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R B Silver

Publications and source records attributed to R B Silver.

51 records · Page 3Linked to original sources
PubMed1992

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule.

Changes in intracellular pH (pHi) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pHi. Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Nai) would alter pHi in a predictable way. Maneuvers designed to alter Nai were without significant effects within a 10-min time frame. Specifically, addition of 100 microM ouabain to increase Nai or exposure of the tubules to 10(-5) M amiloride to decrease luminal Na entry and reduce Nai did not have an effect on pHi. In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pHi. These results suggest that Na/H exchange is a major regulator of pHi in principal cells. However, regulation of Na transport by changes in pHi in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.

Animals↗
PubMed1992

Presynaptic calcium concentration microdomains and transmitter release.

n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.

Aequorin↗
PubMed1990

Calcium and cellular clocks orchestrate cell division.

The results of experiments recently reported from this and other laboratories provide firm support for Heilbrunn's thesis that mitotic events are initiated by transient elevation of intracellular Ca2+, derived from intracellular stores. The ATP-dependent MA Ca2(+)-pump working in concert with an endomembrane Ca2+ channel appears to share the responsibility for regulating these Ca2+ signals. Further results demonstrated a limited time window during which the cell is sensitive to agents that impose mitotic arrest by interfering with transient elevations in intracellular "free" Ca2+ concentration. From this it appears that a discrete, timed increase in cytosolic Ca2+ derived from endomembrane stores is a necessary signal for regulating the onset of NEB, AO, and mitosis. Results from the arrest and release experiments provide support for a model in which Ca2+ is used to coordinate the action of parallel independent and interdependent biochemical pathways whose interaction results in the cytologic events of mitosis. These pathways apparently are operating under the influence of a metabolic "clock" that continues to cycle, at least once, in the absence of a Ca2+ transient sufficient to initiate NEB or AO. The discrete and temporal regulation of this Ca2+ transient through the interaction of the endomembrane Ca2+ pump, an endomembrane Ca2+ channel, and intracellular Ca2(+)-dependent reaction pathways suggest a mechanism incorporating a negative feedback loop to limit the size and duration of the Ca2+ transient and prevent the release of excessive amounts of Ca2+. Deeper understanding of the regulatory mechanism that governs the onset of mitosis requires: (1) quantitative imaging of intracellular Ca2+, especially the Ca2+ signal throughout the cell cycle, with high spatial and temporal resolution; and (2) identifying the molecules responsible for regulating the expression and reception of the Ca2+ signal itself. It is clear that Ca2(+)-dependent pathways are necessary elements of the mitotic process. Molecular candidates for the regulators and regulatees have yet to be identified. The upstream controlling molecules of these transmembrane Ca2+ regulatory elements, as well as the initial mitotic "start" signal, await future identification. Downstream regulation is also clearly indicated, perhaps through regulation of cyclin expression, degradation, or both.

Animals↗
PubMed1989

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

8-BrcAMP-induced capacitance and transport of H2O and Na in skin and urinary bladder of urodele amphibians.

The effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on capacitance (C), osmotic water flow (Jv), and amiloride-sensitive short-circuit current (INa) were studied in bladder and skin derived from the tiger salamander (aquatic and postmetamorphosed terrestrial phase). 8-BrcAMP-dependent increases in C, measured from the transepithelial voltage response to constant current pulses, occurred in aquatic (delta C = 44%) and terrestrial (delta C = 61%) bladders and terrestrial skin (delta C = 19%). Jv (200-mosM gradient, mucosal side hypotonic) was observed in the bladders and was further enhanced by addition of 8-BrcAMP [10(-3) M; delta Jv = 0.42 microliter.min-1.microF-1 (aquatic) and 0.32 microliter.min-1.microF-1 (terrestrial)]. The aquatic and terrestrial skins were relatively impermeable to water, but the terrestrial skin showed a small response to 8-BrcAMP (delta Jv = 0.04 microliter.min-1.microF-1). 8-BrcAMP-mediated natriferic responses were observed in aquatic bladder (delta INa = 62%) and terrestrial skin (delta INa = 105%). Antidiuretic hormone (ADH)-induced Jv was also observed in the aquatic bladder (delta Jv = 0.33 microliter.min-1.microF-1) and was similar to the 8-BrcAMP-mediated Jv measured in this tissue. The terrestrial bladder displayed a more vigorous response to 8-BrcAMP than to ADH (delta JvADH = 0.09 microliter.min-1.microF-1 and delta Jv8-BrcAMP = 0.32 microliter.min-1.microF-1), suggesting that diminished sensitivity to ADH accompanies the transition from water to land in this species.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Effect of induced hypercapnia on anaerobic metabolic rate of anoxic musk turtles.

