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Biomedical subjects

J C Owicki

Publications and source records attributed to J C Owicki.

At least 19 recordsLinked to original sources
PubMed1994

Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.

Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared with values determined by independent means. This technique was found to be quick, simple, reproducible, and accurate.

Antibodies↗
PubMed1994

Using microphysiometry to study the pharmacology of exogenously expressed m1 and m3 muscarinic receptors.

The microphysiometer, an instrument that uses a semiconductor-based sensor to monitor cellular metabolic activity, has been shown to detect the activation of a variety of receptors in living cells, largely irrespective of the signal-transduction mechanism. Using the Cytosensor Microphysiometer, we have studied agonist concentration responses for the activation of CHO-K1 cell lines exogenously expressing rat m1 or m3 receptors. Three levels of receptor expression were investigated for each subtype. Carbachol is more potent for m3 than m1 receptors (0.5 to 1.0 log unit lower EC50); for both, potency correlates positively with receptor density. The results agree well with those obtained by measuring phosphoinositide hydrolysis and intracellular [CA++] in the same cells. We also determined that two subtype-selective antagonists, pirenzepine (for m1) and p-fluoro-hexahydrosila-difenidol (for m3) displayed appropriate differential ability to shift carbachol concentration-response curves in the microphysiometer. This study provides additional evidence that pharmacological results obtained by microphysiometry are consistent with those obtained by more conventional functional assays.

Animals↗
PubMed1994

Direct measurement of extracellular proton flux from isolated gastric glands.

We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Microfabrication in silicon microphysiometry.

Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability. We present results from a research instrument with eight separate assay channels on a 5-cm2 chip. We discuss the planned evolution of the technology toward high-through-put instruments and instruments capable of performing single-cell measurements.

Biosensing Techniques↗
PubMed1993

A metabolic view of receptor activation in cultured cells following cryopreservation.

The effect of cryopreservation on agonist-induced receptor activation in mammalian cells was investigated with the Cytosensor microphysiometer, a biosensor that monitors cellular metabolic activity by measuring changes in extracellular pH. In this study, two different cell types--nonadherent TF-1 cells (from a human erythroleukemia patient) and adherent WT3 cells (CHO-K1 cells transfected with the m1 muscarinic acetylcholine receptor)--were cryopreserved by freezing in a disposable cell capsule used in the microphysiometer. The recovery of metabolic activity by TF-1 cells was observed over approximately 1 h following thawing. Responses of the TF-1 cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet activating factor (PAF) were measured before cryopreservation and 90 min after thawing. The GM-CSF and PAF responses retained 71 +/- 14% and 73 +/- 10% of maximum stimulation, respectively. Post-thaw cholinergic stimulation of WT3 cells was 73 +/- 9% of its level in similarly treated but unfrozen cells. Cryopreservation caused no detectable difference in desensitization of the response due to repeated application of carbachol. These results demonstrate the feasibility of pharmacological studies with cryopreserved cells in the microphysiometer and further suggest that the microphysiometer may be useful in exploring the biological consequences of cryopreservation in the early post-thaw period.

Animals↗
PubMed1993

Cholinergic stimulation of the Na+/K+ adenosine triphosphatase as revealed by microphysiometry.

The activation of a wide range of cellular receptors has been detected previously using a novel instrument, the microphysiometer. In this study microphysiometry was used to monitor the basal and cholinergic-stimulated activity of the Na+/K+ adenosine triphosphatase (ATPase) (the Na+/K+ pump) in the human rhabdomyosarcoma cell line TE671. Manipulations of Na+/K+ ATPase activity with ouabain or removal of extracellular K+ revealed that this ion pump was responsible for 8.8 +/- 0.7% of the total cellular energy utilization by those cells as monitored by the production of acid metabolites. Activation of the pump after a period of inhibition transiently increased the acidification rate above baseline, corresponding to increases in intracellular [Na+] ([Na+]i) occurring while the pump was off. The amplitude of this transient was a function of the total [Na+]i excursion in the absence of pump activity, which in turn depended on the duration of pump inhibition and the Na+ influx rate. Manipulations of the mode of energy metabolism in these cells by changes of the carbon substrate and use of metabolic inhibitors revealed that, unlike some other cells studied, the Na+/K+ ATPase in TE671 cells does not depend on any one mode of metabolism for its adenosine triphosphate source. Stimulation of cholinergic receptors in these cells with carbachol activated the Na+/K+ ATPase via an increase in [Na+]i rather than a direct activation of the ATPase.

Biophysical Phenomena↗
PubMed1993

Aggregation of hapten-bearing liposomes mediated by specific antibodies.

We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.

Animals↗
PubMed1992

PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments.

