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Biomedical subjects

R J Miller

Publications and source records attributed to R J Miller.

At least 253 records · Page 14Linked to original sources
PubMed1988

L-[2,5-H2]phenylalanine, an alternate substrate for rat liver phenylalanine hydroxylase.

The phenylalanine analogue L-[2,5-H2]phenylalanine (1) was found to be a viable substrate (KM = 0.45 mM, kcat = 8 s-1) for L-phenylalanine-activated, rat liver phenylalanine hydroxylase (EC 1.14.16.1) (PAH). The PAH-catalyzed oxidation of 1 was stoichiometric with the oxidation of cofactor, 6-methyl-tetrahydropterin. Spectral and chromatographic data of the product from the oxidation of 1 by PAH were found to be in accord with a 3,4-epoxide. The enzymatic epoxidation of 1 is consistent with the hypothesis of an intermediate arene oxide on the reaction coordinate for PAH hydroxylation of L-phenylalanine.

Animals↗
PubMed1988

Calmodulin- and protein phosphorylation-independent release of catecholamines from PC-12 cells.

Catecholamine secretion from PC-12 cells can be triggered by agents that increase intracellular Ca2+ and is enhanced by phorbol esters and agents that elevate intracellular cAMP concentrations. In mutant PC-12 cells lacking cAMP-dependent protein kinase (PK-A) in which protein kinase C (PK-C) was down-regulated, Ca2+-dependent secretion occurred normally but was no longer enhanced by cAMP or phorbol esters. In digitonin-permeabilized PC-12 cells that lacked PK-C and PK-A, a range of calmodulin (CaM) inhibitors failed to block Ca2+-triggered catecholamine release. Moreover, Mn2+, a CaM activator, failed to trigger catecholamine release whereas Ba2+, which does not activate CaM, supported secretion. These results indicate that the basic mechanism of stimulus/secretion coupling in PC-12 cells does not absolutely require a regulated protein phosphorylation- or calmodulin-dependent step.

Adrenal Gland Neoplasms↗
PubMed1988

Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons.

Multiple types of calcium channels have been found in neurons, but uncertainty remains about which ones are involved in stimulus-secretion coupling. Two types of calcium channels in rat sympathetic neurons were described, and their relative importance in controlling norepinephrine release was analyzed. N-type and L-type calcium channels differed in voltage dependence, unitary barium conductance, and pharmacology. Nitrendipine inhibited activity of L-type channels but not N-type channels. Potassium-evoked norepinephrine release was markedly reduced by cadmium and the conesnail peptide toxin omega-Conus geographus toxin VIA, agents that block both N- and L-type channels, but was little affected by nitrendipine at concentrations that strongly reduce calcium influx, as measured by fura-2. Thus N-type calcium channels play a dominant role in the depolarization-evoked release of norepinephrine.

Animals↗
PubMed1988

Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology.

Fluorescent indicator molecules, such as fura-2, are useful probes for studying the concentrations of ions in single cells. A key feature of these fluorescent dyes is the shift in their excitation spectra upon binding the ion, thus making alternate excitation from two wavelengths desirable. In this report we describe an inexpensive system for making simultaneous electrophysiological and dual excitation fluorescence measurements using equipment much of which is available in a typical biophysical laboratory. In order to synchronize the fluorescence signal with the appropriate excitation wavelength we devised a simple computer program which uses the output of photodiodes placed in the excitation beam to determine which wavelength is illuminating the cell. We also describe the use of a liquid light guide to transmit excitation illumination from the light source to the epifluorescence port of the microscope in order to isolate a perfusion chamber from light, electrical noise and vibration. A sensitive light detection system was assembled using a photomultiplier tube and discriminator that took advantage of sampling single unit events obtained with photon counting rather than the analog of anode current. However, rather than employing a sophisticated and expensive photon counting system, a filter was used to integrate the signal so that an analog output could be presented to a multichannel analog to digital converter commonly used for electrophysiological recordings.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Modifying hypnotic suggestibility with the Carleton Skills Training program.

