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K W Miller

Publications and source records attributed to K W Miller.

At least 73 records · Page 4Linked to original sources
PubMed1993

Stereoselectivity of channel inhibition by secondary alkanol enantiomers at nicotinic acetylcholine receptors.

BACKGROUND: At the nicotinic acetylcholine receptor, long chain alkanols reduce, whereas short chain alkanols augment endplate currents. Using the enantiomers of five members of a homologous series of secondary alkanols (2-butanol through 2-octanol), we tested the hypothesis that these actions occur at a single hydrophobic site in the lumen of the channel. Small alkanols would bind to this site without blocking the channel, stabilizing the open state and enhancing the apparent affinity of the agonist for channel opening. Long chain alkanols would bind the same site and simply inhibit without affecting the agonist's apparent affinity. METHODS: Agonist-stimulated 86Rb+ efflux from acetylcholine receptor-rich vesicles from Torpedo nobiliana was studied by adding agonist and allowing efflux to proceed for 10 s before termination by filtration. RESULTS: All of the 2-alkanols inhibited 86Rb+ efflux elicited by a maximally stimulating concentration of agonist. Inhibitory potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alkanol. The inhibitory potency of the enantiomers of 2-butanol differed twofold, but the other enantiomers exhibited no stereoselectivity. The enantiomers of 2-octanol caused a concentration-dependent depression of carbamylcholine-stimulated 86Rb+ efflux without significantly altering the agonist's apparent dissociation constant. In contrast, the enantiomers of 2-butanol caused: (1) a nonstereoselective decrease in carbachol's apparent dissociation constant and (2) the expected stereoselective decrease in maximal carbamylcholine-stimulated 86Rb+ efflux. CONCLUSIONS: The alkanol site that modulates the apparent agonist affinity for channel opening is distinct from the site that results in inhibition of cation flux through the channel.

Alcohols↗
PubMed1993

Barbiturate action is dependent on the conformational state of the acetylcholine receptor.

BACKGROUND: Barbiturates act on many neuronal ion channels by poorly understood mechanisms. The authors investigated the hypothesis that barbiturates inhibit the transient open-channel conformation of the nicotinic acetylcholine receptor (nAchR) by binding to a discrete site. METHODS: Inhibition curves of the agonist-stimulated efflux of 8bRb+ from nAchR-rich membrane vesicles prepared from the electric tissue of Torpedo nobiliana were obtained for 14 barbiturates using a filter assay. RESULTS: When added simultaneously with agonist, all agents inhibited the ion efflux with half-inhibitory concentrations (IC50), varying from 23 microM for pentobarbital to 880 microM for barbital, and with Hill coefficients of one. The effect of several barbiturates on the agonist concentration-response curve for carbachol-stimulated efflux indicated that this inhibitory action was not competitive. CONCLUSIONS: The IC50s of these agents did not correlate with their octanol/water partition coefficients, nor with general anesthetic potency, although a degree of channel inhibition occurred with many agents at general anesthetic concentrations. The existence of a barbiturate-inhibitory site of action was indicated by the structural specificity. This conclusion was supported by the Hill coefficient of one, and by the high inhibitory potencies, which ruled out membrane perturbations as a mechanism. This site on the transient open-channel conformation exhibits different structure-activity relationships than an allosteric site established by equilibrium barbiturate binding on the resting conformation of the AchR. Thus, barbiturate action depends on the nAchR's conformational state.

Animals↗
PubMed1993

Topical anesthesia during infant eye examinations: does it reduce stress?

We studied the effect of topical anesthesia on infant stress and corneal haze during the routine eye examination for retinopathy of prematurity. Using a double-blind protocol, 55 premature infants weighing less than 1501 g at birth were selected randomly to receive normal saline or proparacaine HCl 0.5% eye drops as a corneal wetting agent at their initial eye examination. Before, during, and after the procedure, infant stress was evaluated by heart rate, respiration rate, blood pressure, and transcutaneous oxygen saturation. Subjective assessment of the infant's cry intensity and corneal haze also were recorded. Adequate data were collected on 42 patients. Using analysis of variance and chi-square tests, we found no difference in any of these parameters between the two patients groups. These data suggest that topical anesthetic agents offer no advantage over normal saline eye drops during the examination of premature infants.

