Biomedical subjects

K L Kirk

Publications and source records attributed to K L Kirk.

At least 19 recordsLinked to original sources

PubMed1992

Regulation of plasma membrane recycling by CFTR.

The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

Base Sequence↗

PubMed1992

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

B-Lymphocytes↗

PubMed1992

Incorporation of 2-fluorohistidine in murine protein in vivo.

The tissue distribution and time course of incorporation into acid insoluble (bound) and acid soluble (free) fractions of [3H]2-fluorohistidine is compared to that of U[14C]Histidine in mouse tissues in vivo. The cycloheximide-sensitive incorporation of 2-FHis is between 9 and 17 percent of that of His. Unlike [14C]His a major fraction, approximately 90% at 72 hrs, of isotope derived from [3H]2-FHis remains in tissues for a prolonged period in an acid soluble form. The excretion of isotope derived from [14C]His (T1/2 = 5 hr) is more rapid than from [3H]2-FHis (T1/2 = 11.4 hrs). 2-FHis, at doses from 100 to 250 mg/kg produce a reversible inhibition of growth in mice.

Animals↗

PubMed1992

Regulated endocytosis in a chloride secretory epithelial cell line.

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.

Cell Line↗

PubMed1992

Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes.

The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.

Amiloride↗

PubMed1992

Quantitation of alveolar distribution of liposome-entrapped antioxidant enzymes.

Liposome-encapsulated Cu,Zn superoxide dismutase (Cu,Zn SOD) and catalase (CAT) were instilled intratracheally in rabbits, and the temporal and spatial distribution of Cu,Zn SOD and CAT within the lung was assessed at the organ and cellular levels. Specific activities of Cu,Zn SOD and CAT were increased in both lung homogenates and isolated alveolar type II pneumocytes. Peak Cu,Zn SOD activities in lung homogenates and alveolar type II cells were observed 4 h after liposome instillation and returned to control levels by 24 h, whereas CAT activities remained significantly above controls. There were no significant differences in liposome distribution or antioxidant enzyme uptake among lung lobes. The distribution of fluorescently labeled Cu,Zn SOD and CAT was assessed with the use of epifluorescence microscopy and digital image processing to determine patterns of cellular incorporation of liposome-entrapped Cu,Zn SOD and CAT within the lung. Although the mean fluorescence intensity of alveoli from rabbits instilled with liposomes containing labeled Cu,Zn SOD and CAT was greater than autofluorescence observed with either no liposome or empty liposome instillation, fluorescence intensity varied between adjacent alveoli. Both fluorescently labeled Cu,Zn SOD and CAT were located cytosolically, and uptake was not limited to alveolar type II pneumocytes. These results demonstrate that a single intratracheal instillation of liposomes can effect increases in Cu,Zn SOD and CAT activities in distal lung cells, including alveolar type I and type II cells and macrophages.

Animals↗

PubMed1992

In vivo imaging of insulin receptors in monkeys using 18F-labeled insulin and positron emission tomography.

We previously described a prosthetic group methodology for incorporating 18F into peptides and showed that 18F-labeled insulin (18F-insulin) binds to insulin receptors on human cells (IM-9 lymphoblastoid cells) with affinity equal to that of native insulin (1). We now report studies using 18F-insulin with positron emission tomography to study binding to insulin receptors in vivo. Positron emission tomography scans were performed in six rhesus monkeys injected with 0.3-1.4 mCi of 18F-insulin (approximately 0.1 nmol, SA 4-11 Ci/mumol). Integrity of the tracer in blood, determined by immunoprecipitation, was 94% of control for the first 5 min and decreased to 31% by 30 min. Specific, saturable uptake of 18F was observed in the liver and kidney. Coinjection of unlabeled insulin (200 U, approximately 1 nmol) with the 18F-insulin reduced liver and increased kidney uptake of the labeled insulin. Liver radioactivity was decreased by administration of unlabeled insulin at 3 min, but not 5 min, after administration of the tracer, while some kidney radioactivity could be displaced 5 min after injection. Clearance of 18F was predominantly in bile and urine. 18F-insulin is a suitable analogue for studying insulin receptor-ligand interactions in vivo, especially in the liver and kidney.

