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R Nuccitelli

Publications and source records attributed to R Nuccitelli.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of tyrosine phosphorylation prevents thrombin-induced mitogenesis, but not intracellular free calcium release, in vascular smooth muscle cells.

alpha-Thrombin, a G-protein-coupled receptor agonist, is mitogenic for neonatal vascular smooth muscle (VSM) cells, but it also causes secretion of the tyrosine kinase-coupled receptor agonist platelet-derived growth factor (PDGF). In order to determine the role of growth factors with tyrosine kinase-coupled receptors in thrombin's mitogenic signal transduction cascade, the synergistic effect of basic fibroblast growth factor (bFGF) in this system was examined. While bFGF itself is a growth factor for VSM cells, it causes a 1.7-fold synergistic effect when added together with thrombin. Herbimycin A, a specific tyrosine kinase inhibitor, both decreases thrombin-induced mitogenesis by greater than 90% and abolishes tyrosine phosphorylation of phospholipase C (PLC)-gamma-1. The magnitude and time course of the increase in intracellular free calcium concentration in response to thrombin is comparable in both the presence and absence of herbimycin A. These results provide evidence that herbimycin A specifically inhibits PLC-gamma-1 tyrosine phosphorylation without affecting VSM cell viability or calcium release. Furthermore, tyrosine phosphorylation is a necessary step in thrombin's mitogenic signal transduction cascade, but it is not essential for thrombin-induced release of calcium from intracellular stores. These data suggest that a tyrosine kinase, possibly supplied by the bFGF receptor, plays an essential role in thrombin-induced mitogenesis.

Animals↗
PubMed1992

Endogenous ionic currents and DC electric fields in multicellular animal tissues.

Through the use of the non-invasive vibrating probe technique for detecting extracellular ionic currents developed in 1974 [Jaffe and Nuccitelli: J Cell Biol 63:614-628, 1974], embryonic currents have been detected in a wide range of animal systems (recently reviewed in [Nuccitelli, Noninvasive Techniques in Cell Biology. New York: Wiley-Liss, 1990, pp 273-310]. In four of these studies, the corresponding electric field has been measured within the animal tissue. Such measurements of internal electric fields are quite challenging because they involve the insertion of microelectrodes into the developing tissue along specific regions of current flow. This paper reviews the evidence for endogenous transembryonic currents and dc electric fields in animal systems and provides the range of values for such physiological fields. These data should provide a guide to the range of imposed electric field strengths that could influence normal biological functions in living organisms.

Animals↗
PubMed1992

Inositol lipid hydrolysis contributes to the Ca2+ wave in the activating egg of Xenopus laevis.

We have used fluorescence ratio-imaging of fura-2 in the activating egg of Xenopus laevis to study the wave of increased intracellular free Ca2+ concentration ([Ca2+]i) while monitoring that of cortical granule exocytosis. Naturally matured eggs were dejellied, injected with fura-2, and activated by the iontophoresis of 1-30 nCoul of inositol-1,4,5-trisphosphate which triggers an immediate increase in free [Ca2+]i at the injection site. The Ca2+ rise spreads throughout the egg, reaching the opposite side in 5-8 min, and is followed by elevation of the fertilization envelope about 20-30 sec behind the [Ca2+]i wave. [Ca2+]i returns to preactivation levels within about 20 min after activation. We further studied the role of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by microinjecting antibodies to PIP2 into the egg. PIP2 antibodies did not alter the propagation velocity of the wave but greatly reduced the amount of Ca2+ released in the egg cortex. These data suggest that PIP2 hydrolysis plays a role in the release of [Ca2+]i in the outer regions of the egg following activation.

Animals↗
PubMed1992

A computerized 2-dimensional vibrating probe for mapping extracellular current patterns.

We describe a computer-assisted 2-dimensional vibrating probe system for mapping endogenous electric current patterns in biological preparations. This system overcomes some of the main limitations of the original 1-dimensional vibrating probe design and adds several new capabilities. Two piezo-electric bender elements mounted perpendicularly are used to vibrate the probe in a circle by applying 2 sine waves (1 to each element) that are 90 degrees out-of-phase with each other. The circular rotation of the probe allows it to detect simultaneously the 2 orthogonal components of a current in the horizontal plane. The voltages measured by the probe are digitized and analyzed by a computer and are used to calculate a current vector. A graphical representation of the current vector is then superimposed on a video image of the experimental preparation. This probe system responds to known currents in the expected manner and exhibits a low inherent noise level. Also included in this paper are some preliminary measurements made with this instrument on neurulating Xenopus embryos and on transected larval sea lamprey (Petromyzon marinus) spinal cords.