The Carleton Skills Training (CST) program was used to investigate (a) whether increases in hypnotic responsiveness obtained at Carleton University could be replicated at a different laboratory, (b) the influence of demand characteristics on suggestibility gains, and (c) whether initial gains are maintained over time. After two screening sessions, a replication and experimental group received the CST program, while a control group was allowed to practice hypnotic responding. All groups were then tested twice. Whereas the replication group was told that training and testing were part of the same experiment, the experimental group was told that training and testing were unrelated. Trained subjects returned after 4 months for a final session. Results indicated that (a) the CST program does increase hypnotic responsiveness, (b) suggestibility gains found in this study were relatively modest, (c) demand characteristics may influence suggestibility gains, and (d) suggestibility gains were not maintained at follow-up.

Adolescent↗
PubMed1988

Guanine nucleotide-binding protein Go-induced coupling of neuropeptide Y receptors to Ca2+ channels in sensory neurons.

Neuropeptide Y (NPY) inhibited the Ca2+ current (ICa) in rat dorsal root ganglion neurons in vitro. NPY inhibited the sustained ICa evoked by steps to 0 mV from a holding potential of -40 mV and the inactivating ICa, which was additionally evoked from a more negative holding potential of -80 mV. The effects of NPY on both phases of the ICa were abolished if cells were first treated with pertussis toxin (PTX). When a combination of GTP and the purified alpha-subunit of the guanine nucleotide-binding protein Go was perfused into PTX-treated cells, the inhibitory effects of NPY on the ICa reappeared in a time-dependent fashion. GTP or alpha-subunit perfused separately was relatively ineffective. The effects of NPY reappeared more rapidly at higher concentrations of alpha o. Chronic treatment of these cells with phorbol ester "down-regulates" protein kinase C (PKC) and reduces inhibition of the sustained current by NPY. In PTX-treated cells in which PKC had been removed by down-regulation, inhibition of ICa was also reconstituted following the perfusion of GTP/alpha o. Under these circumstances, NPY inhibited the transient phase of the ICa more than the sustained phase. These results indicate that Go, the major PTX substrate in the central nervous system, may normally mediate the inhibitory effects of NPY receptors on dorsal root ganglion Ca2+ channels.

Animals↗
PubMed1988

Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca2+ concentration ([Ca2+]i) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH greater than 10 pM elicited increases in [Ca2+]i in identified cells but not in others. In contrast, depolarization induced by 50 mM K+ increased [Ca2+]i in all cells. Ca2+ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca2+]i fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca2+ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca2+]i. Removal of extracellular Ca2+ or addition of the dihydropyridine Ca2+-channel blocker nitrendipine completely blocked the secondary rise in [Ca2+]i but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K+-induced increases in [Ca2+]i in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca2+ influx was permitted either by allowing a secondary rise to occur or by producing a Ca2+ transient by depolarizing the cells with 50 mM K+. It therefore appears that the response to GnRH consists of an initial phase of Ca2+ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca2+ influx through nitrendipine-sensitive Ca2+ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.

Animals↗
PubMed1988

A glutamate receptor regulates Ca2+ mobilization in hippocampal neurons.

We investigated the effect of various excitatory amino acids on intracellular free Ca2+ concentration ( [Ca2+]i) in single mouse hippocampal neurons in vitro by using the Ca2+-sensitive dye fura-2. In normal physiological solution, glutamate, kainate, N-methyl-D-aspartate, and quisqualate all produced increases in [Ca2+]i. When all extracellular Ca2+ was removed, kainate and N-methyl-D-aspartate were completely ineffective, but quisqualate and glutamate were able to produce a spike-like Ca2+ transient, presumably reflecting the release of Ca2+ from intracellular stores. Ca2+ transients of similar shape could also be produced by the alpha 1-adrenergic agonist phenylephrine. After the production of a Ca2+ transient a second addition of quisqualate was ineffective unless intracellular stores were refilled by loading the cell with Ca2+ following depolarization in Ca2+-containing medium. None of the conventional excitatory amino acid receptor antagonists inhibited the Ca2+-mobilizing effects of quisqualate. Furthermore alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unable to produce Ca2+ mobilization in Ca2+-free medium, although it could produce Ca2+ influx in Ca2+-containing medium. Thus, glutamate can produce mobilization of Ca2+ from intracellular stores in hippocampal neurons by acting on a quisqualate-sensitive but AMPA-insensitive receptor. This receptor is therefore distinct from the quisqualate receptor that produces cell depolarization. The possibility that this Ca2+-mobilizing effect is mediated by inositol triphosphate production is discussed.

