Biomedical subjects

J T Russell

Publications and source records attributed to J T Russell.

At least 37 records · Page 2Linked to original sources

PubMed1997

Comparison of type 2 inositol 1,4,5-trisphosphate receptor distribution and subcellular Ca2+ release sites that support Ca2+ waves in cultured astrocytes.

We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP3R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP3Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP3Rs (InsP3R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP3R2 immunofluorescence were compared in the same cell, domains of elevated Ca2+ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP3R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca2+ release sites that support regenerative wave propagation include increased levels of InsP3R2 expression.

Animals↗

PubMed1996

Mitochondria support inositol 1,4,5-trisphosphate-mediated Ca2+ waves in cultured oligodendrocytes.

We have examined the spatial and temporal nature of Ca2+ signals activated via the phosphoinositide pathway in oligodendrocytes and the cellular specializations underlying oligodendrocyte Ca2+ response characteristics. Cultured cortical oligodendrocytes were incubated with fluo 3 or fura 2, and digital video fluorescence microscopy was used to study the effect of methacholine on [Ca2+]i. Single peaks, oscillations, and steady-state plateau [Ca2+]i elevations were evoked by increasing agonist concentration. The peaks and oscillations were found to be Ca2+ wave fronts, which propagate via distinct amplification regions in the cell where the kinetics of Ca2+ release (amplitude and rate of rise of response) are elevated. Staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanine++ + iodide (JC-1) and 3,3'-dihexyloxacarbocyanine iodide revealed that mitochondria are found in groups of three or more in oligodendrocyte processes and that the groups are distributed with considerable distance separating them. Cross-correlation analysis showed a high degree of correlation between sites where mitochondria are present and peaks in the amplitude and rate of rise of the Ca2+ response. Intramitochondrial Ca2+ concentration, measured using rhod 2, increased upon treatment with methacholine. Methacholine also evoked a rapid change in mitochondrial membrane potential as measured by the J-aggregate fluorescence of JC-1. Pretreatment with the mitochondrial inhibitors carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 microM, 2 min) or antimycin (2 microg/ml, 2 min) altered the methacholine-evoked Ca2+ response in most cells studied, responses being either markedly potentiated or inhibited. The results of this study demonstrate that stimulation of phosphoinositide-coupled muscarinic acetylcholinoceptors activates propagating Ca2+ wave fronts in oligodendrocytes and that the characteristics of these waves are dependent on mitochondrial location and function.

Animals↗

PubMed1995

Excitatory amino acid receptors in glia: different subtypes for distinct functions?

It is now well established that expression of voltage- and ligand-gated ionic channels, as well as G protein-coupled receptors, is not a property unique to neurons, but is also shared by macroglial cells (astrocytes and oligodendrocytes). These glial cells can receive a variety of signals from neurons at different stages of their development. Activation of membrane receptors may affect glial cell activity, proliferation, maturation, and survival through a complex cascade of intracellular events leading to long-term changes in glial cell phenotype and functional organization. Here we review the experimental evidence for glutamate receptor expression in glial cells in culture and in situ, and the molecular and functional properties of these receptors. We also describe some experimental models that identify possible functions of glutamate receptors in glia. Now that the existence of glutamate receptors in glia has been unambiguously demonstrated, future research will have to 1) determine which receptor subtypes are expressed in macroglial cells in vivo; 2) analyze, in adequate experimental models, the short- and long-term changes produced by glutamate receptor activation in glia; and 3) establish whether these receptors play a role in neuron-glia communication in the brain.

Animals↗

PubMed1995

AMPA receptors shape Ca2+ responses in cortical oligodendrocyte progenitors and CG-4 cells.

