 microM). 5. We conclude that bradykinin B1 receptors are expressed by cultured BTSM cells and mediate the des-Arg9-bradykinin-induced influx of calcium ions at low agonist concentrations (< 1 microM). At higher concentrations, des-Arg9-bradykinin (1 microM and 10 microM) can stimulate both B1 and B2 receptors to effect intracellular calcium release under calcium-free conditions.

Activation of specific ATP receptors induces a rapid increase in intracellular calcium ions in rat hypothalamic neurons

We have used the real-time dynamic video imaging Fura-2 fluorescence to study the acute effects of external ATP on [Ca 2+] i in cultured rat hypothalamic neurons. The addition of ATP at μM concentrations, but not adenosine, AMP, ADP or GTP, produced a rapid, dose-dependent increase in cytosolic Ca 2+. The hydrolysis-resistant ATP analogues 3-thio-ATP and β,γ-imido-ATP produced a similar response but α,β-methylene ATP had much lower efficacy. The ATP response was inhibited by 10 μM nifedipine, abolished by 50 μM cadmium and by the absence of extracellular Ca 2+, but was unaffected by ryanodine or ω-conotoxin GVIA. The P 2-purinoceptor antagonist suramin reversibly and selectively inhibited the ATP response but had no effect on other neurotransmitter-induced Ca i 2+ responses. Antagonists to muscarinic, nicotinic, NMDA, non-NMDA, GABA, 5-HT and adenosine receptors had no effect on the ATP response. Thus the Ca 2+ response of hypothalamic neurons to ATP is mediated by specific suramin-sensitive ATP-receptors, activation of which is independent of ATP hydrolysis and results in an influx of extracellular Ca 2+ largely through high voltage-gated Ca 2+ channels. These findings support the assertion that ATP acts in the CNS as an excitatory neurotransmitter.