Animals↗
PubMed1988

Effect of pupil size on dark focus accommodation.

A potential concern in some studies of dark focus accommodation is the role of pupil size (and related spherical aberration) in laser optometer measurements. Dark focus measurements were obtained for 25 young adult subjects while they viewed the laser speckle pattern through artificial pupils of 3, 4, 5, 6, 7 and 8 mm diameter. Analysis showed no significant effect of pupil size on dark focus. It would appear that pupil size variations should not be a source of concern in laser optometer dark focus assessment.

Accommodation, Ocular↗
PubMed1988

Tricyclic antidepressants block N-methyl-D-aspartate receptors: similarities to the action of zinc.

1. Using the radioligand [3H]-MK801, we have examined drug interactions with the phencyclidine recognition site of the N-methyl-D-aspartate receptor. 2. The tricyclic antidepressants desmethylimipramine and imipramine inhibited [3H]-MK801 binding with IC50 values of 7.4 and 22.5 microM, respectively. Other related tricyclic antidepressants and neuroleptics were also effective but less potent. 3. Desmethylimipramine, imipramine and chlorimipramine slowed the dissociation rate of [3H]-MK801 in a similar manner to Zn2+. Phencyclidine and related compounds had no effect on the dissociation rate of [3H]-MK801. 4. Desmethylimipramine, imipramine and ketamine also prevented the Ca2+ influx into cultured cortical neurones of the rat produced by N-methyl-D-aspartate. 5. As the actions of tricyclic antidepressants in this system are not competitive with respect to N-methyl-D-aspartate, glycine or MK-801, and as they slow the dissociation of [3H]-MK801, we conclude that tricyclic antidepressants may be acting at the Zn2+ recognition site on the N-methyl-D-aspartate receptor.

Animals↗
PubMed1988

Multiple sites for the regulation of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) receptor consists of a recognition site for NMDA, a cation-selective ion channel, and binding sites for glycine, Zn2+, and phencyclidine-like compounds. In addition, the channel can be blocked by Mg2+. We have studied the NMDA receptor using the potent and specific phencyclidine-like compound [3H]MK-801. Drugs that bind to the NMDA, glycine, Zn2+, and Mg2+ recognition sites profoundly affect both the association and the dissociation rate of [3H]MK-801. NMDA-like agonists, glycine, and Mg2+ all increase the rates of association and dissociation of [3H]MK-801, whereas the NMDA antagonists AP5 and Zn2+ decrease these rates. These data allow the construction of a model of drug interaction at the NMDA receptor that is based on the binding of MK-801 within the NMDA-operated channel. Using this model it is possible to clearly distinguish between drug action at any of the five binding sites proposed.

Animals↗
PubMed1988

125I-neuropeptide Y and 125I-peptide YY bind to multiple receptor sites in rat brain.

We describe the preparation of monoiodinated neuropeptide Y (Tyr1-125I-NPY) and monoiodinated peptide YY (Tyr36-125I-PYY). Using these ligands, we detected high, moderate, and low affinity receptor populations in rat brain. Only high and moderate affinity binding sites were suggested by saturation binding studies. Tyr1-125I-NPY bound to 8 +/- 3% of the sites with a Kd of 54 pM (Bmax = 19.4 fmol/mg of protein) and to 92 +/- 3% of the sites with a Kd of 0.92 nM (Bmax = 220.0 fmol/mg of protein). Tyr36-125I-PYY bound to 14 +/- 3% of the sites with a Kd of 23.5 pM (Bmax = 36.4 fmol/mg of protein) and to 86 +/- 3% of the sites with a Kd of 1.9 nM (Bmax = 220.1 fmol/mg of protein). The fragments NPY 13-36 and PYY 13-36 were able to compete with 10 pM Tyr1-125I-NPY for essentially all the binding sites. The fragments were 1 to 2 orders of magnitude less potent than the native peptides. Approximately 50% of the moderate affinity sites, but not the high affinity sites, were reversibly "lost" in the presence of 5'-guanylyl imidophosphate [Gpp(NH)p], a nonhydrolyzable analog of GTP. Kinetic studies revealed that Tyr1-125I-NPY dissociation could be best described by three dissociation rates. The proportions of slow and intermediate dissociation matched the proportions of moderate and high affinity binding sites, respectively, as suggested by equilibrium studies. There also existed a phase of fast dissociation. When Gpp(NH)p was added during dissociation, the proportion of slow dissociation decreased to the same extent that the fast dissociation was increased. However, the proportion of intermediate dissociation did not change. We propose that rat brain contains a minor population of high affinity NPY binding sites with an intermediate dissociation rate and no sensitivity to Gpp(NH)p. There is also a major population of moderate affinity binding sites with a slow dissociation rate. A component of these sites can convert to a low affinity state with a fast dissociation rate. Gpp(NH)p enhances conversion by stabilizing the low affinity state, thereby producing a "loss" of moderate affinity binding.

