Search PubMed⌕ Search

Biomedical subjects

J A Connor

Publications and source records attributed to J A Connor.

At least 91 records · Page 5Linked to original sources
PubMed1988

Interactive effects of serotonin and acetylcholine on neurite elongation.

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.

Acetylcholine↗
PubMed1988

NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca2+ mobilization.

NIH-3T3 cells transformed by the EJ-ras oncogene synthesize only 10-15% as much inositol 1,4,5-trisphosphate (InsP3) as control cells after stimulation with platelet-derived growth factor (PDGF). This is despite the fact that the basal (unstimulated) levels of InsP3 synthesized in control and EJ-ras-transformed cells are not significantly different. Using the fluorescent indicator fura-2 and digital-imaging techniques, we have visualized and quantified changes in intracellular Ca2+ concentrations in control and EJ-ras-transformed NIH-3T3 cells in response to PDGF. Within 3 min after exposure of control cells to PDGF, intracellular Ca2+ levels are increased 3- to 9-fold, paralleling the increase in InsP3. In contrast, the majority (greater than 90%) of the EJ-ras-transformed cells show no increase in Ca2+ levels after PDGF exposure and the few that did respond exhibited only a small transient increase. Pronounced differences in the intracellular localization of Ca2+ increases in control and the responding EJ-ras-transformed cells were also observed. Despite the inhibition of InsP3 synthesis and subsequent Ca2+ mobilization, the EJ-ras-transformed cells respond mitogenically to PDGF. These data do not support the hypothesis that the EJ-ras gene product (p21) stimulates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in NIH-3T3 cells; instead they suggest that the EJ-ras p21 may uncouple the PDGF receptor from phospholipase C resulting in inhibition of PDGF-stimulated activity of phospholipase C, InsP3 synthesis, and Ca2+ mobilization.

Animals↗
PubMed1988

Inositol trisphosphate releases intracellularly stored calcium and modulates ion channels in molluscan neurons.

Stimulation of the bag cell neurons of Aplysia triggers a long-lasting afterdischarge in these cells. In vivo, such a discharge causes the onset of a sequence of reproductive behaviors. We have found that treatments that trigger discharges in vitro stimulate the hydrolysis of phosphoinositides in the bag cell neurons, as measured by increased incorporation of 3H-inositol into fractions containing membrane lipids and water-soluble inositol phosphates. The electrophysiological effects of inositol trisphosphate, one of the products of phosphoinositide turnover that has been shown to mobilize intracellular calcium in non-neuronal cells, were investigated using isolated bag cell neurons in cell culture. Microinjection of inositol trisphosphate into cultured bag cell neurons caused a transient hyperpolarization of the membrane (approximately 35 sec), together with an increase in conductance. This effect of inositol trisphosphate was abolished by 50 mM tetraethylammonium ions. Inositol trisphosphate also reduced the amplitude of action potentials. Injection of calcium ions directly into bag cell neurons mimicked these responses seen after inositol trisphosphate injection. Using the cell-attached patch-clamp technique in conjunction with inositol trisphosphate microinjection, we observed that inositol trisphosphate evoked increases in the activity of a channel carrying outward current at the resting potential and more positive potentials. The estimated slope conductance of the channel modulated by inositol trisphosphate was approximately 40 pS, and its reversal potential was close to that predicted for potassium ions. The increased opening of this channel in response to inositol trisphosphate injection appeared to result from a transient shift of its voltage-dependence to more negative potentials. In a few cases, inositol trisphosphate injection also elicited an increase in the activity of a channel passing inward current at rest. Direct measurements of changes in intracellular calcium in response to inositol trisphosphate were made using digital imaging of isolated neurons loaded with the fluorescent calcium indicator fura-2. These revealed that injection of inositol trisphosphate significantly elevated intracellular calcium levels, and that this inositol trisphosphate-induced rise in cytosolic calcium was not affected by removal of extracellular calcium. In contrast to the effects of trains of action potentials in calcium-containing media, which produced increases in calcium primarily in neurites, the inositol trisphosphate-induced elevation of calcium appeared more localized to the somata of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques.

