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K L Kirk

Publications and source records attributed to K L Kirk.

At least 73 records · Page 4Linked to original sources
PubMed1989

18F-labeled insulin: a prosthetic group methodology for incorporation of a positron emitter into peptides and proteins.

In the present study we synthesize 18F-labeled insulin of high specific radioactivity. A new prosthetic group methodology, in which [18F]fluoride displaces a bromide group of 4-(bromomethyl)-benzoylamine intermediates, was used. The 4-(fluoromethyl)benzoyl product was chemically stable. 18F-Labeled insulin retains the essential biological properties of native insulin, as measured in vitro by binding to insulin receptors on human cells and stimulation of glucose metabolism in rat adipocytes. The overall process can be carried out speedily to yield a product of sufficient purity to permit in vivo studies. The method appears to be applicable to a wide variety of peptides.

Adipose Tissue↗
PubMed1989

Macromolecular transport in rabbit blastocysts: evidence for a specific uteroglobin transport system.

Though uterine proteins are found within the blastocoel of the rabbit blastocyst, the mechanisms involved in protein entry into the blastocoel have not been studied. To investigate potential avenues of protein entry into the blastocoel, we have monitored uptake of a fluorescent, fluid-phase marker (lucifer yellow) into the rabbit blastocyst trophectodermal cell. In addition, we have measured the transtrophectodermal permeabilities of several uterine proteins (uteroglobin (UTG), rabbit serum albumin, rabbit IgG), and radiolabeled fluid-phase markers (sucrose, polyethylene glycol, dextran) in the 6- and 7-day post-coitus rabbit blastocyst. Extrablastocoelic lucifer yellow (LY) was rapidly endocytosed by the trophectodermal cell and was subsequently trapped in a perinuclear compartment for at least 30 min after removal of the extracellular dye. The slow rate of LY turnover within the endocytic compartments implies that the rate of fluid-phase transcytosis was negligible. Permeability coefficients of the fluid-phase markers and proteins, with the exception of UTG, decreased with increasing molecular weight of each compound tested. These data are consistent with the conclusion that these compounds traverse the trophectoderm via a 'leak' pathway, as opposed to a transcytotic pathway. In contrast, permeability of 125I-UTG when compared to the other compounds, was 10-fold greater than would be predicted based on its molecular weight. Furthermore, the flux of radioiodinated UTG displayed saturation kinetics with a Km of 19.5 micrograms/ml and a Vmax of 132 ng.cm-2.h-1. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the radioiodinated protein recovered from the blastocoel was of a lower molecular weight than native UTG and was not immunoreactive with goat anti-UTG antibody. Our data are consistent with the idea that UTG is transported across the trophectoderm by a receptor-mediated system, and UTG is modified intracellularly during transport to a protein of a lower molecular weight.

Animals↗
PubMed1989

Defective regulation of epithelial Cl- permeability and protein secretion in cystic fibrosis: the putative basic defect.

The search for a basic functional defect in CF appears to be converging on a defect in the regulation of epithelial Cl- permeability and perhaps protein secretion. Fundamental issues that remain unresolved include (1) the identity of the CF gene, (2) the precise role played by the CF gene product in regulating Cl- permeability and protein secretion, and (3) the identities and properties of alternate pathways for regulating Cl- permeability and protein secretion that are not compromised in CF. The first issue should be resolved in the near future as molecular genetic approaches are used to pinpoint the location of the CF locus on chromosome 7. The second issue is more complex and will require the development of generally useful assays of Cl- permeability and protein secretion that can be used to assess the abilities of candidate CF gene products to complement, or correct, the functional defect in CF cells. Characterizing the precise function of the CF gene product may be difficult if the regulatory pathways that control these cellular processes are complex (ie, involve multiple regulatory steps and second messengers) or if the CF gene is a regulatory gene (rather than a structural gene) that represses or induces the synthesis of proteins involved in modulating Cl- permeability and protein synthesis. The third issue relates to the development of therapeutic strategies for treating CF patients that involve elevating epithelial Cl- permeability or modulating protein secretion by pharmacologically activating regulatory pathways that are unaffected in CF. In this regard, it is important to note that the stimulation of the Cl- permeabilities of airway epithelial cells by Ca2+-mediated secretagogues appears not to be compromised in CF. Pharmacological manipulation of this or other regulatory pathways may provide a means to activate the Cl- permeabilities of CF affected cells.

Chlorides↗
PubMed1989

Cl- permeability of human sweat duct cells monitored with fluorescence-digital imaging microscopy: evidence for reduced plasma membrane Cl- permeability in cystic fibrosis.