Animals↗

PubMed1991

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.

Anions↗

PubMed1991

Diffusion resistances between ADH-induced vacuoles and the extracellular space in rabbit collecting duct: evidence that most vacuoles are intracellular, endocytic compartments.

Large vacuoles form in the renal collecting duct following the onset of antidiuretic hormone (ADH)-stimulated water reabsorption. The aim of the present study was to test two alternative hypotheses regarding the origins of these structures: (1) the vacuoles constitute basilar, extracellular spaces that dilate as water flows through these spaces from cells into the peritubular compartment; or (2) the vacuoles represent intracellular, endocytic compartments that dilate during water reabsorption due to enhanced fluid phase endocytosis. Fluorescence-digital imaging microscopy was used to visualize the uptake into vacuoles of a hydrophilic fluorochrome (6 methoxy-N-[3 sulfopropyl] quinolinium) whose fluorescence is markedly quenched by halides. During their formation, most vacuoles (67%) accumulated the fluorochrome from the peritubular bath and trapped the dye well after (greater than 60 min) washing it from the bath. The spatial pattern of fluorescence within individual vacuoles indicated that the dye was trapped within these structures as a fluid-phase marker and was not bound to the vacuole margins. The fluorescence of dye trapped within vacuoles was virtually unaltered by changes in peritubular Cl- or Br- concentration that elicit dramatic quenching of dye-fluorescence in bulk solution, as expected if there exists a high diffusion resistance between the interiors of these structures and the peritubular space. These results indicate that most ADH-induced vacuoles represent endocytic compartments that are not directly connected to the extracellular space.

Animals↗

PubMed1991

A single column, rapid quality control procedure for 6-[18F]fluoro-L-dopa and 6-[18F]fluorodopamine PET imaging agents.

6-[18F]Fluoro-L-dopa and 6-[18F]fluorodopamine are promising PET imaging agents for visualizing cerebral dopaminergic centers and cardiac sympathetic innervation and function. Administration to humans requires a means to determine the purity before injection. We describe such a method using HPLC with u.v. and radioactivity detection and a single high-speed C-18 column with gradient elution. The procedure can resolve within 10 min these fluorinated catechols, their isomers, and dihydroxyphenylalanine. The chemical and radiochemical purity, and specific activity, can be determined before injection.

Chromatography, High Pressure Liquid↗

PubMed1991

Effect of fluorine substitution on the adrenergic properties of 3-(tert-butylamino)-1-(3,4-dihydroxyphenoxy)-2-propanol.

The 2- and 6-fluoro derivatives of the potent beta-adrenergic agonist 3-(tert-butylamino)-1-(3,4-dihydroxyphenoxy)-2-propanol were prepared and their adrenergic properties examined. The order of potency was as follows: beta-adrenergic activity (simulation of cyclic AMP formation in C6 glioma cells), 2-F = parent much greater than 6-F; beta 1-activity (rate of contraction, guinea pig atria), parent greater than 2-F much greater than 6-F; beta 2-activity (relaxation of guinea pig tracheal strip), 2-F greater than parent much greater than 6-F. The affinity of the 2-fluoro analogue for beta 1-adrenergic receptors (inhibition of the specific binding of [3H]dihydroalprenolol, rat cerebral cortical membranes) was 2 times greater, while the 6-fluoro analogue was 1450 times less than the parent. These results suggest that the aromatic rings of phenoxypropanolamine adrenergic agonists and phenylethanolamine adrenergic agonists bind in similar fashion to the adrenergic receptor, and that if interactions between fluorine and the side-chain hydroxyl group are critical in defining beta-adrenergic selectivity, the interactions are similar in both phenoxypropanolamines and phenylethanolamines.

Adrenergic beta-Agonists↗

PubMed1991

Positron emission imaging of cardiac sympathetic innervation and function using 18F-6-fluorodopamine: effects of chemical sympathectomy by 6-hydroxydopamine.

