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R B Silver

Publications and source records attributed to R B Silver.

At least 37 records · Page 2Linked to original sources
PubMed1996

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

Animals↗
PubMed1996

Stimulation of apical H-K-ATPase in intercalated cells of cortical collecting duct with chronic metabolic acidosis.

This study evaluated the role of H-K-adenosinetriphosphatase (H-K-ATPase) with chronic metabolic acidosis (CMA) in intercalated cells (ICs) of rabbit cortical collecting duct (CCD). CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10-14 days. CCDs isolated from CMA and control rabbits were split open and exposed to the intracellular pH (pHi) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions, the resting pHi in ICs was similar for both groups. K-dependent pHi recovery (5 mM K, 140 mM N-methyl-D-glucamine) was monitored in response to a pulse of NH4Cl (10 mM). The K-dependent pHi recovery rate was threefold higher in CMAICs compared with controls and was abolished with the gastric H-K-ATPase inhibitor, Sch-28080 (10(-5) M). Polarity of the H-K-ATPase was studied in microperfused CMA and control CCDs. Luminal K-dependent pHi recovery was monitored in response to an acute pulse of NH4Cl in individual peanut lectin agglutinin (PNA)-binding ICs. The apical Sch-28080-inhibitable K-dependent pHi recovery rate was significantly greater in CMA ICs than control ICs. In summary, CMA enhances functional activity of an apical H-K-ATPase in PNA-binding ICs of rabbit CCD.

Acidosis, Renal Tubular↗
PubMed1995

The concept of calcium concentration microdomains in synaptic transmission.

Ever since the initial measurements of presynaptic calcium currents it has been evident that calcium triggers transmitter release quite rapidly. Several models indicate, as did the initial voltage clamp measurements, that the calcium concentration triggering such release could be very high at the entry site and that this concentration should be very short lasting. In order to determine this time course, calcium entry was studied at the squid giant synapse by imaging light emission from n-aequorin-J, intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per sec. The results indicate that the calcium entry, triggered by action potentials, reaches a peak within 200 musec and has an overall duration of close to 800 musec, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions.

Animals↗
PubMed1995

Time resolved calcium microdomains and synaptic transmission.

The time course for the calcium entry that triggers release was studied at the squid giant synapse by imaging light emission from n-aequorin-J intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per second. The results indicate that the calcium entry triggered by action potentials reaches a peak within 200 microseconds and has an overall duration of close to 800 microseconds, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions.

Aequorin↗
PubMed1995

Feedback regulation of Na channels in rat CCT. III. Response to cAMP.

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1994

Phosphoprotein phosphatase 1 (PP1) is a component of the isolated sea urchin mitotic apparatus.

A protein component of isolated mitotic apparatus having a relative molecular mass of 62,000 (p62) is a substrate of a calcium/calmodulin dependent protein kinase, and the phosphorylation of p62 in vitro correlates directly with microtubule disassembly. In vivo experiments have determined the phosphorylation of p62 increases after fertilization; maximum incorporation of phosphate occurs during late metaphase/early anaphase and decreases thereafter. Because the level of p62 is constant throughout the cell cycle [Johnston and Sloboda, 1992: J. Cell Biol. 119:843-54] the decrease in phosphorylation of p62 observed after anaphase onset is most likely due to the action of a phosphatase. By examination of the relative amount of phosphorylated p62 which remained radiolabeled as a function of time using a standard in vitro phosphorylation assay, the activity of a phosphoprotein phosphatase capable of dephosphorylating p62 in the isolated mitotic apparatus was observed. To characterize the p62 phosphatase, okadaic acid and calyculin A were used to inhibit the dephosphorylation of p62 in vitro. It was found that specific concentrations of okadaic acid (50-500 nM) and of calyculin A (10-100 nM) were effective at inhibiting the dephosphorylation of p62 in vitro. Lower concentrations of either inhibitor had a negligible effect on dephosphorylation of p62. These data indicate the presence of phosphoprotein phosphatase type 1 activity associated with mitotic apparatus isolated from sea urchin embryos using the procedures described here. The implications of these findings relative to our understanding of the regulation of mitosis and cytokinesis are discussed.

Animals↗
PubMed1994

Time-resolved imaging of Ca(2+)-dependent aequorin luminescence of microdomains and QEDs in synaptic preterminals.

Localized elevation of intracellular free calcium [Ca2+]i concentration serves as the trigger for a wide variety of physiological processes, e.g., neurotransmitter release at most chemical synapses (1-3). The details of the mechanisms that regulate these processes are still unresolved (3-6), but they must involve precise temporal sequences of molecular events initiated by a transient localized elevation of Ca2+ concentration (i.e., a Ca2+ microdomain [3,7-15]). A microdomain is defined as an autonomous compartment of minimal spatio-temporal volume within which a signaled process can occur (8, 10, 12). A quantum emission domain (QED) is a quantal signal element (3, 16, 17). The concept of a QED was first applied to Ca2+ signaling at the synaptic preterminal (3, 4) and for large-diameter mitotic cells (16, 17). The concept of Ca2+ microdomains was tested by labeling preterminals of squid giant synapses with low-sensitivity aequorin (a photoprotein that emits a photon upon binding Ca2+ [18, 19]). That work confirmed earlier modeling efforts (10, 16) and showed that, upon depolarization, the [Ca2+]i profile reaches 200-300 microM within the microdomains, and that these [Ca2+]i profiles are composed of groups of short-lived 0.5 microns diameter QEDs. In those records, obtained with 2:1 interlacing devices operating at the RS-170 standard, QEDs appeared as striped dots or chevrons rather than as solid dots, indicating that a QED lasted less than 16.6 ms (one video field), and thus establishing the need for higher sampling rates to better characterize the QED.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1994

High-resolution measurement of the time course of calcium-concentration microdomains at squid presynaptic terminals.