Intracellular calcium signals triggered by glutamate receptor activation were studied in primary cortical oligodendrocyte lineage cells and in the oligodendrocyte cell line CG-4. Glutamate, kainate, and AMPA (30-300 microM) increased [Ca2+]i in both types of cells at the stage of oligodendrocyte progenitors (O-2A; GD3+) or pro-oligodendroblasts (O4+). The peak amplitude of Ca2+ responses to glutamate receptor agonists was significantly larger in cortical cells. In CG-4 and in cortical cells, the majority (more than 90%) of bipolar GD3+ or multipolar O4+ cells responded to kainate. In all the cells analyzed, kainate was more efficacious than AMPA and glutamate. The percentage of bipolar or multipolar cells responding to glutamate was significantly lower in the CG-4 cell line than in primary cultures. Cellular responses typical of metabotropic glutamate receptor activation were observed in 20% of the cortical O-2A progenitors, but in none of the CG-4 cells. The AMPA-selective antagonist GYKI 52466 blocked kainate-induced Ca2+ responses in cortical O-2A cells. The selective AMPA receptor modulator cyclothiazide (30 microM) greatly potentiated the effects of AMPA (30-100 microM) on [Ca2+]i in cortical and CG-4 cells. Our findings indicate that Ca2+ responses in cells of the oligodendrocyte lineage are primarily shaped by functional AMPA receptors.

Animals↗

PubMed1995

A mathematical model of agonist-induced propagation of calcium waves in astrocytes.

In astrocytes, calcium signals evoked by neurotransmitters appear as waves within single cells, which spread to other cells in the network. Recent analysis has shown that waves are initiated at a single invariant site in the cell and propagated within the cell in a nonlinear and saltatory manner by regenerative amplification at specific predestined cellular sites. In order to gain insight into local cellular waves and wave collisions we have developed a mathematical model of cellular wave amplification loci. This model is in good agreement with experimental data which includes: ambient calcium gradients in resting cells, wave origination and local amplification and generation of local waves. As observed in experiments, the model also predicts that different locations in the cell can have different frequencies of oscillation. The amplification loci are thought to be specialized areas of the endoplasmic reticulum membrane containing a higher density or higher sensitivity of IP3 receptors. Our analysis suggests that the cellular loci act as weakly coupled oscillators each with its intrinsic latency and frequency of oscillation. Thus the appearance of the propagated calcium wave may be a reflection of these differences rather than an actual diffusional wave propagation.

Animals↗

PubMed1995

ets-1 in astrocytes: expression and transmitter-evoked phosphorylation.

The ets-1 protein has been primarily studied as a sequence-specific transcriptional regulator that is predominately expressed in lymphoid cells. In this report, we show that ets-1 is also expressed in astrocytes and astrocytoma cells and is regulated during both signal transduction and differentiation. Both isoforms of ets-1, p51 and p42, were found in astrocytes and astrocytoma cells, but whereas expression of p51 was strong, p42, the alternate splice product previously shown to lack the phosphorylation domain, was difficult to detect and was present at a level 10- to 40-fold lower than that of p51. This differed by roughly an order of magnitude from the ratio generally observable in T cells and thymocytes. In two astrocytoma lines of human origin, CCF and 1321N1, ets-1 phosphorylation was stimulated by bradykinin and carbachol, respectively. Glutamate, norepinephrine, and bradykinin elicited phosphorylation of p51 in cultures of primary rat type 1 astrocytes. ets-1 phosphorylation was dramatically blocked by KT5926, an inhibitor of myosin light-chain kinase, suggesting that this kinase may be involved in phosphorylation of ets-1 in vivo. Investigations of retinoic acid-induced differentiation in P19 cells provided further support for a strong correlation of ets-1 with the pathway for astrocyte differentiation.

Alkaloids↗

PubMed1995

Vasoactive intestinal peptide elevates pinealocyte intracellular calcium concentrations by enhancing influx: evidence for involvement of a cyclic GMP-dependent mechanism.

Vasoactive intestinal peptide (VIP) receptor density is high in the pineal gland, which receives VIP innervation and responds to VIP with a relatively small increase in cAMP and cGMP levels. In the present study, we show that VIP (5-200 nM) treatment increased the intracellular calcium concentration ([Ca2+]i) in 64% of isolated individual pinealocytes; in comparison, norepinephrine (NE) elevated [Ca2+]i in 93% of the cells and produced more robust responses. Analysis of the role of second messengers indicated that [Ca2+]i was strongly elevated by cGMP analogs, but not by cAMP analogs. The nitric oxide-releasing agent S-nitro-N-acetylpenicillamine and 2,2-diethyl-1-nitroxyhydraxine also elevated [Ca2+]i. Investigation of the mechanisms revealed that responses to VIP or 8-bromo-cGMP involved Ca2+ influx, as did the plateau component of the response to NE; the large rapid component of the response to NE, however, appeared to reflect release from intracellular stores. Pharmacological studies indicated that the VIP-induced Ca2+ influx was mediated by a retinal rod-type cyclic nucleotidegated cation channel, expression of which was confirmed by reverse transcription-polymerase chain reaction analysis. These observations indicate that fundamentally different mechanisms generate the responses to NE and VIP. The dominant effect of VIP causing transient elevation of [Ca2+]i appears to be through cGMP gating aI-cis-diltiazem-sensitive rod-type cyclic nucleotide-gated cation channel. In contrast, the dominant effect of NE on [Ca2+]i is due to enhanced Ca2+ release from intracellular stores; the plateau component is due to influx through aI-cis-diltiazem-insensitive channel.