Salt reabsorption by the human sweat duct is markedly reduced in cystic fibrosis (CF). We used fluorescence-digital imaging microscopy in combination with a halide-specific fluorescent dye [6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] to determine if this defective salt reabsorption is referable to a reduced plasma membrane Cl- permeability of the epithelial cells that line the sweat duct. Sweat duct cells were cultured from explants of normal and CF reabsorptive duct and loaded with SPQ, the fluorescence of which is specifically quenched by halide ions (Br- greater than Cl-) and provides a relative index of intracellular halide concentration. Two lines of evidence indicate that normal sweat duct cells exhibit a substantial permeability to Cl- and Br-. First, the replacement of extracellular Cl- with an impermeant anion (i.e., gluconate) resulted in a rapid and reversible increase in the intracellular fluorescence, as expected if the cells rapidly lost Cl- to the extracellular media. Second, the replacement of extracellular Cl- with Br- resulted in a rapid and reversible quenching of the intracellular fluorescence, as expected if the cells accumulated Br- (a more effective quencher of SPQ fluorescence) in exchange for Cl-. The rate of fluorescence change that was induced by either maneuver was inhibited by the Cl- channel blocker, diphenylamine-2-carboxylate (10 microM). Moreover, CF cells exhibited markedly reduced rates of fluorescence change in response to either maneuver. Our results document the utility of this imaging strategy for assessing the Cl- permeabilities of individual epithelial cells that are affected in cystic fibrosis and indicate that the defective salt reabsorption by the CF sweat duct is referable, at least in part, to a reduced plasma membrane Cl- permeability of sweat duct cells.

Adolescent↗
PubMed1989

Quality control procedure for 6-[18F]fluoro-L-DOPA: a presynaptic PET imaging ligand for brain dopamine neurons.

The goal of the current study was to establish a quality control procedure for clinical use of 6-[18F]fluoro-L-DOPA (6-[18F]-DOPA) as a selective presynaptic positron emission tomographic (PET) imaging ligand for brain dopamine neurons. A high performance liquid chromatographic procedure using a 5-mu C-18 reverse phase column and ion-pairing mobile phase was used for the quantification of 6-[18F]-DOPA. The radiochemical purity of 6-[18F]-DOPA was measured by 18F radioactivity in HPLC fractions while the chemical purity was determined by an amperometric electrochemical detector with a sensitivity of 25 pg. Quality control of eight consecutive batches of highly purified 6-[18F]-DOPA sample used in a pre-clinical trial revealed that the chemical and/or radiochemical purity of the PET imaging ligand, 6-[18F]-DOPA was greater than 97 +/- 0.5% with a specific activity of 365 +/- 31 mCi/mmol. The knowledge and assurance of radiochemical purity of PET ligands are essential for the interpretation of clinical PET imaging results. The assurance of such quality control would enable comparisons of 6-[18F]-DOPA/PET data obtained from various medical centers using different radiopharmaceutical procedures.

Brain↗
PubMed1989

Neuronal uptake and metabolism of 2- and 6-fluorodopamine: false neurotransmitters for positron emission tomographic imaging of sympathetically innervated tissues.

The neuronal uptake and metabolism of 2-fluorodopamine (2F-dopamine), 6-fluorodopamine (6F-dopamine) and tritium-labeled dopamine were compared in heart, submaxillary gland and spleen of rats to assess the utility of 18F-labeled 2F- or 6F-dopamine for positron emission tomographic imaging of sympathetically innervated tissues. Tritiated dopamine with and without 2F- or 6F-dopamine, or tritiated 2F-dopamine alone, were injected i.v. into rats that were or were not pretreated with desipramine to block catecholamine neuronal uptake or with reserpine to block vesicular translocation of catecholamines. Tissue and plasma samples were obtained at intervals up to 1 hr after injections. At 1 hr after injection of tritiated dopamine, tritium-labeled norepinephrine, dopamine, dihydroxyphenylacetic acid and dihydroxyphenylglucol accounted for less than 2% of the tritium in plasma but up to 92% of that in tissues; tritiated norepinephrine accounted for 70% or more of the tritium in tissues. In contrast, at 1 hr after injection of tritiated 2F-dopamine, tritiated 2F-norepinephrine accounted for 30 to 46% of the tritium in tissues. Desipramine and reserpine pretreatment blocked the tissue accumulation of tritiated and fluorinated dopamine as well as their dihydroxy-metabolites, indicating that accumulation of exogenous norepinephrine and dopamine analogs was within sympathetic storage vesicles. Relative to the doses of dopamine precursors, less 2F- and 6F-norepinephrine accumulated in tissues than tritiated norepinephrine, due largely to inefficient beta-hydroxylation of fluorinated dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

8-Substituted xanthines as antagonists at A1- and A2-adenosine receptors.