The response of cytosolic calcium [Ca2+]i to angiotensin II (AII) and potassium (K+) in individual rat glomerulosa cells was determined using the calcium-sensitive fluorescent dye, fura-2 and digital imaging. Control (4 mM K+) cytosolic calcium levels were generally in the 80-120 nM range and increased monotonically as [K+] was increased from 4 to 12 mM. There was no delay in the onset of the response. In most cells the [Ca2+]i decreased from its peak after 3-4 min, even in the presence of superfusate containing elevated K+. The time course of the change in [Ca2+]i in response to AII stimulation, on the other hand, was more variable. It was most often characterized by an early decrease followed by a large delayed increase. The response also was observed to decline during sustained AII stimulation. The majority of the cells showed some response to one or the other secretagogue with a sizeable minority (25%) having an increase in [Ca2+]i in excess of 200%. While the majority showed a response, the cell to cell variation was substantial. Finally, the pattern of cytosolic calcium increase sometimes showed a marked dependence on the secretagogue used, with different regions of the same cell being more strongly affected by one agent or the other. A few cells (10%) responded to AII only at one pole, establishing a large concentration gradient of calcium across the cell. Because of differences in time course, pattern, and degree of responsiveness, it is likely that the mechanisms underlying the Ca2+ elevation with K+ and AII are different.

Angiotensin II↗
PubMed1987

Immunocytochemical and electrophysiological differentiation of rat cerebellar granule cells in explant cultures.

We have used a combination of immunocytochemical and electrophysiological measurements to monitor the differentiation of cerebellar granule cells in vitro. We present immunocytochemical evidence showing that several characteristic features of developing rat cerebellar tissue were retained in postnatal explant cultures. Most notably the cultures expressed radiating GFAP-positive (Bergmann) glia processes, proliferating NSE-negative neuroblasts, and migrating NSE-positive granule cells. The latter were subdivided into 3 developmental stages--i.e., immature, intermediate, and mature granule cells, based upon cell differences in location from the explant, intensity of NSE staining, excitability, and the amplitude of voltage-dependent conductances. Immature cells were identifiable during the first week in culture and were located up to 140 micron from the explant. These cells stained lightly for NSE and displayed conductances of insufficient magnitude to generate action potentials. Intermediate cells were present after 1-2 weeks in culture and were located up to 500 micron from the explant. These cells were also NSE positive and were characterized by the presence of soma action potentials. Intermediate cells displayed 3 large voltage-dependent conductances: a transient, TTX-sensitive inward current; a delayed, TEA-sensitive outward current; and a transient, 4AP-sensitive outward current. Mature cells were present after 1 month in culture and, like intermediate cells, were no more than 500 micron from the explant. However, mature cells stained more intensely for NSE, and the somata of these cells were devoid of voltage-dependent conductances (although axonal currents were usually present). These results indicate that granule cells undergo a stereotypic sequence of differentiation in postnatal explant cultures. These stages may correspond to developmental changes in granule cells during migration into the (internal) granular cell layer in vivo.

4-Aminopyridine↗
PubMed1987

Depolarization- and transmitter-induced changes in intracellular Ca2+ of rat cerebellar granule cells in explant cultures.

Digital imaging of the Ca indicator fura-2 has been used to study the responses of developing granule cells in culture to depolarization and transmitter action. Unstimulated cells bathed in Krebs saline exhibited cytoplasmic Ca ion concentrations, [Ca2+], that were generally in the 30-60 nM range. Exposure of cells to high-potassium (25 mM) saline depolarized the membrane potential and produced an immediate rise in [Ca2+] that recovered within 2-3 min in normal saline. The response grew progressively larger over the first 20 d in culture. Transient increases in [Ca2+] to levels greater than 1 microM were observed after 12-14 d in vitro, at which time the cells displayed intense electrical activity when exposed to high K. At this stage, the increases were attenuated by blocking action potential activity with TTX. In TTX-treated or immature cells, in which the transient phase of the Ca change was relatively small, a second exposure to high K typically produced a much larger Ca response that the initial exposure. The duration of this facilitation of the response persisted for periods longer than 5 min. Application of the neurotransmitter GABA induced a transient increase in membrane conductance, with a reversal potential near resting potential (approx. -60 mV), and caused an intracellular Ca2+ increase that outlasted the exposure to GABA by several minutes. Glutamate, or kainate, induced an increase in membrane conductance but with a reversal potential more positive than spike threshold. These agents also elevated intracellular Ca2+, but unlike the case with GABA, this Ca response reversed rapidly upon removal of the transmitter. The facilitatory effect of repeated exposures to high-K saline, as well as the persistent Ca elevation following a brief GABA application, suggests that granule cells possess the capability of displaying activity-dependent changes in Ca levels in culture.

Animals↗
PubMed1987

Electrically and chemically mediated increases in intracellular calcium in neuronal growth cones.

