Biomedical subjects

D Rodbard

Publications and source records attributed to D Rodbard.

At least 55 records · Page 3Linked to original sources

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

Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion.

We have developed a new computer program for detection of "peaks" in sequential hormone measurements in longitudinal studies of episodic hormone secretion. The program provides: (a) several statistically based approaches to the estimation of the random measurement error as a function of hormone level; (b) peak detection based on analysis of first derivatives with logic that has been optimized for asymmetrical peaks with exponential decays; (c) several approaches to the estimation of tolerances for the first and second derivatives; (d) a sensitive curve-fitting approach, to distinguish between upstrokes, exponential decays, and flat baselines; (e) ability to detect multiple overlapping peaks; (f) analysis of "robustness" by systematically varying the threshold around the most-likely value; (g) superimposition of detected peaks, to evaluate "average peak shape"; (h) analysis of the "decay rate," to obtain an estimate of the disappearance rate constant and half-life; (i) use of a "discrete deconvolution" approach, to solve for the apparent instantaneous rate of secretion, and provision of an error analysis to obtain estimates of the precision of these derived values; and (j) correlation with other relevant series as a means of cross validating. The program has been tested extensively on real and synthetic data, and appears to perform well. The frequency of "false positive" peaks can be held at any desired low level, and can be prevented from increasing as sampling frequency increases. The number of arbitrary assumptions, approximations, or thresholds is held to an absolute minimum. These methods are natural, logical, and follow from first principles of statistics.

Biometry↗

PubMed1986

Studies of thyroxine binding to plasma proteins in health and disease.

Thyroxine binding protein characteristics were defined in 6 normal subjects, 4 with thyroxine binding globulin (TBG) deficiency, 3 with TBG increase, one with hyperthyroxinemic dysalbuminemia, 7 with severe non-thyroidal illness, and 3 with chronic renal failure. Free thyroxine was measured by Sephadex partition in plasma to which increasing thyroxine concentrations were added. Deconvolution of the resultant titration data was performed by computer modelling. Abnormalities of thyroxine binding capacities or of binding affinities occur in non-thyroidal illness, chronic renal failure, and sporadically. The patient with hyperthyroxinemic dysalbuminemia had increased thyroxine binding affinity to thyroxine binding prealbumin as well as to albumin. "Free-T4 assays" or estimates of Free-T4 by calculation from total thyroxine and measures of protein binding such as T3-uptake must be expected to be perturbed by these binding protein abnormalities unless such interferences are explicitly demonstrated to be absent.

Adult↗

PubMed1986

Identification and characterization of a vasopressin isoreceptor in porcine seminal vesicles.

Neurohypophysial hormones stimulate the motility of tunica albuginea, epididymis, and vas deferens acting through oxytocin (OT) and V1 vasopressin receptors. To test the hypothesis that these hormones are involved also in the regulation of seminal vesicle physiology, we studied binding of [3H]OT and [3H] arginine vasopressin ([3H]AVP) to porcine seminal vesicle membranes. Neurohypophysial hormones bind to two different classes of sites. The first class shows low capacity (35 fmol per mg of protein) and a very high affinity (Kd less than 1 nM) for both the labeled ligands. The second class is characterized by a high capacity (2000 fmol per mg of protein) and a high affinity for AVP (Kd approximately equal to 2.5 nM), whereas OT has 160 times lower affinity. Lysine vasopressin and the V1 antagonist [1-deaminopenicillamine, 2-(O-methyl)tyrosine]Arg8-vasopressin compete with high affinity with [3H]AVP binding, whereas the V2 agonist [1-deamino,4-valine]D-Arg8-vasopressin (dVDAVP) is 110 times less potent than AVP. The OT agonist [Thr4,Gly7]OT and the OT antagonist [1(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)tyrosine, 8-ornithine]vasotocin failed to affect [3H]AVP binding. These findings seem to suggest that AVP interacts with the V1 vasopressin isoreceptor in porcine seminal vesicle membranes. However, AVP stimulates adenylate cyclase activity in a dose-dependent fashion with an EC50 of 14 nM, whereas OT or dVDAVP has no effect at 100 nM. Moreover, a well-characterized V1 vasopressin antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin [d(CH2)5Tyr(Me)AVP], competes with [3H]AVP binding with an IC50 of 0.17 microM. These pharmacological properties are distinct from the previously described V1 and V2 vasopressin receptors and indicate the presence of a new class of AVP receptors. Although this vasopressin isoreceptor shares some pharmacological characteristics with the V1 (pressor) isoreceptor, it has low affinity for the V1 antagonist d(CH2)5-Tyr(Me)AVP and is linked to the adenylate cyclase system. The extremely high density of AVP receptors in porcine seminal vesicles (2 pmol per mg of protein) is comparable to the density of V2 vasopressin receptors in porcine renal medulla, suggesting a physiological role for vasopressin in the seminal vesicle.