Animals↗

PubMed1994

Glutamate regulates intracellular calcium and gene expression in oligodendrocyte progenitors through the activation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages.

Animals↗

PubMed1994

Vasoactive intestinal peptide increases intracellular calcium in astroglia: synergism with alpha-adrenergic receptors.

In type I astrocytes from rat cerebral cortex, vasoactive intestinal peptide (VIP) at concentrations below 1 nM evoked an increase in intracellular calcium ion concentration. This response, however, was observed in only 18% of the astrocytes examined. alpha-Adrenergic stimulation with phenylephrine or norepinephrine also resulted in an intracellular calcium response in these cells and the threshold sensitivity of astrocytes to phenylephrine was vastly different from cell to cell. Treatment of these astrocytes with VIP (0.1 nM) together with phenylephrine at subthreshold concentrations produced large increases in intracellular Ca2+ concentration ([Ca2+]i) and oscillations. The continued occupation of the alpha-adrenergic receptor was required for sustained synergism. Both alpha-receptor stimulation and stimulation with the mixture of agonists induced the cellular calcium response by triggering release of calcium from cellular stores, since the response persisted in the absence of extracellular calcium. Furthermore, thapsigargin pretreatment, which depletes intracellular stores, abolished the agonist-induced [Ca2+]i response. VIP (0.1 nM) and phenylephrine were found to increase cellular levels of inositol phosphates; however, there was no apparent additivity in this response when the agonists were added together. These observations suggest a calcium-mediated second messenger system for the high-affinity VIP receptor in astrocytes and that alpha-adrenergic receptors act synergistically with the VIP receptor to augment an intracellular calcium signal. The synergism between diverse receptor types may constitute an important mode of cellular signaling in astroglia.

Animals↗

PubMed1994

Nonlinear propagation of agonist-induced cytoplasmic calcium waves in single astrocytes.

In astrocytes in primary culture, activation of neurotransmitter receptors results in intracellular calcium signals that propagate as waves across the cell. Similar agonist-induced calcium waves have been observed in astrocytes in organotypic cultures in response to synaptic activation. By using primary cultured astrocytes grown on glass coverslips, in conjunction with fluorescence microscopy we have analyzed agonist-induced Ca2+ wave initiation and propagation in individual cells. Both norepinephrine and glutamate elicited Ca2+ signals which were initiated focally and discretely in one region of the cell, from where the signals spread as waves along the entire length of the cell. Analysis of the wave propagation and the waveform revealed that the propagation was nonlinear with one or more focal loci in the cytoplasm where the wave was regeneratively amplified. These individual loci appear as discrete focal areas 7-15 microns in diameter and having intrinsic oscillatory properties that differ from each other. The wave initiation locus and the different amplification loci remained invariant in space during the course of the experiment and supported an identical spatiotemporal pattern of signalling in any given cell in response to multiple agonist applications and when stimulated with different agonists which are coupled via InsP3. Cytoplasmic Ca2+ concentration at rest was consistently higher (17 +/- 4 nM, mean +/- S.E.M.) in the wave initiation locus compared with the rest of the cytoplasm. The nonlinear propagation results from significant changes in signal rise times, amplitudes, and wave velocity in cellular regions of active loci. Analysis of serial slices across the cell revealed that the rise times and amplitudes of local signals were as much as three- to fourfold higher in the loci of amplification. A phenomenon of hierarchy in local amplitudes of the signal in the amplification loci was observed with the wave initiation locus having the smallest and the most distal locus having the largest amplitude. By this mechanism locally very high concentrations of Ca2+ are achieved in strategic locations in the cell in response to receptor activation. While the average wave velocity calculated over the length of the cell was 10-15 microns/s, in the active loci rates as high as 40 microns/s were measured. Wave velocity was fivefold lower in regions of the cell separating active loci. The differences in the intrinsic oscillatory periods give rise to local Ca2+ waves that show the properties of collision and annihilation. It is hypothesized that the wave front provokes regenerative Ca2+ release from specialized areas in the cell where the endoplasmic reticulum is endowed with higher density of InsP3 receptor channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Single-cell [Ca2+]i analysis and biochemical characterization of pinealocytes immobilized with novel attachment peptide preparation.