Hypotheses concerning the pathophysiology of hypertension, cardiac failure and other cardiovascular disorders have imputed abnormal cardiac sympathoneural activity. Here we describe a technique to examine cardiac sympathetic innervation and function using positron emission tomographic (PET) scanning after systemic intravenous injection of 18F-6-fluorodopamine, and the effects of chemical sympathectomy by the neurotoxin, 6-hydroxydopamine (6-OHDA). Uptake of 18F-6-fluorodopamine by the heart of anesthetized dogs resulted in striking delineation of the left ventricular myocardium. Myocardial radioactivity declined bi-exponentially, with a half-life of approximately 2 h during the longer phase. In 6-OHDA-treated animals, the ventricular myocardium was barely distinguishable from the chamber; myocardial radioactivity declined rapidly and was virtually absent within 30 min after injection of 18F-6-fluorodopamine. The rates of decline in myocardial radioactivity in dogs treated with 6-OHDA were similar to those in dogs treated with reserpine, but the mechanisms of sympatholysis by these drugs were distinguished by arterial plasma levels of 6-fluorodihydroxyphenylacetic acid (6-FDOPAC). Plasma 6-FDOPAC levels were diminished in 6-OHDA-treated dogs and elevated in reserpinized dogs. The results confirm that, after injection of 18F-6-fluorodopamine, cardiac sympathetic nerve endings are radiolabeled, allowing visualization of sites of sympathetic innervation. Combined assessments of PET time-activity curves and plasma levels of metabolites of 18F-6-fluorodopamine constitute a new, potentially clinically applicable means by which to examine cardiac sympathetic function.

Animals↗

PubMed1991

Direct electrophilic fluorination of tyrosine in dermorphin analogues and its effect on biological activity, receptor affinity and selectivity.

In a preliminary communication we reported [(Tetrahedron Lett. 31, 619 (1990)] that acetyl hypofluorite can be used efficiently to introduce fluorine regiospecifically (ortho to OH) into the phenolic ring of tyrosine-containing peptides. This procedure has been applied to the fluorination of a number of mu-selective opioid peptides derived from dermorphin. While the procedure can be used even when the side chains of Arg, Lys, and Tyr are left unprotected, the sulfoxide of a Met(O)-containing analogue was oxidized to sulfone faster than fluorination of the phenolic ring. This method can also be used when the peptide is attached to Merrifield resin. Thus, Tyr(3-F)-D-Ala-Phe-Gly-NH2 and Tyr(3-F)-D-Arg-Phe-Lys-NH2 (F-DALDA) have been prepared, purified, and characterized. Affinities of these fluorinated peptides for both mu- and delta- opioid receptors are reduced (two- to nine-fold) relative to their nonfluorinated analogues, but their selectivity for mu-opioid receptors is not significantly altered. Similarly, the in vitro biological potencies (GPI and MVD assays) of the fluorinated analogues are reduced (two- to seven-fold) relative to their nonfluorinated parent peptides. Thus, F-DALDA, which has high affinity (Ki mu = 15.2 nM) and selectivity (Ki delta/Ki mu = 5390) for mu-opioid receptors, has potential use in biochemical studies which utilize 19F or 18F- labeled compounds.

Amino Acid Sequence↗

PubMed1991

Histamine-induced chloride channels in apical membrane of isolated rabbit parietal cells.

The electrical properties of the apical membrane of isolated rabbit parietal cells were studied using the patch-clamp technique. The apical membrane of the parietal cells plated on Matrigel and maintained in culture conditions was identified by lectin-binding studies. Cell-attached and excised inside-out patches from 10(-4) M cimetidine-treated parietal cells infrequently contained Cl- channels (9% of the patches). A single class of voltage-dependent outwardly rectifying Cl- channels with 24 +/- 1-pS conductance was observed in 75% of the patches from cells stimulated (acid secreting) by 10(-4) M histamine. Other anions passed through these channels with a permeability sequence of I- (1.2) greater than Br- (1.1) greater than or equal to Cl- (1.0) greater than NO3- (0.7) greater than SO4(2-) (0.1), but there was a very low permeability for Na+ or K+ (PCl-/PNa+ or PCl-/PK+ greater than 5). In inside-out patch configurations the Cl- channel was insensitive to Ba2+ and stilbene derivatives but was inhibited by diphenylamine-2-carboxylic acid in a manner characteristic of a reversible open-channel blocker. It is concluded that H2-receptor agonist stimulation of acid secretion by rabbit parietal cells activates Cl- channels in the apical cell membrane.