We have used microphysiometry and antisense methodology to show that the epsilon isoenzyme of protein kinase C (PKC) is involved in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a human bone marrow cell line, TF-1. These cells require GM-CSF or a related cytokine for proliferation. When the cells are appropriately exposed to GM-CSF, they exhibit a burst of metabolic activity that can be detected on the time scale of minutes in the microphysiometer, a biosensor-based instrument that measures the rate at which cells excrete protons. These cells express PKC alpha and -epsilon, as determined by Western blot analysis. Treatment with isoenzyme-specific antisense oligonucleotides inhibits expression appropriately, but only inhibition of PKC epsilon appreciably diminishes the burst of metabolic activity induced by GM-CSF. Consistent with the involvement of PKC epsilon, GM-CSF appears to activate phospholipase D and does not cause a detectable increase in cytosolic [Ca2+].

Alkaloids↗
PubMed1992

The cytosensor microphysiometer: biological applications of silicon technology.

A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

Animals↗
PubMed1992

Biosensors based on the energy metabolism of living cells: the physical chemistry and cell biology of extracellular acidification.

The silicon microphysiometer is a biosensor-based instrument that detects changes in the physiological state of cultured living cells by monitoring the rate at which the cells excrete acidic products of metabolism. This paper discusses the chemical and biological factors that determine the performance and applications of such a system. Under typical culture conditions, extracellular acidification is dominated by the excretion of lactic and carbonic acids formed during the energy metabolism, using glucose and glutamine as carbon sources. The maintenance of transmembrane ionic gradients is an important use of energy, as is cell growth. The activation of cellular receptors usually causes transient or sustained increases in acidification rate. The energetic cost of generating second messengers is probably too small to account for either change, so events more distal to the receptor-activation process must be responsible. The opening of ion channels may cause the increases in some cases. In others, changes in intracellular pH and loose coupling between ATP hydrolysis and synthesis may be involved; models for these processes are presented.

Acids↗
PubMed1992

Effects of excitotoxin exposure on metabolic rate of primary hippocampal cultures: application of silicon microphysiometry to neurobiology.

Increasing evidence implicates glutamate receptor over-stimulation in the neurotoxicity associated with a host of metabolic insults, including seizures and hypoxia-ischemia. To begin to understand more completely the role of energy metabolism in the mechanism of neuron death following excitatory amino acid exposure, we investigated the effects of kainic acid exposure on metabolic rate in cultured hippocampal cells using a recently developed silicon microphysiometer. The device gives a continual real-time measure of metabolism in relatively small numbers of cells, as assessed by efflux of protons generated at least in part by ATP hydrolysis and lactic acid production. In the first half of this report, we characterize the feasibility of using this device for measuring cellular metabolism in hippocampal cultures. Metabolic rate in both astrocytes and neurons was readily detectable, with a high signal-to-noise ratio. The rate was proportional to the number of cells and was sensitive to metabolic enhancement or depression. We then utilized this device to study metabolic responses to the excitotoxin kainic acid. We observed a receptor-mediated, dose-dependent increase in metabolic rate upon stimulation by kainic acid, with an EC50 of approximately 100 microM. Exposure to toxic levels of kainic acid for 10 min produced an initial elevation (for 2 hr) in metabolic rate and then a gradual decline in metabolism over the next 8 hr that preceded a measurable loss of cell viability. This study further delineates a time window for the onset of kainic acid-induced damage. The results clearly show the feasibility of using silicon microphysiometry for assessing metabolism of brain cultures and for exploring the relationship between metabolism and synaptic activation.

Animals↗
PubMed1990

Continuous monitoring of receptor-mediated changes in the metabolic rates of living cells.

Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal growth factor receptors in human keratinocytes produces 15% to 200% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several factors including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta 2-adrenergic receptors, isoproterenol produces a 20-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 12 min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.

Animals↗
PubMed1990

Biosensors for directly measuring cell affecting agents.

Cellular perfusion chambers have been constructed from the Light Addressable Potentiometric Sensor (LAPS) previously described. The authors have used these chambers to measure the effects of a variety of agents on the metabolic rates of cells. The chambers are used in a stopped flow mode. When flow is on, samples may be introduced to the chamber. When flow is stopped, acidification of the very small volume of medium in the chamber is used to determined the metabolic rate of the cells. Using a variety of types of mammalian cells the authors have demonstrated the following. The triggering of cellular receptors can be determined in minutes. Metabolic inhibition of normal human cells by a test compound can be correlated with the compounds in vivo ocular irritancy. And the efficacy of chemotherapeutic agents on tumor cells exhibiting multidrug resistance can be determined in a few hours.

Biosensing Techniques↗
PubMed1989

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Biosensing Techniques↗
PubMed1989

Concentration effects on reactions in membranes: rhodopsin and transducin.

The reaction rate of two laterally-diffusing species in a biological membrane shows a maximum at some concentration of reactants, because an increase in the concentration of reactants tends to increase the reaction rate by the law of mass action but decreases the diffusion rate of the reactants. The activation of transducin by rhodopsin in the disk membrane of the rod outer segment is described in terms of a steady-state diffusion model with concentration-dependent diffusion coefficients. The optimum concentrations of reactants are obtained from contour plots of the reaction rate as a function of rhodopsin and transducin concentrations, and the sensitivity of the results to the assumed values of the variables is examined. To determine whether the observed concentrations are in fact those yielding the maximum reaction rate, several variables must be known more accurately.

Diffusion↗