In the present report we used the calcium indicator fura-2 to compare intracellular levels of free calcium in growth cones of isolated Helisoma neurons under a variety of experimental conditions. We tested whether 2 different signals that inhibit growth cone motility--action potentials and serotonin--changed calcium levels in growth cones. Electrical stimulation of the cell body caused a rise in calcium levels at the growth cone. After brief stimulation, calcium levels quickly recovered to normal values, whereas longer stimulation periods required longer recovery times. The application of serotonin to growth cones caused an increase in calcium levels that was selective for growth cones of neurons whose outgrowth was inhibited by serotonin, but not for neurons whose outgrowth was not affected. We also found that motile growth cones had higher free calcium levels than growth cones that had spontaneously stopped growing. Furthermore, the distribution of calcium in neurons that contained motile growth cones was heterogeneous; calcium levels were always higher in the growth cone than in the neurite or soma. These data indicate that calcium levels in growth cones vary in different states of outgrowth and that calcium levels can be modulated by both electrical and chemical signals.

Action Potentials↗
PubMed1986

Expression of membrane currents in rat diencephalic neurons in serum-free culture.

The whole-cell gigaseal voltage clamp technique has been used to investigate the timing of expression and type of voltage-dependent ionic currents in dissociated primary cultures of fetal rat (E17) diencephalic neurons grown in a serum-free defined medium. The expression of membrane currents varied among cells at any particular time in culture. Despite this variability, certain characteristics of the appearance of ionic currents emerge from this study. These are: (i) the earliest appearing membrane current is a voltage-dependent outward current carried by K+. In some cells, it is the classical delayed rectifier current, whereas in others it is the transient outward current (IA). (ii) The earliest appearing inward current is carried by Na+. In some cells the channels are first expressed in the neurites and then in or near the cell body. The early neuritic Na+ channels are blocked by cobalt or cadmium as well as by tetrodotoxin (TTX). In others, the early Na+ channels appear in or near the cell body and are only blocked by TTX. (iii) With additional time in culture, a majority of cells exhibit a Ca2+ current at the time of Na+ channel appearance in or near the cell body as well as a transient Ca2+-dependent outward current. The Ca2+ current is only a small fraction of the total inward current. These inward currents show the classical pharmacologic profile. The complex pattern of expression of ionic current may reflect multiple populations of neurons with different developmental sequences resulting from differences in cell age and lineage.

Action Potentials↗
PubMed1986

Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells.

Intracellular free calcium levels have been measured in cultured central nervous system (CNS) cells by using the fluorescent indicator fura-2 and digital imaging techniques. Cells were plated from rat embryo diencephalon (embryonic day 17 or 18), with nearly all of the cells surviving dissociation having undergone final mitosis within the previous 24 hr. The initially spherical cells were observed within the first 24 hr in culture when they were extending processes but had not established a network of fibers that would prevent the identification of the origin of a given fiber. Cells that were rapidly extending showed high Ca2+ levels in the regions of growth. Where processes had just emerged from the soma or where growth was proceeding from more than one pole, Ca2+ levels were uniform and estimated levels of 500 nM were commonly seen. In active growth cones distant from the soma, Ca2+ levels exceeded 200 nM, whereas the soma levels were in the 60-80 nM range. Nonextended and extended cells that had stalled had uniform Ca2+ levels in the range of 30-70 nM. The results show that high Ca2+ levels are at least a correlate of extension in CNS cells and that under some conditions the region of high calcium can be localized to a small part of the cell.

Animals↗
PubMed1986

Calcium levels measured in a presynaptic neurone of Aplysia under conditions that modulate transmitter release.

We have utilized the Ca2+ indicator dye, Arsenazo III, to examine the role of presynaptic Ca2+ concentration in two types of synaptic plasticity observed at the synapses of cell L10 in Aplysia californica; post-tetanic potentiation (p.t.p. - the increased transmitter release which follows high frequency stimulation), and resting membrane potential modulation of release. Intracellular Ca2+ was monitored in the cell body and main neurites of L10 injected with Arsenazo III. Tetanic stimulation caused an increase in intracellular Ca2+ concentration that decayed, after tetanus, with fast and slow time constants which paralleled the time course of decay of p.t.p. When the voltage-sensitive Ca2+ current was reduced by removing external Ca2+ (0 mM-Ca2+, 4 mM-EGTA) or by blocking Ca2+ channels with divalent cation channel blocker (4 mM-Cd2+), tetanic stimulation did not cause increases in Arsenazo absorbance even when Na+ currents were not blocked. This finding suggests that Ca2+ entering the cell through voltage-dependent Ca2+ channels was the major source of Ca2+ which accumulated during the tetanus. Transmitter release is increased when L10 is maintained at a depolarized membrane potential, and is decreased when L10 is hyperpolarized. We found that the base-line Arsenazo absorbance signal in L10 increased when L10 was depolarized from -60 to -40 mV and decreased when L10 was hyperpolarized. This finding supports the idea that the steady-state Ca2+ concentration contributes to the membrane-potential modulation of transmitter release. These results support the idea that transmitter release can be modulated by the residual or resting Ca2+ concentration of the presynaptic cell.

