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Biomedical subjects

K R Gee

Publications and source records attributed to K R Gee.

32 records · Page 2Linked to original sources
PubMed1998

Control of NO concentration in solutions of nitrosothiol compounds by light.

We studied the thermal and photolytic decomposition of two S-nitrosothiols, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP), in water or propanol solutions. A "concentration clamp" (relatively constant concentration of NO as a function of time) could be implemented in a closed volume by varying the pH, concentration of nitrovasodilator and intensity of the light source. Depending on the conditions, the light either stimulated NO release or sharply decreased NO concentration in the test solutions. Changes in the absorption spectra of GSNO solutions were monitored as a function of light exposure. Generation of superoxide as a product of a photolytic decomposition reaction of S-nitrosothiols and further oxidation of NO is the most likely mechanism for light suppression of NO concentration.

Glutathione↗
PubMed1997

Caged nicotinic acid adenine dinucleotide phosphate. Synthesis and use.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADP as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 micro;M when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.

Animals↗
PubMed1997

Chemical two-photon uncaging: a novel approach to mapping glutamate receptors.

Functional mapping of neurotransmitter receptors requires rapid and localized application of transmitter. The usefulness of caged glutamate for this purpose has been limited, because photolysis by unfocused light above and below the target cell limits depth resolution. This problem is eliminated by using a double-caged glutamate that requires absorption of two photons for conversion to active glutamate, resulting in a substantial improvement in spatial resolution over conventional caged glutamate. This method was used to map the distribution of glutamate receptors on hippocampal pyramidal neurons. A higher density of AMPA receptors was found on distal apical dendrites than on basal or primary apical dendrites, suggesting that synaptic efficacy is locally heterogeneous. Such "chemical two-photon uncaging" offers a simple, general, and economical strategy for spatially localized photolysis of caged compounds.

2-Amino-5-phosphonovalerate↗
PubMed1996

Activation and inactivation of Ca2+ release by NAADP+.

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is a recently identified metabolite of NADP+ that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP+ in intact sea urchin eggs elicits long term Ca2+ oscillations. On the other hand, uncaging threshold amounts of NAADP+ produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP+ receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP+-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization.

Animals↗
PubMed1996

Synthesis and photochemical properties of a kainate precursor and activation of kainate and AMPA receptor channels on a microsecond time scale.

Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolylpropionate (AMPA) receptors are transmembrane proteins that can form ion channels upon binding a specific ligand. The receptors are located at major excitatory synapses in the mammalian central nervous system. Kainate and AMPA receptors participate in many physiological activities of the brain, including learning and memory, and are involved in many neurological disorders. Elucidation of the mechanisms of receptor transmembrane channel formation, inhibition, and regulation is important in understanding fundamental central nervous system function and in designing potential therapeutic agents. Kainate can activate both kainate and AMPA receptors, leading to channel opening in the microsecond to millisecond time region. A newly developed laser pulse photolysis technique, with a microsecond time resolution, has been successfully used to study the chemical reactions of receptor proteins in the microsecond to millisecond time region. To apply the technique to kainate and AMPA receptors, a photolabile kainate precursor in which a caging group, the alpha-carboxy-2-nitrobenzyl group, is attached to the gamma-carboxyl group of kainic acid has been synthesized. The photolytic release of free kainate from the caged kainate on the microsecond time scale, initiated by a pulse of laser light at 308 nm, was measured. The quantum yield is 0.34 at pH 6.8 and room temperature. The half-life of the major component (approximately 86%) of the photolytic reaction is 45 microseconds, while that of the minor component (approximately 14%) is 0.7 ms. The effects of the caged kainate on kainate and AMPA receptors endogenously expressed in rat hippocampal neurons were also evaluated. Caged kainate (750 microM) did not activate the receptor channels, nor did it potentiate or inhibit the kainate response. Photolysis of the caged kainate by a pulse of 333-nm laser light resulted in a rapid rise (with a t1/2 of 0.4 ms) in the whole-cell current due to the opening of kainate-activated receptor channels. The results presented demonstrate that this kainate precursor is suitable for rapid chemical kinetic investigations of the kainate and AMPA receptors in the microsecond to millisecond time region.

Animals↗
PubMed1996

Combining 'caged-dopamine' photolysis with fast-scan cyclic voltammetry to assess dopamine clearance and release autoinhibition in vitro.

We have developed a methodology for inducing a rapid rise in extracellular dopamine concentrations. The clearance of the applied dopamine, as well as its effect on the endogenous dopamine release (i.e., autoinhibition), was then examined using fast scan cyclic voltammetry. In a recording chamber mounted on a Nikon Optiphot epifluorescence microscope, coronal rat brain slices containing either the caudate nucleus or prefrontal cortex were perfused with ACSF containing 100-200 microM 'caged-DA.' UV illumination (100-200 ms) focused at the tip of the recording electrode produced a peak DA concentration of 1-2 microM within 100-200 ms of terminating the illumination. The caudate nucleus exhibited a faster clearance rate for photo-released DA compared to the prefrontal cortex. Cocaine reduced the clearance rates in both the caudate nucleus and prefrontal cortex. In the prefrontal cortex a combination of desipramine/clomipramine also reduced dopamine clearance, suggesting heterologous uptake of the applied DA by noradrenergic and/or serotonergic terminals. Photo-released dopamine inhibited release of endogenous caudate DA release evoked by single electrical stimulation. The advantages of this methodology are discussed.