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

PubMed1990

Effect of ring fluorination of epinephrine on its cardiovascular adrenoceptor activities.

Effects of fluorine (F) substitution on the 2- and 6-positions of the catechol ring of epinephrine (Epi) on its alpha- and beta-adrenoceptor agonist activities were studied in anesthetized dogs. Increments in heart rate and contractile force were used as measures of beta 1-adrenoceptor activity, while increases and decreases in blood pressure and decreases and increases in femoral blood flow were used as measures of alpha- and beta 2-adrenoceptor activities, respectively. F substitution on the 2- and 6-positions of the catechol ring yielded compounds with opposite receptor selectivities: 2-FEpi was a selective beta-adrenoceptor agonist with little agonist activity at alpha-adrenoceptors, while the 6-F analog was a selective a-adrenoceptor agonist with no significant beta-adrenoceptor effects. Of added significance, 2-FEpi was more potent than Epi as a beta 1-adrenoceptor agonist, while 6-F Epi was more potent than the parent compound as an alpha-adrenoceptor agonist. The possible mechanisms for the effects of ring fluorination on the adrenoceptor activities of Epi and other sympathomimetic amines are discussed.

Animals↗

PubMed1990

Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

B-Lymphocytes↗

PubMed1990

Oxygenation of fluorinated tyrosines by mushroom tyrosinase releases fluoride ion.

The reactions of 2-fluoro- and 3-fluoro-L-tyrosine with mushroom tyrosinase have been investigated. Both fluorinated tyrosines are good substrates for tyrosinase, with Vmax and Vmax/Km values similar to those for L-tyrosine. Oxygenation of 2-fluorotyrosine is regioselective, and only 6-fluorodopa was detected by HPLC in reaction mixtures. Oxygenation of both isomers of monofluorotyrosine results in fluoride ion production in the absence of ascorbic acid; however, 2-fluorotyrosine also produces fluoride in the presence of ascorbic acid. These results are consistent with previous studies demonstrating rapid intramolecular cyclization of nascent 6-fluorodopaquinone (M.E. Rice, B. Moghaddan, C.R. Creveling, and K.L. Kirk, 1987, Anal. Chem. 59, 1534-1538), which is competitive with reduction by ascorbate, resulting in elimination of the aromatic fluorine as fluoride ion.

Basidiomycota↗

PubMed1990

Metabolic fate of injected radiolabelled dopamine and 2-fluorodopamine in rats.

In evaluating positron-emitting analogs of dopamine (DA) as possible imaging agents for visualizing tissue sympathetic innervation and function, the metabolic fate of systemically injected [3H]-DA or [14C]-DA was compared with that of [3H]-2-fluoroDA in plasma and in sympathetically innervated tissues (left ventricle, spleen and salivary glands) of rats. By 60 min after the injection of [3H]-DA or [3H]-2-fluoroDA, concentrations of [3H]-DA. [3H]-2-fluoroDA, [3H]-norepinephrine ([3H]-NE) and [3H]-2-fluoroNE in tissue exceeded concentrations in plasma by up to several thousand-fold. Whereas most of the radioactivity in tissue was in catechols, radioactivity in plasma was due to O-methylated metabolites of DA, including homovanillic acid (HVA) and of NE, including normetanephrine (NMN) and methoxyhydroxphenylglycol (MHPG). Estimated ratios of tissue: blood radioactivity at 60 min after injection of [3H]-2-fluoroDA were 4.10 for the heart, 1.91 for the spleen and 2.10 for the salivary glands. The patterns of levels of catechol metabolites and analogs of HVA in plasma and effects of blockade of neuronal uptake with desipramine suggested that [3H]-2-fluoroDA was not as efficiently removed by neuronal uptake and not as efficiently beta-hydroxylated as the non-fluorinated compound. Concurrent administration of [3H]-DA and large amounts of non-radioactive 2-fluoroDA did not substantially alter the pattern of metabolites of [3H]-DA in plasma. After injection of [18F]-fluoroDA, visualization of sympathetic innervation of tissue should be feasible by position emission tomography.

Animals↗