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R A Lutz

Publications and source records attributed to R A Lutz.

At least 19 recordsLinked to original sources
PubMed1992

Opioid receptors and their pharmacological profiles.

Opioid receptors can be divided into three major classes, which are called mu, delta and kappa-receptors. The molecular basis of the receptors is discussed and a hypothesis of the binding of bivalent ligands to the receptor is presented. Furthermore the mechanism of action, the distribution and the probable function of these classes is overviewed. Increasing evidence is accumulating that the classical binding model cannot explain completely the interaction of opioids with their receptors. In addition to the mu-receptors, high affinity mu 1 binding sites have been demonstrated. Similarly, the delta receptors may be divided in delta 1 and delta 2. The significance of these subclasses is not yet fully understood. The high affinity mu 1-binding sites, however, represent probably an activated receptor complex, e.g. the complex between the receptor and the guanine-nucleotide-binding protein.

Amino Acid Sequence↗
PubMed1991

A computer controlled device to facilitate studies of the kinetics of ligand-binding: binding of diazepam to bovine brain membranes.

A device to facilitate kinetic receptor filtration assays is described. The receptor containing membranes and the labeled ligand are placed in two separate syringes and are rapidly mixed into a collecting syringe using a pneumatic ram. Shortly after the start of mixing, a pneumatically controlled valve switches the collecting syringe containing the receptor-ligand mixture to the filtration unit. Filtration is performed on glass/microfiber filters or equivalent by pushing the plunger of the collecting syringe by a stepper motor. A valve positioner controlling several valves allows the filtered membranes to be washed and dried by pressure in any user programmable sequence. Further filtration of the receptor-ligand mixture can be programmed at selected time points. The entire system is controlled by an IBM-PC. With this system, the association and dissociation of diazepam from crude bovine-brain membranes has been studied at 4 degrees C. The dissociation shows a biphasic pattern with half lifes of 1.3 and more than 23 minutes respectively. Association appears to be into a single compartment.

Animals↗
PubMed1989

Binding characteristics of a series of dimeric tripeptide enkephalins for delta opiate receptors in rat brain and NG108-15 cells.

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.

Animals↗
PubMed1989

A bumetanide-sensitive, potassium carrier-mediated transport system in excitable tissues.

The binding of [3H]-bumetanide to rat brain synaptosomes revealed the existence of two binding sites. The high affinity site (R1 = 46.6 fmoles/mg protein) binds bumetanide and furosemide with Kd1 of 13 nM and 1.5 microM respectively, while the low affinity site (R2 = 1.37 nmoles/mg protein) is characterized by Kd2 of 200 microM and 680 microM for bumetanide and furosemide, respectively. Bumetanide sensitive 86Rb uptake was 34 +/- 14.5, 38.3 +/- 1.4, 18.6 +/- 1.3 and 29.0 +/- 6.1% of total 86Rb uptake in synaptic plasma membrane vesicles, rat brain synaptosomes, Neuroblastoma N1E115 cell line and chick chest muscle cells, respectively. Furosemide and bumetanide inhibited 86Rb uptake to rat brain SPM- vesicles in a dose dependent fashion. Half maximal inhibition (IC50) was observed at 20 nM and 4 microM for bumetanide and furosemide, respectively. Bumetanide-sensitive transport was dependent on extravesicular sodium and chloride concentrations with a Km of 21 and 25 mM for the two ions, respectively. These results demonstrate the existence of a "loop diuretic" sensitive carrier-mediated K+ transport system in brain and other excitable cells.

Animals↗
PubMed1988

Multiple interactions of unsaturated fatty acids with opiate and ouabain binding sites and beta-adrenergic sensitive adenylate cyclase system.

