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R Nuccitelli

Publications and source records attributed to R Nuccitelli.

17 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↗
PubMed1977

Relations between ameboid movement and membrane-controlled electrical currents.

We have studied the pattern of electrical currents through amebas (mainly Chaos chaos) with an ultrasensitive extracellular vibrating probe. Amebas drive both steady currents and current pulses through themselves. Relatively steady current with an average surface density of 0.1-0.2 muA/cm2 enters the rear quarter of an ameba and leaves its pseudopods. Streaming reversals are preceded by changes in this current pattern and the region with the largest new inward current becomes the new tail. Ion substitution studies suggest that some of the steady inward current is carried by calcium ions. Characteristic stimulated pulses of current sometimes follow the close approach of the vibrating probe to the side of an advancing pseudopod. Such a pulse enters the cytoplasm through a small patch of membrane near the probe (and seems to leave through the adjacent membrane), is usually followed by hyaline cap and then by pseudopod initiation, is calcium dependent, lasts about 5-10 s, and has a peak density of about 0.4 muA/cm2. Spontaneous pulses of similar shape and duration may enter or leave any part of an animal. They are much less localized, tend to have higher peak densities, and occur in physiological salt solutions at about 0.2-4 times per minute. Retraction of a pseudopod is always accompanied or preceded by a spontaneous pulse which leaves its sides.

Amoeba↗
PubMed1975

The pulse current pattern generated by developing fucoid eggs.

Using a newly developed extracellular vibrating electrode, we have made the first study of the spatial distribution of the growth currents around a single developing egg. This pattern was studied during the current pulses wihic traverse two-celled Pelvetia embryos. These pulses can be stimulated to occur with a periodicity of 70 min by mild acidification of the dea water medium. Current enters only at the growing rhizoid's tip while leaving both the base of the rhizoid cell and the whole outer membrane of the thallus cell. The field in front of the rhizoid cell falls off as the inverse cube of the distance from the rhizoid cell's center in the manner of a dipole field. The total inward and outward currents are equal, agreeing with theory. The current density at the rhizoid cell's base is twice that at the top of the thallus cell and this probably represents a change in the outer membrane's properties. There are no significant differences in the durrent density over the thallus cell. These results suggest a model in which the pulse current leaks in through newly opened channels in the growing tip and leaks out elsewhere due to the resultant fall in the membrane potential.

Electrodes↗
PubMed1975

Large electrical currents traverse growing pollen tubes.

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro. From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself. Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end. After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain. The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow. This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B. After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component. These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink. In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.

Cytochalasin B↗