Two classes of 8-substituted analogs of theophylline (1,3-dialkylxanthines), having 8-cycloalkyl, 8-cycloalkenyl or 8-(para-substituted aryl) groups, were shown to be potent and, in some cases, receptor subtype selective antagonists at A1- and A2-adenosine receptors. New analogs based on a functionalized cogener approach and on classical medicinal chemical approaches were prepared. Affinity at A1-adenosine receptors was evaluated by inhibition of binding of [3H]N6-phenylisopropyladenosine to rat brain membranes. Activity at A2-adenosine receptors was measured by the reversal of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated production of cyclic AMP in membranes from rat pheochromocytoma PC12 cells. Cycloalkenyl analogs containing rigid olefinic bonds differed greatly in potency from the saturated analogs. The selectivity of phenylsulfonamide analogs depended on distal structural features. Novel xanthine analogs include diamino-, thiol-, aldehyde, and halogen-substituted derivatives, peptide conjugates of 8-[4-[2-aminoethylaminocarbonylmethyloxy]phenyl]1,3-dipropylxan thi ne (XAC), and a hydroxyethylamide analog of XAC.

Adenosine↗
PubMed1988

Autoradiographic localization of mouse brain adenosine receptors with an antagonist ([3H]xanthine amine congener) ligand probe.

A [3H]xanthine amine congener (XAC), a potent adenosine receptor antagonist, binds in a saturable and reversible fashion to high affinity binding sites in mouse brain (Bmax = 323 +/- 17 fmol/mg protein, Kd = 1.4 +/- 0.4 nM). Adenosine receptor agonists and antagonists are more potent than adenosine uptake inhibitors in displacing the binding of [3H]xanthine amine congener ([3H]XAC). The anatomical distribution of [3H]XAC binding sites is consistent with its being a ligand probe for adenosine receptors. High binding site densities were observed in the hippocampus (stratum oriens and radiatum, molecular layer), superior colliculus (superficial gray), cerebellum (molecular layer), cerebral cortex and substantia nigra. The availability of a high affinity antagonist radioligand probe like [3H]XAC for adenosine receptors allows the comparative quantitative autoradiographic analysis of agonist and antagonist binding to adenosine receptors, e.g. under varying in vitro incubation conditions (presence and absence of guanine nucleotides and cations).

Animals↗
PubMed1988

Radiosynthesis of NCA [carbonyl-11C]6-fluoromelatonin.

A method is described for the preparation of [carbonyl-11C]6-fluoromelatonin for intravenous injection and potential study of the melatonin system in vivo by positron emission tomography. The preparation is based on the acetylation of 6-fluoro-5-methoxytryptamine with NCA [1-11C]acetyl chloride (itself prepared from cyclotron-produced [11C]carbon dioxide) and purification by HPLC. It gives chemically and radiochemically pure [carbonyl-11C]6-fluoromelatonin in 35% radiochemical yield (from [11C]CO2, decay-corrected) within 35 min from the end of radionuclide production and with high specific activity e.g. 1.6 GBq/mumol (43 mCi/mumol) at the end of synthesis from 1.1 GBq (30 mCi) of [11C]carbon dioxide.

Carbon Radioisotopes↗
PubMed1988

Syntheses and adrenergic activities of ring-fluorinated epinephrines.

The study of chemical and biological effects of fluorine substitution on the aromatic ring of catecholamines has now been extended to epinephrine (Epi). 2- and 6-fluoroepinephrines (2-FEpi and 6-FEpi) have been synthesized. Fluorine substitution on the 2- or 6-carbon of the aromatic ring alters the selectivity of epinephrine toward alpha- and beta-adrenergic receptors, similar in manner to the change in selectivity seen with norepinephrine (NE). Thus, 2-FEpi is a relatively selective beta-adrenergic ligand, while 6-FEpi is a relatively selective alpha-adrenergic ligand. Fluorine substitution of Epi also can markedly increase potency at either alpha- or beta-adrenergic receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Complex interactions of agonists with alpha 1-adrenoceptors in intact cells.

1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

Adrenergic alpha-Agonists↗
PubMed1988

In vitro and in vivo studies of the effects of halogenated histidine analogs on Plasmodium falciparum.

The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by these two compounds, 50% inhibition resulting at 200 microM concentration. Morphological analysis of parasite development proved a sensitive assay, since development of mature trophozoites was inhibited 50% by 25 microM 2-F-HIS or 100 2-I-HIS. Electron microscopy studies suggested different mechanisms of action of 2-F-HIS and 2-I-HIS on P. falciparum. 2-F-HIS produced a decrease in knob number at the erythrocyte surface and accumulation of electron-dense material under the parasite membrane. 2-I-HIS had no obvious effect on knobs or electron-dense material but affected parasite morphology. Surprisingly, 2-chloro-L-histidine and 2-bromo-L-histidine did not inhibit P. falciparum in vitro, even though their halogen atom substituents are intermediate in size between F and I atoms. 2-F-HIS and 2-I-HIS were tested in vivo against P. falciparum in owl monkeys (Aotus sp.) but were ineffective at doses that were nontoxic.