Single-cell image analysis of rat pinealocytes has been difficult because they do not attach readily to coated or uncoated surfaces and typically adhere in clusters to fibroblast-like cells. In the present report, a new method for the rapid attachment of rat pinealocytes is described. Cells were prepared using papain digestion and density centrifugation and then were placed on coverslips or slides coated with PepTite-2000, a preparation containing the attachment peptide sequence Arg-Gly-Asp. Cells immobilized with this preparation responded to norepinephrine treatment with an increase in cyclic AMP and melatonin production. Single-cell analysis of Fura-2-loaded cells revealed that norepinephrine increased [Ca2+]i. This development makes it possible to conduct routine single-cell image analysis and other studies of freshly isolated rat pinealocytes.

Amino Acid Sequence↗

PubMed1993

Accommodation of mouse DRG growth cones to electrically induced collapse: kinetic analysis of calcium transients and set-point theory.

Electrical stimulation causes growth cones of mouse dorsal root ganglion neurons to collapse. During chronic stimulation, however, growth cones resume motility. In addition, these growth cones are now resistant to the collapsing effects of subsequent stimulation, a process we term accommodation. We compared the kinetics of electrically induced Ca2+ transients in naive and accommodated growth cones in order to determine whether the accommodation process results from a change in the Ca2+ transient, or a change in the Ca2+ sensitivity of the growth cones. Three kinetics were determined: (1) the initial increase to peak Ca2+ levels produced by 10 Hz stimulation; (2) recovery from peak Ca2+ levels during stimulus trains lasting 15 min; and (3) clearing of Ca2+ from growth cones after terminating the stimulus. These kinetics were analyzed using single exponential fits to changes in fura-2 fluorescence ratios. The electrically evoked increase in Ca2+ was significantly slower in accommodated growth cones (tau = 6.0 s) compared to naive growth cones (tau = 1.4 s). Despite the slower increase of [Ca2+]i in accommodated growth cones, peak [Ca2+]i was similar to that reached in naive growth cones, and the steady-state Ca2+ level was significantly elevated after chronic stimulation. Thus, accommodated growth cones maintained outgrowth at [Ca2+]i that caused collapse initially. Time course experiments show that accommodation is a slow process (t 1/2 = about 3 h). Accommodation did not induce measurable changes in the rates of Ca2+ homeostasis during or after stimulus trains. The kinetics of Ca2+ recovery during (tau = 90 s) and after 15 min of stimulation (tau = 8.5 s) was not significantly different in accommodated versus naive growth cones. Rates of 45Ca2+ efflux were also similar in both types of growth cones. These results suggest two regulatory processes contributing to growth cone motility during chronic stimulation: (1) recovery of [Ca2+]i to levels permissive to neurite outgrowth, and (2) an increase in the range of optimal [Ca2+]i for growth cone motility. These adaptive responses of mammalian growth cones to chronic stimulation could be involved in the modulation of CNS development by electrical activity of neurons.

Adaptation, Physiological↗

PubMed1992

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.

Calcium↗

PubMed1992

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca2+.

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]o, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]o), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]o) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]o became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.

Animals↗

PubMed1992

Spontaneous changes in intracellular calcium concentration in type I astrocytes from rat cerebral cortex in primary culture.