Animals↗
PubMed1992

Development of functional calcium channels in cultured avian photoreceptors.

Vertebrate photoreceptors are unusual neurons in that they are capable of continuous calcium-mediated release of neurotransmitter (Trifonov, 1968; Hagins et al., 1970). In this study, we have examined the development and characteristics of calcium currents in chick cone cells placed in culture on embryonic day 8. Cone cells were identified by their lectin-binding properties, rhodopsin-like immunoreactivity, and the presence of an oil droplet. Using the whole-cell patch-clamp method, we have seen calcium currents in these cells after three days in culture, slightly before the appearance of synapses (Gleason & Wilson, 1989). Because cone calcium currents are blocked by cadmium and nifedipine but are enhanced by Bay K 8644, they most closely resemble L-type current (Nowycky et al., 1985). An unexpected feature of these currents is that their gating ranges varied widely between cells so that some cells showed the foot of their activation range at -70 mV and others as positive as -25 mV. Calcium imaging of fura-2 loaded cells was used to confirm the time course of calcium current development and describe the distribution of cytosolic calcium. As expected, depolarization of young cells failed to increase cytosolic calcium but in older cells an increase of threefold to fourfold was usually observed. Both at rest and during depolarization, most cone cells showed regional differences in internal calcium concentration. In the most mature cones, depolarization strongly elevated cytosolic calcium at the terminal end of the cell while producing a lesser change around the oil droplet and the ellipsoid region, suggesting that calcium channels are localized to the terminal.

Animals↗
PubMed1992

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres.

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.

Animals↗
PubMed1991

Neural crest cell galvanotaxis: new data and a novel approach to the analysis of both galvanotaxis and chemotaxis.

The galvanotaxis response of neural crest cells that had migrated out of the neural tube of a 56-hr-old quail embryo onto glass coverslips was observed using time-lapse video microscopy. These cells exhibit a track velocity of about 7 microns/min and actively translocate toward the negative pole of an imposed DC electric field. This nonrandom migration could be detected for fields as low as 7 mV/mm (0.4 mV/cell length). We find that this directional migration is independent of the speed of migration and have generated a rather simple mathematical equation that fits these data. We find that the number of cells that translocate at a given angle, phi, with respect to the field is given by the equation N(phi) = exp(a0 + a1cos phi), where a1 is linearly proportional to the electric field strength for fields less than 390 mV/mm with a constant of proportionality equal to KG, the galvanotaxis constant. We show that KG = (150 mV/mm)-1, and at this field strength the cellular response is approximately half maximal. This approach to cellular translocation data analysis is generalizable to other directed movements such as chemotaxis and allows the direct comparison of different types of directed movements This analysis requires that the response of every cell, rather than averages of cellular responses, is reported. Once an equation for N(phi) is derived, several characteristics of the cellular response can be determined. Specifically, we describe 1) the critical field strength (390 mV/mm) below which the cellular response exhibits a simple, linear dependence on field strength (for larger field strengths, an inhibitory constant can be used to fit the data, suggesting that larger field strengths influence a second cellular target that inhibits the first); and 2) the amount of information the cell must obtain in order to generate the observed asymmetry in the translocation distribution (for a field strength of 100 mV/mm, 0.3 bits of information is required).

Animals↗
PubMed1991

The effects of inositol trisphosphates and inositol tetrakisphosphate on Ca2+ release and Cl- current pattern in the Xenopus laevis oocyte.