Animals↗
PubMed1988

[3H]MK801 binding to the N-methyl-D-aspartate receptor reveals drug interactions with the zinc and magnesium binding sites.

We examined the ability of a range of tricyclic antidepressants (TCADs) and phenothiazine derivatives and their metabolites to inhibit the N-methyl-D-aspartate (NMDA) receptor complex using a [3H]MK801 binding assay. Desmethylation of the side chain of both TCADs and phenothiazines increased their potency against [3H]MK801 binding, as did removal of Cl substituted on the conjugated ring. Other side chain modifications further increased the potency of phenothiazines such as in the case of ethopropazine. Generally, the increase in potency of drugs at the NMDA receptor complex was associated with a decrease in the potency at other sites of action of these compounds. This finding suggests that it may be possible to separate the established actions of these compounds from their NMDA inhibitory effects. We also examined the mechanism of action of a number of compounds by monitoring drug effects on the dissociation rate of [3H]MK801 in the presence of Mg++. Phenothiazines and TCADs generally slow the dissociation of [3H]MK801, although to differing extents. Drugs such as 9-aminoacridine, cyproheptadine and ethopropazine also slowed the dissociation rate. These findings suggest a Zn++-like action of these compounds. In contrast, mecamylamine, methapyrilene and procyclidine had very little effect on the dissociation rate, suggesting a competitive action at the [3H]MK801/phencyclidine binding site. Chlorpromazine at low concentrations slowed the dissociation rate, while increasing it at higher concentrations. Thus, chlorpromazine demonstrated both Zn++ and Mg++-like effects. These studies demonstrate novel inhibitory actions of TCAD and phenothiazine derivatives at the NMDA receptor complex that are apparently mediated by the Zn++ binding site.

Animals↗
PubMed1988

Voltage-dependent binding of dihydropyridine calcium channel blockers to guinea pig ventricular myocytes.

The voltage dependence of the binding of dihydropyridine Ca channel blockers (+)-[3H]PN200-110 and [3H]nitrendipine to enzymatically isolated guinea pig ventricular myocytes was examined. The equilibrium saturation binding of (+)-[3H]PN200-110 could be well described by a simple 1:1 binding scheme under all conditions tested. The results demonstrated that the effect of depolarization induced by high extracellular K+ (50 mM) compared to normal K+ (4.8 mM) was an increase in the observed number of binding sites (Bmax) with no change in the measured affinity of binding (Kd). Similarly, depolarization induced by a combination of Na channel toxins in the presence of normal extracellular K+ caused an increase in the observed binding of (+)-[3H]PN200-110 comparable to that observed with high K+. [3H]Nitrendipine binding was likewise increased by depolarization in the presence of 50 mM K+ compared to 4.8 mM K+. Percoll density gradient centrifugation was found to enrich the cell preparation with viable myocytes and increased the measured voltage-dependent change in binding. In agreement with the predictions from previous studies (Kamp and Miller, 1987a), the magnitude of the observed change in Bmax was directly related to the fraction of cells which were viable in a given experiment. These results are discussed in comparison to the modulated receptor hypothesis proposed from electrophysiological studies.

Animals↗
PubMed1988

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons.

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.

Animals↗