Action Potentials↗
PubMed1984

Diffusion of ions and indicator dyes in neural cytoplasm.

The dispersion of dye molecules and small cations injected from a point source in the cytoplasm of molluscan neurons has been measured photometrically and compared with dispersion in aqueous solution. The diffusion of phenol red and arsenazo III was at least a factor of five slower in the cytoplasm than in saline. Movement of both dyes was slowed by about the same factor in a given cell. The dispersion rate of arsenazo III was not significantly affected by preloading the cytoplasm to dye concentrations up to 0.5 mM. Calcium and barium dispersion was measured in neurons and saline droplets preloaded with arsenazo III, while phenol red absorbance changes were used to follow the dispersion of injected protons. Ba2+ and H+ moved very slowly in the cytoplasm compared to aqueous solution. Ca2+ movement in all probability underwent a similar retardation in the neurons but high-affinity buffering of the cytoplasm severely restricted the spread of detectable amounts of this ion away from the injection site.

Animals↗
PubMed1984

Alteration of calcium conductances and outward current by cyclic adenosine monophosphate (cAMP) in neurons of Limax maximus.

Membrane responses to cyclic adenosine monophosphate (cAMP) injections have been studied by means of voltage clamp, Ca-indicator dye, and ion substitution techniques in identified neurons from the abdominal ganglion of Limax maximus. The ventral abdominal giant cell (AGC) displayed a response consisting of a decrease in outward current usually accompanied by a smaller enhancement of voltage-gated Ca2+ influx. Both responses were eliminated by external Cd2+ or Mn2+ and required membrane voltages more positive than -40 mV for expression. The enhanced influx persisted in Ba2+-substituted saline, while the decrease in outward current was blocked. A group of dorsal neurons (RD1-3, LD1) showed a mixed Na-Ca influx induced by cAMP that could be activated over a wide range of membrane potentials (less than -100 to greater than -20 mV). This flux caused a measurable increase in internal Ca2+. The influx was insensitive to Cd2+ and Mn2+ but was reduced by prolonged exposure to Co2+. The relative magnitude of the Na-Ca flux ratio showed considerable variation between specimens. In immature animals the Ca component was absent. The results demonstrated that elevation of intracellular cAMP can cause cell-specific changes of membrane conductance within closely associated neurons.

Animals↗
PubMed1984

A novel membrane sodium current induced by injection of cyclic nucleotides into gastropod neurones.

Injection of cyclic AMP (cAMP) or cyclic GMP into identifiable neurones from several different gastropod species immediately depolarized the cell membranes in a dose-dependent manner. Doses were monitored photometrically and evidence is presented for depolarizing effects following nucleotide injections of as little as 30-35 mumol. The depolarizing effect was reversible and was demonstrated under voltage clamp to be primarily the result of a nucleotide-induced, transient increase in a membrane Na current, INa (cAMP). The charge-carrying species was identified by using ion-substituted salines, reversal potential in low-Na saline, and intracellular ion-sensitive electrode measurements. The current was resistant to tetrodotoxin, ouabain and amiloride. Substituting Trisma, tetramethylammonium or bis-tris propane for Na prevented the induced current, whereas Li substitution did not. Duration of the induced current was greatly prolonged in neurones bathed in the phosphodiesterase inhibitor isobutylmethylxanthine, or following injection of any of several cAMP analogues, indicating that the reversible nature of the current stems primarily from in situ hydrolysis of the injected dose and not current inactivation. Amplitude of the induced current either remained constant or decreased over the voltage range where it could be easily measured, i.e. -30 greater than Vm greater than -100 mV, reflecting a voltage as well as a chemical sensitivity of INa (cAMP).