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and beta-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on beta-adrenergic sensitive adenylate cyclase was more complex. 0.2-0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid, inhibited basal, beta-agonist- and NaF-stimulated activities in membranes of A431 and C6 cells. To evaluate which aspects of the unsaturated fatty acid molecules might be responsible for the observed effects, myristic acid, monoolein and taurodeoxycholic acid were studied. They also inhibited binding to the opiate receptor. Myristic acid, did not inhibit ouabain binding, binding to beta-receptor, nor adenylate cyclase activity. Monoolein, had no inhibitory effect on ouabain binding but behaved similar to oleic acid in the beta-receptor/adenylate cyclase system. Taurodeoxycholic acid inhibited binding to all three receptors as well as adenylate cyclase activity. We conclude that the effects of unsaturated fatty acids on ligand binding and adenylate cyclase activity are the result of their multiple interactions with various molecular processes rather than any unique property of long chain unsaturated fatty acids, per se.

Adenylyl Cyclases↗
PubMed1987

Uptake of norepinephrine and related catecholamines by cultured chromaffin cells: characterization of cocaine-sensitive and -insensitive plasma membrane transport sites.

Norepinephrine and its closely related analogues, dopamine and epinephrine, are transported into chromaffin cells in culture by two distinct types of sites on the plasma membrane: one is sensitive to cocaine while the other is not. The cocaine-sensitive site has a high affinity for catecholamines and depends on sodium in the medium. The apparent Km for norepinephrine uptake by the cocaine-sensitive site is 5.8 microM when determined in the presence of 118 mM NaCl, obtained using nonlinear least-square curve fitting. Detailed kinetic analysis has also shown cocaine to be a competitive inhibitor of norepinephrine uptake with an apparent Ki of ca. 1 microM. This site is blocked by a series of tricyclic antidepressant drugs with relative potencies characteristic of norepinephrine transport sites in neurons. In contrast, the cocaine-insensitive site(s) have a low affinity for norepinephrine (apparent Km, approximately 88 microM) and are also able to transport catecholamine analogues such as dimethyl-epinephrine and isoproterenol, which have bulky groups attached to the amine moiety. Transport of norepinephrine at both sites is blocked by low temperature, by mitochondrial uncouplers, and by other metabolic inhibitors. Both of these transport sites in the chromaffin cell plasma membrane, therefore, appear to be different from the well-characterized catecholamine transport sites in the chromaffin granule membrane on the basis of substrate specificity and their sensitivity to inhibitors.

Adrenal Medulla↗
PubMed1987

Demonstration and characterization of two classes of cardiac glycoside binding sites to rat heart membrane preparations using quantitative computer modeling.

Cardiac glycoside binding to rat heart membrane preparations was measured by rapid filtration technique. The binding data were analyzed using quantitative computer analysis. The experimental results using [3H]-ouabain as the labeled ligand were consistent with a model in which cardiac glycoside specific binding occurs at two independent classes of sites. The high affinity sites were characterized by a dissociation constants of 40 nM, 50 nM, and 61 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 1.3 pmoles/mg protein. The lower affinity sites for ouabain were characterized by dissociation constants of 2.3 microM, 67 nM and 71 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 3 pmoles/mg protein. Potassium ions inhibit [3H]-ouabain binding in a dose dependent manner with an IC50 of 500 microM. Quantitative computer modelling indicated that potassium inhibits ouabain binding at both binding sites.

Animals↗
PubMed1987

The ouabain receptor in animal tissues and its endogenous ligand.

Cardiac glycosides bind to the Na+,K+-ATPase and inhibit its activity. Low concentrations (less than 10(-7) M) of ouabain stimulate the activity of Na+,K+-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentration of ouabain which induces maximal stimulation is also highly variable and ranges between 10(-9) to 10(-7) M. This stimulation may be explained by the presence of an endogenous ouabain-like compound (OLC) in the brain homogenate. Mammalian tissues and body fluids including brain, heart, kidney, plasma, urine and cerebrospinal fluid contain a unidentified OLC. An endogenous OLC was also demonstrated in toad skin and plasma. This compound was purified to homogeneity and identified using UV, NMR and Mass spectroscopies to be 3-hydroxy-14, 15-epoxy-20,22-dienolide glycoside (resibufogenin). Several reports have suggested that unsaturated fatty acids are the ouabain-like regulators of the Na+,K+-ATPase. Furthermore, Saline infusion to WKY rats, which was shown to increase OLC in the plasma causes also an elevation of free fatty acids. Thus, the interaction of fatty acids with several plasma membrane components was studied. Ouabain binding, opiate binding and binding to the beta-adrenergic receptor were all inhibited by micromolar concentrations of the unsaturated fatty acids, linoleic, oleic and arachidonic. Binding to the opiate receptor was inhibited with IC50 of 40-90 microM and binding to beta-adrenergic receptor with IC50 of 350-450 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Naloxonazine effects on the interaction of enkephalin analogs with mu-1, mu and delta opioid binding sites in rat brain membranes.