Adenylyl Cyclases↗

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

Naloxone administration does not affect gonadotropin secretion in agonadal men either basally or during testosterone treatment.

Naloxone administration has no effect on plasma gonadotropin levels of agonadal men. The present study was designed to evaluate whether testosterone replacement therapy could restore LH responsiveness to naloxone in such men. We measured plasma LH and FSH levels at 15-min intervals during naloxone infusion (8 mg in 1 min followed by 12 mg in 3 h) and for the following 3 h in a group of agonadal men both before and after at least 2 months of three different schedules of testosterone replacement therapy: 1) testosterone undecanoate, 40 mg three times a day by mouth; 2) testosterone enanthate 200 mg im every 2 weeks; and 3) testosterone enanthate 100 mg im once a week. Mean plasma gonadotropin levels as well as LH pulse frequency did not vary during naloxone infusion vs. placebo either basally or during each testosterone regimen. These results suggest that long term testosterone therapy does not affect the altered opioid modulation of gonadotropin secretion which is present in agonadal men.

Adolescent↗

PubMed1986

Adrenal steroid responses to continuous intravenous adrenocorticotropin infusion compared to bolus injection in normal volunteers.

We compared the adrenal steroid responses after synthetic ACTH-(1-24) (Cosyntropin) administration given by either continuous iv infusion or bolus injection in 11 normal women and 6 normal men. Each subject received 250 micrograms Cosyntropin as a bolus iv injection on 1 occasion and as a continuous 2-h iv infusion on another occasion, in random order. There was a 1-week interval between the studies. We measured the plasma levels of cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, progesterone, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, delta 5-androstenediol, androstenedione, and testosterone by RIA 15 and 0 min before and 30, 45, 60, and 120 min after administering ACTH. The steroid concentrations and their increments, ratios, or areas above baseline did not differ significantly between the bolus injection and the continuous infusion. Thus, at the dose of 250 micrograms, a bolus ACTH injection stimulates adrenal steroid secretion as effectively as a 2-h continuous ACTH infusion.

Adrenal Cortex Hormones↗

PubMed1986

Opioid receptors of bovine posterior pituitary neurosecretosomes are exclusively kappa.

Intact neurosecretosomes (NSS) from bovine posterior pituitary were prepared and characterized. Ligand binding studies were performed, using 3H-[D-Ala2-D-Leu5]enkephalin (DADL), 3H-etorphine and 3H-ethylketocyclazocine (EKC). The absence of specific binding of 3H-DADL and the inability of DADL to displace 3H-etorphine, implies the absence of mu, delta, or DADL-suppressible benzomorphan ("kappa-2") sites. Self- and cross displacement studies for etorphine and EKC suggested receptor heterogeneity. EKC fails to displace - 20% of specific binding of etorphine. Mathematical modelling indicates the presence of three classes of sites. The present findings imply that bovine posterior pituitary opioid receptors are exclusively of the kappa type.

Analgesics, Opioid↗

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

Characterization of histamine H-1 receptors on human peripheral lung.