Measurement of fura-2 fluorescence in type I astrocytes from rat cerebral cortex showed that the intracellular calcium ion concentration undergoes very large spontaneous changes. These spike-like changes ranged from resting levels of calcium of 50-250 nM to as high as 1-2 microM. The spikes were found to be irregular in frequency and amplitude and were frequently synchronous in confluent cultures. The synchronous events appeared as propagating waves that spread over many cells. The spontaneous spikes persisted when the extracellular calcium concentration was reduced to below micromolar levels suggesting that the source for the increases in [Ca2+]i was intracellular. Treatment of the astrocytes with tetrodotoxin did not abolish the spontaneous changes, nor did blockade of voltage-dependent calcium channels with nimodipine and D-600. Ryanodine, a blocker of the sarcoplasmic reticulum calcium-induced calcium release channel, was also without effect. These changes in [Ca2+]i were different in character from both agonist-induced oscillations and depolarization-induced increases in intracellular calcium concentration. Depolarization using 25-100 mM [K+]o resulted in a prompt rise in intracellular calcium concentration, which returned to near resting levels, and this response was sensitive to removal of extracellular calcium and voltage-gated calcium channel antagonists. L-glutamate (0.5-100 microM) caused large increases in [Ca2+]i that were associated with discrete periodic oscillations in some cells. The cellular trigger for the spontaneous spikes is currently not understood. We conclude that spontaneous changes in [Ca2+]i in astrocytes are distinct from agonist-induced and membrane potential depolarization-induced changes.

Adrenergic Antagonists↗

PubMed1991

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis.

1. We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of 45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes). 2. Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 +/- 0.23- and 3.71 +/- 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P less than or equal to 0.1). 3. Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 +/- 5- and 20 +/- 3-fold, respectively, and A23187-induced secretion was increased 1.3 +/- 0.4- and 1.3 +/- 0.1-fold (P less than or equal to 0.01 for both ionophores). 4. The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P less than or equal to 0.01 for sodium greater than or equal to 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1. 5. The rate of 45Ca efflux from neurosecretosomes showed 2.7 +/- 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P less than or equal to 0.01). 6. Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.

Animals↗

PubMed1991

Activation and inactivation of oxytocin and vasopressin release from isolated nerve endings (neurosecretosomes) of the rat neurohypophysis.

Neurosecretory terminals (neurosecretosomes, NSS) were isolated from rat neurohypophyses. High [K+]o or veratridine stimulated secretion of vasopressin and oxytocin by up to approximately 100-fold. Stimulated secretion was dependent on calcium and temperature, and could be elicited from NSS maintained in culture for 4 days. After overnight culture of the NSS, secretion was still inhibited by calcium channel blockers (cobalt, dihydropyridines, omega-conotoxin, D 600) and kappa opiates (dynorphin and U50488). Ionomycin evoked dose- and calcium-dependent hormone release, with a Hill coefficient for calcium of 1.74. High [K+]o enhanced the 5 microM ionomycin-induced secretion, apparently through calcium entry rather than depolarization, as the increase in secretion was abolished by 100 microM D 600. During prolonged depolarization the hormone secretion peaked within 2 min, then declined to near basal levels. Depolarization for 25 min without calcium neither activated secretion nor prevented subsequent secretion on readdition of calcium, suggesting that the decline in secretion was not due to membrane depolarization. Indeed, the rates of decline in secretion were similar for different levels of depolarization (0.070 +/- 0.003 and 0.081 +/- 0.003 min-1 for 25 and 45 mM [K+]o, respectively). Four minutes after the onset of continuous depolarization (45 mM [K+]o) in the presence of calcium, the declining secretion was still dependent on voltage-activated calcium influx through channels sensitive to D 600 and nitrendipine. The results presented here suggest that the decline in secretion during prolonged depolarizing stimuli may be due to exhaustion, inactivation, or desensitization of a calcium-triggered event.

Animals↗

PubMed1990

High concentration of inositol 1,4,5-trisphosphate in sea urchin sperm.

We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozoon contains 4 x 10(-19) to 1 x 10(-18) moles of InsP3 before the acrosome reaction. Since the acrosome reaction has previously been shown to increase the InsP3 content of sperm severalfold, our measurement indicates that a spermatozoon contains at least 2 x 10(-18) moles of InsP3 at fertilization, corresponding to a concentration in the spermatozoon of about 1 mM. The threshold for activation of eggs by injection of InsP3 dissolved in a much larger volume of solution has been found to be about 3 x 10(-18) moles, corresponding to a concentration in the injectate of 1 microM. This suggests that sea urchin sperm may contain enough InsP3 to activate eggs. With an electroporation method, we also showed that sperm extract acts on eggs only from inside, consistent with a primary messenger role for InsP3.

Acrosome↗