Administration, Topical↗
PubMed1993

Cycloalkanemethanols discriminate between volume- and length-dependent loss of activity of alkanols at the Torpedo nicotinic acetylcholine receptor.

Primary normal alcohols (1-n-alkanols) exert two effects on the nicotinic acetylcholine receptor when added simultaneously with agonist. First, propanol through decanol inhibit the open channel. Second, methanol through butanol, but not higher homologs, increase the apparent affinity of the agonist for inducing cation flux. To test the hypothesis that the length or volume of the alcohols might account for the fact that some members of the 1-n-alkanol homologous series lack activity, we have studied in parallel 11 members of another homologous series, i.e., the cycloalkanemethanols, c(CnH(2n-1)CH2OH. With steadily increasing potency, agents from cyclopropanemethanol to cyclodecanemethanol completely inhibited carbachol-stimulated 86Rb+ efflux from nicotinic acetylcholine receptor-rich postsynaptic vesicles from the electroplaques of Torpedo nobiliana, but even 90% saturated solutions of cycloundecanemethanol inhibited only part of the flux and neither cyclododecanemethanol nor cyclotetradecanemethanol caused any inhibition. Comparison of these results with those previously obtained for 1-n-alkanols indicates that as both series are ascended the cut-off in the inhibitory action on the channel occurs when the volume of the compounds exceeds approximately 340 A3. The apparent affinity for carbachol-induced flux was enhanced only by cyclopropanemethanol through cyclooctanemethanol, consistent with the hypothesis that a critical length of approximately 6.3 A cannot be exceeded. Thus, the sites mediating the two effects have different steric requirements and may be physically distinct.

Alcohols↗
PubMed1992

Probing conformational changes in the nicotinic acetylcholine receptor by Fourier transform infrared difference spectroscopy.

We have developed a Fourier transform infrared (FTIR) difference method for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR). Our approach is to deposit reconstituted nAChR membranes in a thin film on the surface of a germanium internal reflection element, acquire FTIR spectra in the presence of bulk aqueous solution using attenuated total reflection, and then trigger conformational changes by sequentially flowing a buffer either with or without an agonist past the film surface. Using the fluorescent probe, ethidium bromide, it is demonstrated that the method of nAChR film deposition does not affect the ability of the receptor to undergo the resting-to-desensitized state transition. The difference of FTIR spectra of nAChR films recorded in the presence and absence of agonists reveal highly reproducible infrared bands that are not observed in the difference of spectra recorded with only buffer flowing past the film surface. Some of the bands are assigned to changes in protein secondary structure and to changes in the structure of individual amino acid residues. Bands arising from the vibrations of the agonist bound to the receptor are also observed. The results demonstrate that FTIR difference spectroscopy can detect structural changes in the nAChR that occur upon the binding of ligands. The technique will be an effective method for investigating nAChR structure and function as well as receptor-drug interactions.

Animals↗
PubMed1992

Incorporation of the nicotinic acetylcholine receptor into planar multilamellar films: characterization by fluorescence and Fourier transform infrared difference spectroscopy.

A method for preparing thin, planar films of nicotinic acetylcholine receptor (nAChR) membranes that retain the ability to undergo the resting to desensitized state transition and that are suitable for spectroscopic studies has been developed. Native, alkaline-extracted nAChR membranes from Torpedo are dried under nitrogen on either a plastic microscope coverslip or a germanium internal reflection element (IRE) and then equilibrated with buffer. The drying procedure has no effect on the functional state of the nAChR as judged by a fluorescence assay using the probe ethidium bromide. The times required for an acetylcholine analogue (carbamylcholine), a local anesthetic (dibucaine), and a fluorescent probe (ethidium bromide) to penetrate films of varying degrees of thickness, interact with the receptor, and then to be washed from the films have been established. Under these conditions, the nAChR films can be repetitively cycled between the resting and desensitized states. Both fluorescence and infrared spectroscopy show that the films adhere strongly to either support even with buffer flowing continuously past the film surface. Fourier transform infrared difference spectra calculated from spectra recorded in the presence and absence of carbamylcholine show small, reproducible bands which reflect changes in nAChR structure upon desensitization.