Histamine H-1 receptors in human peripheral lung were characterized by radioligand and biochemical assays employing binding of the H-1 receptor antagonist [3H]pyrilamine to plasma membrane preparations. Simultaneous computerized analyses of the data from fourteen separate equilibrium-binding assays indicated the presence of three distinct classes of binding sites with Kd values of 81 +/- 35 pM, 7 +/- 3 microM, and 320 +/- 167 microM and binding capacities of 23 +/- 3 pmoles, 10 +/- 5 nmoles, and 297 +/- 119 nmoles/mg protein respectively. Dissociation kinetics of [3H]pyrilamine binding also supported the presence of three binding sites or states. Further, competition binding curves for histamine receptor agonists and antagonists also indicated the presence of multiple binding sites for the H-1 receptor. The effect of exogenous stimulation of histamine H-1 receptors on human cyclic nucleotides was also examined. Both histamine and the H-1 agonist 2-methyl histamine caused dose-related increases in the cyclic guanosine monophosphate (GMP) content of human lung. The effects of 2-methyl histamine were selective for cyclic GMP. The histamine-induced increase in cyclic GMP peaked within 1.0 min and was effectively prevented by the H-1 antagonist pyrilamine. Thus, human lung possesses a large number of H-1 receptors which exhibit three binding states and produce cyclic GMP, but not cyclic adenosine monophosphate (AMP), when stimulated.

Binding, Competitive↗

PubMed1985

Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates.

We have studied the effects of methotrexate (MTX-Glu1) and the polyglutamate derivatives of methotrexate (MTXPGs) with 2, 3, 4, and 5 glutamyl residues on the catalytic activity of thymidylate synthase purified from MCF-7 human breast cancer cells and on the kinetics of the ternary complex formation by 5-fluoro-2'-deoxyuridine 5'-monophosphate, folate cofactor, and thymidylate synthase. MTX-Glu1 exhibited uncompetitive inhibition of thymidylate synthase when reaction kinetics were analyzed by either double reciprocal plots or a computerized mathematical model based on nonlinear least-squares curve fitting. The Ki for MTX-Glu1 inhibition was 13 microM and the I50 was 22 microM, irrespective of the degree of polyglutamation of the folate. In contrast, the polyglutamated derivatives of MTX all acted as noncompetitive inhibitors. The MTXPGs had 75-300-fold greater potency than MTX-Glu1 as inhibitors of thymidylate synthase catalytic activity, with Ki values from 0.17 to 0.047 microM for MTX-Glu2 to MTX-Glu5, respectively. Neither MTX-Glu1 nor MTXPGs promoted the formation of a charcoal-stable ternary complex with thymidylate synthase and 5-fluoro-2'-deoxyuridine 5'-monophosphate. CH2-H4PteGlu5 (where PteGlu represents pteroylglutamic acid) was found to be 40-fold more potent than CH2-H4PteGlu1 in participating in the formation of a ternary complex, and 10 microM MTX-Glu5 significantly inhibited the formation of a ternary complex containing this folate as cofactor. The inhibition was determined to be due to a reduction in the kon. The potency of this inhibition was markedly greater in the presence of CH2-H4PteGlu1 as compared to CH2-H4PteGlu5. This finding suggests that the degree of interference with complex formation in intact cells would depend on the state of polyglutamation of available folate cofactor. Ternary complex formation with H2PteGlu5 as the folate cofactor was also investigated, and a 50% reduction in complex formation was found in the presence of a 2 microM concentration of MTX-Glu5. These findings have significant implications regarding the mechanism of action of MTX-Glu1 and contribute to an understanding of the complex interactions of MTX-Glu1 and 5-fluorouracil.

Breast Neoplasms↗

PubMed1985

Studies on spinal opiate receptor pharmacology. III. Analgetic effects of enkephalin dimers as measured by cutaneous-thermal and visceral-chemical evoked responses.