We report that Ins(1,3,4,5)P4 releases calcium from intracellular stores of intact Xenopus laevis oocytes, as indicated by two different techniques, Ca2(+)-sensitive microelectrodes and a fura-2 imaging system. Ins(1,3,4,5)P4 releases only 20% as much Ca2+ as the same amount of Ins(1,4,5)P3. This effect is not due to the conversion of the injected Ins(1,3,4,5)P4 to Ins(1,4,5)P3, which is known to release Ca2+, because the amount of [3H]Ins(1,3,4,5)P4 that is converted to Ins(1,4,5)P3 is extremely small, as determined using HPLC. Examination of the different current patterns induced by Ins(1,4,5)P3 and Ins(1,3,4,5)P4, when injected into voltage-clamped oocytes, provided further evidence that the Ins(1,3,4,5)P4 was not being converted back to Ins(1,4,5)P3. We investigated the effects of four compounds, three inositol trisphosphates (Ins(1,4,5)P3, Ins(2,4,5)P3, and Ins(1,3,4)P3), and Ins(1,3,4,5)P4, on Cl- current conductance in order to examine (1) the possible role of Ins(1,3,4,5)P4 in cell activation and (2) the relationships between intracellular Ca2+ and the activation of Cl- currents. Immature stage VI Xenopus laevis oocytes were voltage-clamped and injected with Ins(1,4,5)P3, Ins(2,4,5)P3, and Ins(1,3,4)P3. Ins(1,4,5)P3 and Ins(2,4,5)P3 triggered Ca2(+)-dependent Cl- currents, but Ins(1,3,4)P3 did not trigger currents nor did it release intracellular Ca2+. Ins(2,4,5)P3 was fourfold less effective at inducing the immediate Cl- current pulse than Ins(1,4,5)P3. The Cl- current pattern was quite dependent on the amount of Ins(1,4,5)P3 injected into the oocyte. Low amounts of Ins(1,4,5)P3 triggered only an immediate single Cl- current pulse, whereas large amounts triggered the immediate single pulse, followed by a quiescent period, followed by oscillating Cl- currents. In contrast to the response of Ins(1,4,5)P3, injection of Ins(1,3,4,5)P4 triggered only oscillating Cl- currents whose magnitude, but not pattern, was dependent on the amount injected into the cell. The currents generated by Ins(1,3,4,5)P4 resemble the oscillating Cl- currents triggered by large amounts of Ins(1,4,5)P3 and Ins(2,4,5)P3. Ins(1,3,4,5)P4, unlike Ins(1,4,5)P3 and Ins(2,4,5)P3, rarely caused an immediate Cl- current pulse, but caused an immediate release of calcium. Therefore, we suggest that the oscillating currents are only indirectly dependent on calcium. These [Ca2+]i and conductance measurements suggest that both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 have roles in intracellular Ca2+ regulation.

Animals↗
PubMed1991

Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones.

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

The effect of myo-inositol 1,4,5-trisphosphorothioate on Cl- current pattern and intracellular Ca2+ in the Xenopus laevis oocyte.

Microinjection of myo-inositol 1,4,5-trisphosphate into voltage-clamped Xenopus laevis oocytes or the stimulation of the phosphatidylinositol cycle elicits a complex Ca2(+)-dependent Cl- current pattern. Microinjection of myo-inositol 1,3,4,5-tetrakisphosphate causes an immediate release of Ca2+, but elicits a different Cl- current pattern than myo-inositol 1,4,5-trisphosphate. We have studied the effects of myo-inositol 1,4,5-trisphosphorothioate, which can not be converted to myo-inositol 1,3,4,5-tetrakisphosphate. Myo-inositol 1,4,5-trisphosphorothioate caused an immediate release of intracellular Ca2+, as measured by fura-2 imaging. Myo-inositol 1,4,5-trisphosphorothioate generated a Cl- current pattern similar to myo-inositol 1,3,4,5-tetrakisphosphate, not myo-inositol 1,4,5-trisphosphate.

Animals↗
PubMed1990

Activation of protein kinase C triggers premature compaction in the four-cell stage mouse embryo.

During mouse preimplantation development, the cells of the mouse embryo undergo a progressive subcellular reorganization at compaction, which eventually results in the formation of two distinct cell types. We have investigated the effect that activators of the Ca2(+)-phospholipid-dependent protein kinase (PKC) have on mouse compaction. Phorbol ester activation of PKC caused premature compaction of four-cell embryos within a few minutes of addition followed by a prolonged decompaction phase after 1 hr. This response was dose-dependent to concentrations as low as 250 pg/ml. Diacylglycerides also caused compaction; however, it was more sustained than with phorbol esters and was not followed by a phase of decompaction. Inhibition of PKC with sphingosine blocks induced compaction in a dose-dependent manner and also blocks normal compaction of eight-cell embryos. A monoclonal antibody to the cell adhesion molecule, E-cadherin, which mediates mouse embryo compaction, completely blocks compaction induced by these activators of PKC. Indirect immunofluorescence with a monoclonal antibody to E-cadherin indicates that PKC activation causes a rapid shift in the localization of this cell adhesion molecule, which coincides with the observed compaction. These results suggest that PKC plays a role in the initiation of compaction through its effect either directly or indirectly on E-cadherin.

Animals↗
PubMed1990

Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis.

Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)-sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER-enriched layer exhibited the largest release of Ca2+ in a dosage-dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis.

Animals↗
PubMed1988

Ionic currents in morphogenesis.