To evaluate the possible effect of induced hypercapnia on anaerobic metabolic rate during anoxia, musk turtles (Sternotherus odoratus) were submerged in N2-equilibrated water at 10 degrees C for 3 days either with (anoxic hypercapnic) or without (anoxic normocapnic) elevated aquatic PCO2 (30-40 Torr). Control animals had access to air at 10 degrees C. Plasma [lactate] was significantly higher (P less than 0.01) in the normocapnic [59.4 +/- 7.4 (SD) mM; n = 22] than in the hypercapnic (47.4 +/- 8.5 mM; n = 19) anoxic turtles, although the hypercapnic turtles had lower blood pH (P less than 0.05). Plasma ion concentrations (Na, K, Cl, Ca, and Mg), however, were no different in the two groups, although all values other than Na were different from control. In some of the animals, [lactate] and [glycogen] (per g wet wt) of skeletal muscle, heart, and liver were measured in addition to blood acid-base values and lactate. Tissue lactates, although significantly elevated from control, and glycogens, although (with the exception of skeletal muscle) significantly reduced from control, were no different in the two anoxic groups. We suggest that these tissue data are more valid indicators of anaerobic metabolic rate than is plasma lactate and therefore conclude that induced hypercapnia does not significantly depress anaerobiosis in musk turtles at 10 degrees C.

Acid-Base Equilibrium↗
PubMed1986

Mitosis in sand dollar embryos is inhibited by antibodies directed against the calcium transport enzyme of muscle.

Monospecific antibodies to the calcium transport enzyme (alpha-Ca pump) inhibit mitosis when microinjected into sand dollar embryos. Immunoglobulins were raised against the calcium transport enzyme (Ca pump) of sarcoplasmic reticulum (SR) from rat skeletal muscle and guinea pig ileum smooth muscle. Specific antibodies were further isolated from IgG fractions by using electrophoretically purified SR Ca-pump protein as the immobilized ligand for immunoaffinity chromatography. ELISA demonstrated that common antigenic determinants are shared by SR, SR Ca pump (of rat skeletal and guinea pig ileum smooth muscle), and isolated membrane containing "native" mitotic apparatus (MA). Preimmune sera gave negative results in identical control assays. Triton X-100 extraction of MA removes the Ca-pump antigen. SR Ca pump and the MA Ca pump have nearly identical molecular masses as determined by NaDodSO4/PAGE. These alpha-SR Ca-pump IgGs inhibit ATP-dependent Ca2+ sequestration by purified SR and MA membranes. Indirect immunofluorescence of isolated native MA demonstrated coincident localization of the MA Ca pump, sequestered calcium, and membrane vesicles. Fluorescent foci were regionally concentrated within the volumes of the asters and spindle. Microinjection of the anti-Ca-pump IgGs into one of two sister blastomeres at second metaphase resulted in mitotic arrest of the injected cell accompanied by a rapid loss of spindle birefringence. Karyomeres formed and fused to form nuclei either at the site of the metaphase plate or at the position the chromosomes occupied during anaphase A. The cleavage furrow did not develop in the injected cell, while the sister and neighbor cells continued normal mitotic cycling. Injection later in mitosis yielded cells with two nuclei whose cleavage furrow relaxed completely. Routine control injections of boiled immune IgG, preimmune IgG, Wesson oil, buffer, or goat anti-rabbit IgG did not affect mitosis, birefringence of the MA, or cleavage furrow activity.

Animals↗
PubMed1986

Ionic compensation with no renal response to chronic hypercapnia in chrysemys picta bellii.

The ionic compensatory response to CO2 breathing for 3 days was studied on intact and cystectomized turtles at 10 and 20 degrees C. Arterial blood gases, pH, ionized calcium, and the plasma concentrations of Na+, K+, Cl-, total Ca2+, and total Mg2+ were measured periodically. At 20 degrees C, ureteral urine was also collected from bladderless turtles and was analyzed for pH, ions, NH3+, total CO2, osmolality, and titratable acid. When CO2 was breathed there was a compensatory change in the strong-ion difference as manifest by an increase in plasma [HCO3-] that was approximately 10 meq/l both in the 10 and 20 degrees C turtles. The only significant associated strong-ion changes observed consistent with the ionic compensatory response were increases in total and ionized Ca2+ and total Mg2+. These results were unaffected at either temperature by surgical removal of the urinary bladder. Urine collected from cystectomized turtles showed no compensatory increase in acid excretion during hypercapnia; in fact, changes occurred in the opposite direction. Urinary excretion of HCO3- and urine pH increased significantly, whereas titratable acidity decreased significantly. No significant change occurred in ammonia excretion over the three days of hypercapnia. These data argue against compensatory roles for the kidneys and urinary bladder in this species and point to internal ionic exchanges involving bone and shell.