D-Ala2-D-Leu5-enkephalin (DADL) along with dimers formed from tetra(DTE: des-Leu5-enkephalin)- or pentapeptide (DPE: Leu5-enkephalin) coupled by methylene bridges of various lengths (n) have been shown in in vitro systems to possess varying degrees of mu/delta-receptor selectivity. In the present studies we have systematically compared the intrathecal effect of these agents and morphine on the cutaneous stimuli (hot plate (HP) and tail flick (TF) and visceral-chemical (writhing) tests in the rat. The following observations were made. (1) Dimers with high delta-receptor selectivity were active in the TF(DPE2 greater than or equal to DADL greater than or equal to DTE2 greater than or equal to morphine greater than DPE12 much greater than DTE12 = 0) and HP(DPE2 greater than or equal to DTE2 greater than or equal to DADL greater than or equal to morphine greater than DPE12 greater than or equal to DTE12 greater than 0. To examine cross-tolerance, the intrathecal ED50 for morphine, DPE and DADL were determined in rats rendered tolerant by subcutaneous morphine pellets. The TF ED50 tolerant/TF ED50 naive was 18.4, 5.4 and 1.3, respectively. The ratio of activity on the HP was 14.0, 4.7 and 2.2 (2) On the visceral-chemical test, only morphine was active. The dimers or DADL in doses which totally blocked the TF or HP are at higher doses which were just below those producing motor dysfunction had no effect on the writhing response. (3) At high intrathecal doses (40 X TF ED50), morphine produced a motor rigidity which blocked the placing and stepping reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗

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

Plasma and red blood cell beta-endorphin immunoreactivity in normal and complicated pregnancies: gestational age variation.

Recent observations suggest that there may be two pools of beta-endorphin-like immunoreactivity in mammalian circulation. One of these pools is present in plasma and the other is detected in association with erythrocytes. Elucidation of an erythrocyte-associated pool may explain some of the wide variability of plasma beta-endorphin levels reported in the literature. We measured beta-endorphin immunoreactivity levels in 85 normal and 33 complicated pregnancies to delineate a possible correlation between gestational age and beta-endorphin immunoreactivity levels in plasma and in erythrocytes. Our results indicate that beta-endorphin immunoreactivity levels in both plasma and erythrocytes vary systematically throughout the gestational period, reaching a peak at 31 to 32 weeks of gestation. Amniotic fluids at midgestation were also analyzed and no correlation was observed between the levels of beta-endorphin immunoreactivity and fetal sex. Compared to normal patients, diabetic patients had significantly lower levels of beta-endorphin immunoreactivity in plasma and higher levels in erythrocytes although the total beta-endorphin immunoreactivity was not statistically different from that in normal subjects. We conclude that (1) the total beta-endorphin immunoreactivity level in whole blood is much higher than that reported in plasma, (2) both plasma- and erythrocyte-associated beta-endorphin immunoreactivity levels vary with gestational age, with a peak level at 24 to 32 weeks of gestation, (3) amniotic fluid beta-endorphin immunoreactivity levels are unrelated to fetal sex, and (4) diabetic patients may have a different distribution of beta-endorphin immunoreactivity pools than normal individuals.

Amniotic Fluid↗

PubMed1985

Receptor binding and biological activity of bivalent enkephalins.

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH2)n-X, where X is H-Tyr-D-Ala-Gly-Phe-Leu-NH-(n = 0, 2, 4, 6, 8, 10, 12), for the first series of dimeric pentapeptide enkephalins (DPEn), and H-Tyr-D-Ala-Gly-Phe-NH-(n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTEn). Comparison of biological activities with binding affinities revealed that: (1) the DPE series with n = 2-8 showed increased potency in the MVD assay relative to monomeric [D-Ala2, Leu5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay then predicted on the basis of binding affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2-12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta binding affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponding monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and mu-binding but not with MVD and delta-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and binding assays. Comparison of these compounds with the corresponding dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.

Analgesia↗