1-Methyl-3-isobutylxanthine↗
PubMed1984

Intracellular pH changes induced by injection of cyclic nucleotides into gastropod neurones.

Injections of cyclic AMP (cAMP) or cyclic GMP (cGMP) into identifiable gastropod neurones under voltage clamp induced reversible cytoplasmic pH changes which were measured using the indicator dye Arsenazo III and pH micro-electrodes. Similar injections of 5' AMP or 5' GMP did not induce such effects. In about one-half of the population of neurones examined, cAMP injections (0.1-10 mM) induced pH decreases with latencies of 1-5 min and maximum response times 12-25 min post-injection. Comparable injections of cGMP into these cells resulted in decreases with latencies less than 1 min and maximum response times 5-10 min. The amplitude and duration of these pHi decreases were dose dependent. In the other half of the population of neurones tested, cAMP injection induced an immediate alkalinization lasting 5-10 min followed by an acidification which displayed a maximum response time within the same range as those in the first group. cGMP injection into these cells induced only acidifications with faster peak responses than with cAMP. Since cGMP did not elicit alkalinizing responses, the slower response time of the first group of cells with cAMP may reflect an always present underlying alkalinization. The nucleotide-induced acidification was potentiated in neurones bathed in the phosphodiesterase inhibitor IBMX. Injections of non-hydrolysable cAMP analogues also induced acidifications which were longer lasting than comparable doses of cyclic AMP. These results indicate that the elevated [H+]i was not simply due to hydrolysis of the injected cyclic nucleotide. The pHi changes were invariably of much longer duration than a nucleotide-induced inward current (Connor & Hockberger, 1984) and persisted in Na-free salines which eliminated the current response. These results support the notion that cyclic nucleotide elevation can affect cellular metabolic processes distinct from effects on ion transport mechanisms.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Physiological role of electrogenic pumps in smooth muscle.

Rhythmic electrical activity in the small intestine of cat appears to be initiated by small pacemaker potentials originating in the longitudinal muscle layer. These depolarizing potentials (slow waves) have durations of from 2 to 5 sec and are generally less than 15 mV in amplitude. Frequency depends on the region of intestine and varies between 3 and 10 waves per min. Electrical activity of the intact muscle coat is the summation of this pacemaker activity with triggered activity and is of larger amplitude and more complicated waveform. Exposure to ouabain (10(-5) M) or K-free saline causes a rapid block of pacemaker slow-wave activity and depolarization to levels more positive than the slow-wave crest. Repolarization does not restore spontaneity. Amplitude and frequency are relatively insensitive to changes in extracellular Na and Cl concentrations, and conductance changes, if they occur during the slow-wave cycle, are too small to be detected in the double-sucrose gap. Changes in extracellular Ca have pronounced effects on frequency, being decreased in low Ca and increased in high Ca. Calcium channel blockers have the same effects as low Ca saline, implying that the changes in frequency are mediated through internal Ca levels. Experiments using chlorotetracycline fluorescence provided data that intracellular Ca2+ levels oscillate even under conditions where measurable contractile activity is absent. We have proposed that oscillations in the activity of the Na-K pump are responsible for the pacemaker slow waves and that internal Ca2+ is an important cycle-to-cycle controlling factor in the oscillation.

Animals↗
PubMed1983

Intracellular calcium measurements with arsenazo III during cyclic AMP injections into molluscan neurons.

Injections of cyclic adenosine monophosphate into molluscan neurons often produce a transient membrane depolarization. By using the calcium indicator dye arsenazo III, it was found that cyclic nucleotide injections into neurons of Archidoris montereyensis resulted in elevation of internal calcium concentrations. However, this was demonstrated to be a secondary consequence of an induced increase in membrane sodium permeability, and not due to any direct effect of cyclic adenosine monophosphate on cellular calcium influx or internal calcium regulating processes.

Animals↗
PubMed1983

Theory of electromechanical effects in nerve.

The electromechanical transduction mechanisms operating in nerve membranes are considered theoretically. For mechanical-to-electrical transduction (mechanical generator potentials), a model is proposed in which the surface charge on the membrane mediates stress-induced changes in the intramembrane electric field, thus opening transmembrane ion conductance channels or reducing the ion selectivity of the membrane via leak conductance pathways. For electrical-to-mechanical transduction (axon diameter change with excitation), an investigation into two well-known electrostatic properties of dielectrics, electrostriction and piezoelectricity, in the context of the nerve membrane is undertaken which predicts a few percent change in axon dimensions for voltage- and space-clamped axons.

Axons↗