The authors have characterized the opioid receptors of rat brain membranes using self- and cross-displacement studies with both tritiated and unlabeled [D-Ala2, D-Leu5]-enkephalin and [D-Ala2, MePhe4, Gly-ol5]-enkephalin. Mathematical modeling demonstrated the presence of three classes of binding sites, corresponding to mu, delta and the putative mu-1 classes of site. Unlabeled naloxonazine shows high affinity for all three classes of sites, with highest affinity for the mu-1 sites. Membranes were preincubated with 50 nM naloxonazine or with controls (50 nM naloxone or buffer) for 30 min. Preincubation of membranes with 50 nM naloxonazine resulted in a dramatic, nearly 2-fold reduction in the binding of [3H][D-Ala2, D-Leu5]-enkephalin and [3H][D-Ala2, MePhe4, Gly-ol5]-enkephalin relative to the controls. Quantitative analyses using mathematical modeling with program "LIGAND" suggested that this effect was primarily "competitive," i.e., attributable to changes in affinity, with no apparent or detectable noncompetitive or irreversible effects on binding capacities for the three classes of sites.

Animals↗
PubMed1986

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells. A kinetic analysis.

The binding of the enkephalin dimer [D-Ala2, Leu5-NH-CH2-]2 (DPE2) is characterized by its high affinity for receptors on NG108-15 hybrid cells, the affinity constant K = 4.7 X 10(9) M-1 is up to 8-fold that of monomers (0.6 to 1.0 X 10(9) M-1), and a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE2 binds with a 2-fold higher rate, k1 = 6.3 X 10(7) M-1min-1, than monomers (2.4 to 3.8 X 10(7) M-1min-1), and dissociates at a slower rate than monomers. Dissociation of DPE2 was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE2 binds by cross-linking two subunits of one receptor.

Animals↗
PubMed1986

Computer analysis of enzyme-substrate-inhibitor kinetic data with automatic model selection using IBM-PC compatible microcomputers.

A weighted nonlinear least-squares curve-fitting program, implemented in compiled BASIC for the IBM-PC is described to estimate the parameters of enzyme kinetics obeying Michaelis-Menten kinetics and seven inhibition models. The effects of the inhibitor on the maximal velocity (Vm) and the Michaelis-Menten constant (Km) are used to select automatically the most plausible model of inhibition and to calculate initial estimates of parameters. The program is used to demonstrate that the inhibition of carbamyl-phenylalanine hydrolase by the product phenylalanine is consistent with the pure mixed noncompetitive model.

Binding, Competitive↗
PubMed1986

Computer analysis of radioligand data: advantages, problems, and pitfalls.

Mathematical modeling combined with nonlinear least-squares curve fitting provides a systematic, objective, reproducible, and consistent method to aid the interpretation of ligand-binding data. It forces the experimentalist to formulate hypotheses in an unambiguous manner and to consider alternative, closely related models as plausible counter-hypotheses. Modeling provides estimates of the "goodness-of-fit" of the theory to the data and estimates of the minimal uncertainty of the parameters. With the availability of many programs for micro- and mini-computers, as well as mainframe computers, these methods are now becoming widely used. Accordingly, we must emphasize a number of potential problems and limitations, based on our experience. Interpretation of results of modeling study should be made, in light of the following points: no amount of computer analysis will compensate for "bad" or insufficient data, or for poor experimental design; the interpretation of the computer analysis is subject to the caveat that all underlying assumptions must be satisfied; one must examine the data graphically in several coordinate systems (e.g., "raw data," as well as standardized residuals); one must continuously search for possible systematic biases or artifacts; one must closely examine the reproducibility of results between multiple experiments; and one must recognize that all of the "test tubes" in an experiment are not necessarily "independent observations" in a statistical sense. In view of these potential problems and limitations, one should always seek to corroborate results and interpretations of "modeling" studies of ligand binding by independent biochemical, biophysical, or structural evidence. In this context, ligand-binding studies, appropriately analyzed, can play a useful and constructive role.