Morphogenetic fields must be generated by mechanisms based on known physical forces which include gravitational forces, mechanical forces, electrical forces, or some combination of these. While it is unrealistic to expect a single force, such as a voltage gradient, to be the sole cause of a morphogenetic event, spatial and temporal information about the electrical fields and ion concentration gradients in and around a cell or embryo undergoing morphogenesis can take us one step further toward understanding the entire morphogenetic mechanism. This is especially true because one of the handful of identified morphogens is Ca2+, an ion that will not only generate a current as it moves, but which is known to directly influence the plasma membrane's permeability to other ions, leading to other transcellular currents. It would be expected that movements of this morphogen across the plasma membrane might generate ionic currents and gradients of both electrical potential and intracellular concentration. Such ionic currents have been found to be integral components of the morphogenetic mechanism in some cases and only secondary components in other cases. My goal in this review is to discuss examples of both of these levels of involvement that have resulted from investigations conducted during the past several years, and to point to areas that are ripe for future investigation. This will include the history and theory of ionic current measurements, and a discussion of examples in both plant and animal systems in which ionic currents and intracellular concentration gradients are integral components of morphogenesis as well as cases in which they play only a secondary role. By far the strongest cases for a direct role of ionic currents in morphogenesis is the polarizing fucoid egg where the current is carried in part by Ca2+ and generates an intracellular concentration gradient of this ion that orients the outgrowth, and the insect follicle in which an intracellular voltage gradient is responsible for the polarized transport from nurse cell to oocyte. However, in most of the systems studied, the experiments to determine if the observed ionic currents are directly involved in the morphogenetic mechanism are yet to be done. Our experience with the fucoid egg and the fungal hypha of Achlya suggest that it is the change in the intracellular ion concentration resulting from the ionic current that is critical for morphogenesis.

Animals↗
PubMed1988

A highly localized activation current yet widespread intracellular calcium increase in the egg of the frog, Discoglossus pictus.

Sperm entry in the egg of the painted frog, Discoglossus pictus, occurs only at a specialized region of the animal hemisphere called the animal dimple, a structure not found in other species of frog. An extracellular vibrating electrode was used to measure the activation current to determine if the ion channels that open to generate the fertilization potential are localized in this region. Eggs that were activated by microinjecting inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) exhibited activation potentials very similar to those of fertilized eggs. There was a delay between the time of Ins(1,4,5)P3 injection and the initiation of the activation potential that was proportional to the distance between the site of the activating stimulus and the animal dimple, similar to the delay previously observed in prick-activated eggs (R. Talevi, B. Dale, and C. Campanella (1985). Dev. Biol. 111, 316-323). The delay lasted 30 sec when the stimulus site was 20 degrees (300 micron) from the animal dimple and 14 min when it was 150 degrees C from the dimple. Once the activation potential was initiated, there was an excellent temporal correlation between the time of depolarization and the time of the first detectable current entering the dimple region. This inward current was typically 60 microA/cm2 in amplitude and was found only in the central 200 micron of the dimple region. The outward current was distributed over the remainder of the egg surface and was much smaller in amplitude. The activation current was carried by Cl- efflux in the animal dimple region, and was reduced by DIDS and reversed by high external Cl- or I-. The occurrence of inward current only at the dimple region indicates that Cl- channels which open to produce the activation potential are localized there. Using Ca2+-specific microelectrodes, we found that [Ca2+]i increased from 0.25 to 2 microM following both fertilization and activation and returned to the unactivated level after about 37 min. Immature oocytes of D. pictus were also studied with the vibrating probe and the inward current in these cells was much less localized than that in the activating egg. A steady transcellular current of up to 4 microA/cm2 entered the entire animal hemisphere of the oocyte and exited the vegetal hemisphere.

Animals↗
PubMed1987

The wave of activation current in the egg of the medaka fish.

An extracellular vibrating electrode was used to measure the ring-shaped wave of inward current, the activation current, that propagates at 10 micron/sec across the egg of the medaka fish, Oryzias latipes, from the site of sperm-egg fusion at the animal pole to the vegetal pole. This activation wave is due to a localized increase in the conductance to Na+, K+, and Ca2+ and reflects the propagated opening of these ion channels. The earliest detectable current begins to enter the animal pole 20 sec after the initiation of the fertilization potential, so the first ion movements responsible for the fertilization potential are below the resolution of the vibrating probe system. These channels are present in both the animal and vegetal hemispheres, but the magnitude of the activation current is about seven times greater in the animal hemisphere. An outward current of smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of fertilization where the current is first inward. The current direction is dependent on the external Na+ concentration, and in the more physiological solution of 10% NaCl-Yamamoto's Ringer's, its direction reverses to become outward, apparently carried by K+ efflux. Raising the external Ca2+ in this same low-Na+ medium reverses the current so that it becomes inward again and increases the propagation velocity of the wave, suggesting a Ca2+ component to the inward current. Current enters a given region on the egg's surface about 16 sec before any vesicle fusion occurs in that region. Iontophoresis of inositol-1,4,5,-trisphosphate immediately triggers egg activation with a minimum activating charge of 0.6 nC.

Animals↗