Animals↗
PubMed1994

Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale.

Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -CO2- group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. gamma-O-(alpha-Carboxy-2-nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14. The half-life of the major component of the photolytic reaction, as judged by the transient absorbance change at 430 nm, is 21 microseconds (approximately 90%); the half-life of a minor component (approximately 10%) is 0.2 ms. The amino-linked derivatives have half-lives in the millisecond region and a 4-fold lower quantum yield. The potential of the newly synthesized compound for use in rapid chemical kinetic investigations of glutamate receptors is demonstrated. (i) The caged glutamate at 1 mM concentration does not desensitize glutamate receptors in rat hippocampal neurons. (ii) Caged glutamate (1 mM) does not inhibit activation of the receptors by 50 microM glutamate. (iii) Photolysis of the compound induces rapid onset of transmembrane currents in rat hippocampal neurons.

Animals↗
PubMed1993

10,5-(Iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene and derivatives. Potent PCP receptor ligands.

IDDC (3, 10,5-(iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene++ +) and a series of substituted derivatives were synthesized and evaluated in vitro for their ability to displace tritiated MK-801 ([3H]-2) from its specific binding site in guinea pig brain homogenate. Substitution at the 3-position of 3 with bromine, chlorine, and fluorine led to increased binding affinity. In contrast, substitution of donor groups at the 3-position gave decreased binding affinities, as did all substitutions at the 7-position and on nitrogen. Where racemic mixtures were resolved, the (+)-optical antipodes were more active than their enantiomers or racemates. The most active ligand found in this study was (+)-13e (IC50 = 15.5 +/- 4.5 nM). The affinity of (+)-13e for the PCP receptor makes it among the most potent ligands known. In vitro neuroprotection was demonstrated by 3, (+)-3, and (+)-6 (N-Me-IDDC) against glutamate-induced cell death in rat hippocampal cells.

Animals↗
PubMedDate not supplied

Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin.

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.

Avidin↗
PubMedDate not supplied

N-(3-azidophenyl)-N-methyl-N'-([4-1H]- and [4-3H]-1-naphthyl)guanidine. A potent and selective ligand designed as a photoaffinity label for the phencyclidine site of the N-methyl-D-aspartate receptor.

A novel radiolabeled photoaffinity ligand has been synthesized for the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor. N-(3-Azidophenyl)-N-methyl-N'-([4-3H]-1-naphthyl)guanidine (13) was prepared with a specific activity of 25 Ci/mmol by diazotization of N-(3-aminophenyl)-N-methyl-N'-([4-3H]-1-naphthyl)guanidine (12) followed by treatment with sodium azide. Guanidine 12 was obtained by catalytic tritiation of N-(4-bromo-1-naphthyl)-N'-methyl-N'-(3-nitrophenyl)guanidine (11). The nontritiated analog 5 of 13 was prepared beginning with N-methyl-N'-1-naphthyl-N-(3-nitrophenyl)guanidine (9). The guanidines 9 and 11 were prepared in moderate yield by the aluminum chloride-catalyzed reaction of N-methyl-3-nitroaniline hydrochloride with 1-naphthylcyanamide and 4-bromo-1-naphthylcyanamide, respectively. Azide 5 showed high selectivity and affinity (IC50 = 100 nM vs [3H]MK801; 3000 nM vs [3H]ditolylguanidine) for the PCP site of the NMDA receptor in guinea pig brain homogenate. Photolabeling experiments with 13, however, failed to radiolabel a significant amount of receptor polypeptide.

Affinity Labels↗
PubMedDate not supplied

Chlorinated phenyl azides as photolabeling reagents. Synthesis of an ortho,ortho'-dichlorinated arylazido PCP receptor ligand.

The enhanced photolabeling properties of chlorinated phenyl azides are demonstrated by the synthesis and photolysis of methyl 4-azido-2,3,5,6-tetrachlorobenzoate (3) and methyl 4-azido-3,5-dichlorobenzoate (4). Photolysis of azide 3 in 1 M diethylamine/cyclohexane as the trapping medium gave 34% NH-insertion product. Similar photolysis of azide 4 gave 35% NH insertion product. These results demonstrate that chlorinated phenyl azides are significantly better at undergoing NH insertion than nonhalogenated analogs and suggest that improvement of existing aryl azide-based photolabels might be achieved by introduction of chlorine atoms on either side of the azide group. As an application, 3-azido-2,4-dichloro-10,5-(iminomethano)-10,11-dihydro-5H- dibenzo[a,d]cycloheptene (19), an analog of the potent PCP receptor ligand IDDC (14), was synthesized and its affinity for the PCP receptor was determined to be 6.3 +/- 0.7 microM (IC50 against [3H]MK801).

Affinity Labels↗