Amino Acids↗
PubMed1988

Binding and internalization of a fluorescent vasopressin analogue by collecting duct cells.

The kinetics of binding and internalization of a fluorescent vasopressin analogue [1-desamino-8-rhodamine lysine vasopressin (rhoda LVP)] by principal cells within the microperfused rabbit cortical collecting tubule were quantitatively assessed with computer-assisted video microscopy. At 25 degrees C, binding of rhoda LVP exhibited saturation kinetics with half-maximal binding at 2 nM and maximal binding at concentrations greater than 5 nM. Rhoda LVP binding could be prevented by the simultaneous addition of a 10-fold higher concentration of arginine vasopressin (AVP) or the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (desmopressin). No obvious internalization or rhoda LVP was detected at 25 degrees C, i.e., the rhoda LVP fluorescence remained localized to the basal pole of each principal cell for at least 100 min after rhoda LVP addition and could be largely reversed by the subsequent addition of AVP. Conversely, warming the cells to 38 degrees C after binding was initiated resulted in a rapid (less than 30 min) migration of the fluorescence into the cell interior and a loss of AVP-displaceable binding from the cell surface. These results document the utility of this noninvasive optical strategy for quantitatively monitoring hormone binding to intact collecting tubule cells and demonstrate that rhoda LVP binds reversibly and with high affinity to V2 receptors on principal cells in the collecting tubule. The internalization (and presumed inactivation) of the hormone-receptor complex at 38 degrees C may contribute to the desensitization of collecting tubule cells to vasopressin at physiological temperature.

Animals↗
PubMed1988

Origin of ADH-induced vacuoles in rabbit cortical collecting tubule.

The origin of the vacuoles that form in the mammalian collecting duct during antidiuretic hormone (ADH)-mediated water reabsorption was examined using two computer-assisted, light microscopic methods. First, differential interference-contrast microscopy was used in combination with a simple morphometric procedure to quantitatively characterize the time course, magnitude, and cell specificity of vacuole formation in the microperfused rabbit cortical collecting tubule. Second, video-intensified fluorescence microscopy was used to visualize the basolateral endocytosis of a fluorescent, fluid-phase marker (i.e., lucifer yellow) during vacuole formation. In the presence of a lumen-to-bath osmotic gradient, ADH addition induced the rapid (less than 10 min) formation of large (1- to 3-micron diam) vacuoles in principal cells and, to a lesser extent, in a subpopulation of intercalated cells. The vacuoles subsequently shrank and disappeared over the course of 60-90 min in the continued presence of the hormone and osmotic gradient. The vacuoles collapsed very slowly after elimination of the osmotic gradient at the peak of the vacuolation response, which implies that these structures are intracellular compartments rather than dilated extracellular spaces. During their formation the vacuoles could be loaded with peritubular (but not luminal) lucifer yellow, which remained trapped within most of these structures well after the dye was removed from the bath (greater than 30 min). These results indicate that most vacuoles that form during ADH-mediated water reabsorption are intracellular, endocytic compartments that communicate with the peritubular space via endocytosis of basolateral cell membrane.

Animals↗
PubMed1987

Adenosine analogs with covalently attached lipids have enhanced potency at A1-adenosine receptors.

Chemically functionalized congeners of N6-phenyladenosine and 1,3-dipropyl-8-phenylxanthine have been covalently coupled to fatty acids, diglycerides, and a phospholipid. The lipid-drug conjugates inhibit R-[3H]-phenylisopropyladenosine binding to A1-adenosine receptors in rat cerebral cortex membranes. A xanthine-phosphatidylethanolamine conjugate bound with a Ki value of 19 nM. Various xanthine esters of low potency are potential prodrugs. Amides of an adenosine amine congener (ADAC) with 18-carbon fatty acids exhibited Ki values at A1-adenosine receptors of 70 pM, representing a 130-fold enhancement over the affinity of the corresponding acetyl amide. The very high affinity of adenosine-lipid conjugates may be due to stabilization of these adducts in the phospholipid microenvironment of the receptor protein.

Adenosine↗
PubMed1987

Molecular probes for extracellular adenosine receptors.

Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The "functionalized congener" approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki approximately 10(-9)-10(-8) M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed.

Affinity Labels↗