Transmitter release is considered to be a secretory event triggered by localized calcium influx which, by binding to a low-affinity Ca2+ site at the presynaptic active zone, initiates vesicular exocytosis (1-7). In previous experiments with aequorin-loaded presynaptic terminals we visualized, upon tetanic presynaptic stimulation, small points of light produced by calcium concentration microdomains of about 300 microM (5). These microdomains had a diameter of about 0.5 microns (5) and covered 5-10% of the total presynaptic membrane with an average density of 8.4 microns2 per 100 microns2, corresponding closely to the size and distribution of the active zones in that junction (6, 7). To understand in more detail the nature of these concentration microdomains, we obtained rapid video images (400/s) after injecting the photoprotein n-aequorin-J into the presynaptic terminals of squid giant synapses. Using that experimental approach, we determined that microdomains evoked by presynaptic spike activation had a duration of about 800 microseconds. Spontaneous quantum emission domains (QEDs) observed at about the same locations as the microdomains were smaller in amplitude, shorter in duration, and less frequent. These results illustrate the time course of the calcium concentration profiles responsible for transmitter release. Their extremely short duration compares closely with that of calcium current flow during a presynaptic action potential and indicates that, as theorized in the past (6-8), intracellular calcium concentration at the active zone remains high only for the duration of transmembrane calcium flow.

Action Potentials↗
PubMed1993

Regulation of Na channels of the rat cortical collecting tubule by aldosterone.

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell-attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1-2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.

Aldosterone↗
PubMed1993

Functional identification of H-K-ATPase in intercalated cells of cortical collecting tubule.

A K-dependent proton extrusion mechanism was investigated by means of fluorescence techniques in rabbit cortical collecting tubules. These experiments were performed in split opened tubules from normal animals exposed to the intracellular pH (pH(i)) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. This preparation permitted the separate study of the intercalated cells (IC) from the principal cells (PC). In IC pH(i) recovery in response to an acute acid load was observed under Na-free conditions on addition of 5 mM K. This K-dependent recovery of pH(i) in the IC was only partial, but was Sch 28080 inhibitable (10(-5) M) and ouabain insensitive. This suggests the process is mediated by an H-K-adenosinetriphosphatase similar to that of gastric cells. The PC were capable of recovering from the acid load, but this Na-independent response was not blocked by the Sch 28080, suggesting some other mechanism for this result. In both cell types reintroduction of Na into the superfusate resulted in full recovery back to the initial pH(i), presumably the result of Na/H exchange.

Alkalies↗
PubMed1993

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump.

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry.

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

Amiloride↗
PubMed1993

2,3,7,8-tetrachlorodibenzo-p-dioxin increases cardiac myocyte intracellular calcium and progressively impairs ventricular contractile responses to isoproterenol and to calcium in chick embryo hearts.

Binding by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor leads to transcriptional activation of several genes and a toxicity syndrome that includes tumor promotion, wasting, hormonal and immune system dysfunction, and death. Recent findings indicate that TCDD may also affect cardiac function. Here, we used the chick embryo, a TCDD-sensitive species, to further characterize the effects of TCDD on ventricular muscle contraction and on cardiac myocyte [Ca2+]i assessed with fura 2. The results show that TCDD causes an evolving sequence of contractile defects, independent of changes in diet, first impairing cAMP-modulated contraction (after 48 hr) and later (by seven days) decreasing responses to [Ca2+]o. Phenobarbital, even at high doses, failed to affect the inotropic response to isoproterenol, supporting the specificity of the ventricular contractile effects of TCDD. TCDD treatment also depressed inotropic responses to theophylline and forskolin, indicating that it has a post-beta-adrenergic receptor effect on cAMP action. In contrast to its depression of responses to beta-adrenergic stimuli and to [Ca2+]o, TCDD did not affect initial tensions of ventricular muscle stimulated at 1 Hz or the force-frequency response up to 1 Hz, indicating that TCDD-treated ventricles can respond normally at slow rates of stimulation. TCDD treatment depressed lusitropic (relaxation) responses to isoproterenol and to increasing [Ca2+]o indicating that it impairs the ability of the sarcoplasmic reticulum to sequester Ca2+. Fura 2-based measurements showed that [Ca2+]i was nearly doubled after TCDD treatment. The increase in [Ca2+]i is consistent with the decrease in the contractile response to [Ca2+]o, amelioration of the response to isoproterenol by subphysiologic concentrations of [Ca2+]o, and intermittent lack of response to electrical stimulation in high K+ observed in ventricles from TCDD-treated embryos. TCDD treatment also depressed the initial increase in [Ca2+]i by isoproterenol, consistent with the decreased contractile response to isoproterenol. The findings show that TCDD causes well defined, progressive impairment of avian ventricular responses to inotropic stimuli, providing new evidence that the heart is a target of TCDD action and that TCDD disturbs intracellular calcium processing.

Animals↗
PubMed1992

Changes in intracellular calcium during the development of epithelial polarity and junctions.

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 2',7'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events.

Animals↗
PubMed1992

Microdomains of high calcium concentration in a presynaptic terminal.

Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.

Aequorin↗