Computer Simulation↗
PubMed1986

Characterization of two classes of cardiac glycosine binding sites in rat heart and brain membrane preparations, using quantitative computer modelling.

Cardiac glycoside binding to rat heart and brain membrane preparations was measured by a rapid filtration technique. Data were analysed using quantitative computer analysis. The experimental results were consistent with a model in which cardiac glycoside-specific binding occurs at two independent classes of sites. The high-affinity sites in heart membranes were characterized by dissociation constants (Kd) of 40, 50, and 61 nmol/l for ouabain, digoxin and digitoxin, respectively, and the lower-affinity sites were characterized by Kd of 2.3 mumol/l, 67 nmol/l and 71 nmol/l for ouabain, digoxin and digitoxin, respectively. Comparable results were obtained using brain membranes. Potassium ions inhibit [3H]-ouabain binding in a dose-dependent manner with an IC50 of 500 mumol/l. Quantitative computer modeling indicated that potassium inhibits ouabain binding to approximately the same extent at both classed of binding sites, consistent with the hypothesis that the two classes of binding sites for cardiac glycosides might be associated with the Na+K+-ATPase.

Animals↗
PubMed1985

Mu1: a very high affinity subtype of enkephalin binding sites in rat brain.

Displacement studies of [3H]-[D-Ala2-MePhe4-Gly-ol5]-enkephalin ([3H]-DAGO) and [3H]-[D-Ala2-D-Leu5]-enkephalin ([3H]-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the mu, delta and mu 1 sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively.

Animals↗
PubMed1985

Increased affinity and selectivity of enkephalin tripeptide (Tyr-D-Ala-Gly) dimers.

The binding of alkylendiamide dimers of the three N-terminal residues of [D-Ala2,D-Leu5]enkephalin (DADL) to rat brain and Ng108-15 neuroblastoma-glioma cell membranes was compared with that of DADL, Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO) and morphiceptin. Tritiated DADL and DAGO were used as labeled ligands for delta- and mu-receptors, respectively. Dimerization of the tripeptides resulted in dramatic increases in both mu and delta binding. The binding to mu-receptors showed two peaks at an alkyl chain length of n = 2 and approximately n = 16. In contrast, delta binding (NG108-15 cells) increased steadily with increasing chain length. The dimers with n less than 18 were mu-preferential, and the one with n = 2 showed the most dramatic increase in mu selectivity with a 400 fold higher affinity to mu- than to delta-receptors. For long-chain alkyl spacers the compounds became delta selective.

Animals↗
PubMed1985

Increased affinity of dimeric enkephalins is not dependent on receptor density.

The equilibrium binding and dissociation kinetics of the enkephalin dimer bis-(D-Ala2-D-Leu5-enkephalin)-ethylenediamide (designated DPE2) to neuroblastoma glioma NG108-15 cells were investigated and compared with the monomers D-Ala2-D-Leu5-enkephalin (DADL) and D-Ala2-Leu5-enkephalinamide (DALEA). Binding was studied after exposure of the membrane to increasing concentrations of the irreversible delta receptor selective ligand FIT in order to decrease the density of binding sites on the cell membrane. The increased affinity of DPE2 did not revert to that of the monomer DADL by this reduction of binding sites. Similarly, the dissociation of DPE2 did not approach that of the monomer DALEA in the presence of 1 microM DALEA. These data strongly suggest that crosslinking does not occur, and fail to confirm the hypothesis that dimers with short spanning chain length aid the clustering of receptors. We postulate: 1) If the dimer binds to a bivalent binding site, the monovalent binding state of our bivalent ligand may not exist to an appreciable extent, and 2) the bivalent ligand cannot bind when the binding site is irreversibly blocked by a monovalent ligand.

Animals↗