Acid-Base Equilibrium↗
PubMed1985

Studies on the role of Ca++ in cell division with the use of fluorescent probes and quantitative video intensification microscopy.

It is clear that QVIM systems, when combined with appropriate fluorescent probes can be utilized to perform quantitative cytochemical studies on living and fixed cells. They also have the potential to facilitate studies of substances which like Ca++ are not easily studied by other means. The preliminary studies we have described support the idea that calcium ions and calcium transport enzymes may indeed play important roles in cell division and indicate that the tools we have at hand should help us further our understanding of the mitotic process.

Animals↗
PubMed1984

Early prediction of the need for hospitalization in children with acute asthma.

We studied 133 asthmatic children in the emergency room who were aged 4 to 17 years and who were not receiving ongoing training in pulmonary function testing, to determine whether spirometry correlated with the outcome of the emergency treatment of the acute attack and with relapse during the next four days. A clinical score derived from the physical exam was obtained at the time of spirometry. Patients were hospitalized or discharged home based solely on their clinical response to therapy. The pediatrician making that decision was blinded to the results of the spirometry. The initial pretreatment clinical score or spirometry alone identified the majority of patients hospitalized, but each falsely identified many discharged patients who did not relapse, 20 percent and 15 percent, respectively. Predictive criteria for hospitalization, combining spirometry with a careful clinical evaluation prior to the start of emergency treatment, substantially reduced the number of falsely identified patients. With use of the predictive criteria, the majority of children admitted to the hospital would have been identified hours before that decision was made on clinical grounds alone (5.5 +/- 0.7 hrs). This study suggests a role for spirometry as an adjunct to clinical evaluation in the early identification of the need for hospitalization of acutely ill asthmatic children.

Acute Disease↗
PubMed1983

Identification of nonmitochondrial creatine kinase enzymatic activity in isolated sea urchin mitotic apparatus.

ATP-dependent calcium sequestration was previously localized in vesicles of mitotic apparatus isolated from sea urchins. We now demonstrate that the mitotic apparatus contains an ATP-regenerative system characterized as creatine kinase (EC 2.7.3.2). Mitotic apparatus isolated with vesicles intact converted ADP to ATP if phosphocreatine was present. Omission of ADP or phosphocreatine gave negligible ATP. When mitotic apparatus were washed with detergent-containing buffer to remove vesicles, their ability to produce ATP from ADP and phosphocreatine was reduced. Assays of creatine kinase activity using NADP+:glucose-6-phosphate dehydrogenase indicated that 70% of the creatine kinase activity was extractable with 0.5% Triton X-100. The insoluble residue containing the skeleton of the mitotic apparatus had the rest of the activity. Experiments with a luciferin/luciferase assay showed that Triton removed above 82% of the activity. Preparations of intact mitotic apparatus were free of cytochrome c oxidase (EC 1.9.3.1) activity and therefore free of mitochondria. About 10(8) mitotic apparatus (total volume about 1 liter) could produce 17 mmol of ATP/min when substrates were not limiting. The creatine kinase enzyme activity described herein and the previously described membrane vesicular calcium sequestration system are nonmitochondrial, integral constituents of the sea urchin mitotic apparatus.

Creatine Kinase↗
PubMed1980

Isolation of mitotic apparatus containing vesicles with calcium sequestration activity.

We present the first report of isolate mitotic apparatus with vesicular calcium sequestration. Phase-contrast, differential interference contrast and polarized light microscopy as well as transmission and scanning electron microscopic examinations revealed structures comparable to mitotic apparatus in vivo. Numerous membrane-bound vesicles which retained their osmotic activity were present throughout. Microtubules, yolk, ribosomes and condensed chromatin were also present. The protein composition of mitotic apparatus was not dramatically altered by treatment with 0.5% Triton X-100, even though vesicles were destroyed and yolk was extracted. Calcium sequestration was demonstrated with ATP-dependent accumulation of 45Ca by mitotic apparatus whose vesicles were left intact. Compared with controls for which no nucleotide was added, accumulation by mitotic apparatus with intact vesicles was enhanced to 184% when it was present. When ATP was supplemented with the divalent ionophore A23187, the calcium retention level was comparable to that of the control to which no nucleotide was added. Finally, the calcium accumulation by mitotic apparatus treated with either of the nonhydrolyzable ATP analogs AMPPCP or AMPPNP resulted in calcium retention levels similar to those of controls. The solubilization of vesicles with Triton X-100 abolished calcium accumulation in the presence or absence of any of the above additives. Resolution of vesicles on sucrose step gradients after 45Ca-oxalate loading with ATP or AMPPCP indicates that a specific vesicular fraction sequesters 45Ca.

Animals↗