Animals↗
PubMed1992

[Cholinergic receptors and anesthesia].

Postsynaptic chemically controlled ion channels are a possible location for the action of anaesthetics. The nicotinic acetylcholine receptor (AcChoR) is a member of a superfamily of chemically controlled ion channels, which have a large number of structural similarities in common. This receptor provides an excellent model for studying the effects of anaesthetics on excitable membrane proteins since it is well characterised and available in large quantities. The AcChoR consists of five subunits that surround a central ion channel. Anaesthetics can inhibit the function of the AcChoR by several different mechanisms: 1. By directly blocking the ion channel, 2. by an allosteric inhibition of the ion channel, 3. by changing the properties of the cell membrane surrounding the receptor. In high concentrations, anaesthetics stabilize the receptor in a desensitized state. Investigation of the mode of action of anaesthetics on the AcChoR provides important information as to the mechanisms of anaesthesia.

Anesthetics↗
PubMed1992

The general anesthetic potency of propofol and its dependence on hydrostatic pressure.

Although plasma concentrations of propofol during anesthesia are well known, the free concentration remains unknown because of uncertainties regarding plasma protein binding, interaction with other protein-bound substances, the level of binding to its lipid carrier, and the use of adjuvants. At elevated surrounding pressure, all general anesthetics require higher concentrations to reach adequate levels of anesthesia. To determine the anesthetic potency of propofol at equilibrium conditions and to study the effects of pressure on propofol-induced anesthesia, Rana pipiens tadpoles were exposed to different concentrations of pure, not emulsified, propofol in aqueous solution. Anesthesia was defined as loss of the righting reflex. Ten animals per concentration were used, and each experiment was conducted twice. Pressure experiments were performed with nonanesthetized tadpoles and urethane-anesthetized tadpoles as control groups. Propofol concentrations were measured spectrophotometrically. At 1 atmosphere absolute (atm abs), a semilogarithmic sigmoidal concentration-response curve was obtained with a half-maximal effect of propofol at 2.2 +/- 0.22 microM (EC50; mean +/- SE). Increased pressure shifted the concentration-response curve to the right. The EC50 increased linearly with increasing pressure up to 121 atm abs (EC50 at 121 atm abs = 4.1 +/- 0.41 microM). For pressure greater than 121 atm abs, an increased excitability of the tadpoles made it difficult to distinguish the righting reflex from involuntary movements. The saturated solubility of propofol in aqueous solution was found to be 1.0 +/- 0.02 mM (mean +/- SD), and the octanol/water partition coefficient was 4,300 +/- 280. Propofol adhered to the correlation between anesthetic potency and octanol/water partition coefficient exhibited by other general anesthetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, General↗
PubMed1992

Reductive desulfurization of dibenzyldisulfide.

Dibenzyldisulfide was reductively degraded by a methanogenic mixed culture derived from a sewage digestor. Toluene was produced with benzyl mercaptan as an intermediate in sulfur-limited medium. Toluene production was strictly associated with biological activity; however, the reducing agent for the culture medium, Ti(III), was partially responsible for production of benzyl mercaptan. Sulfide was not detected. Additions of sodium sulfide did not inhibit toluene production. Additions of 2-bromoethane sulfonic acid prevented methanogenesis but did not adversely affect toluene yields.

Journal Article↗
PubMed1992

Can nicotine self-inhibition account for its low efficacy at the nicotinic acetylcholine receptor from Torpedo?

Nicotine, a partial agonist, has a very low efficacy at the nicotinic acetylcholine receptor from Torpedo, but it is not clear whether this is because it is intrinsically poor at opening the ion channel or because, at concentrations that open the channel, it is also capable of blocking it. In this study, we exploited the action of ethanol, which increases the apparent affinity of cholinergic agonists for channel activation, and demonstrated that the weak action of nicotine is consistent with simultaneous activation and inhibition of the receptor. The presence of ethanol increased the efficacy of nicotine, producing an increase in the initial rate of cation efflux from acetylcholine receptor-rich membrane vesicles, as measured by a rapid quench-flow tracer ion assay. The initial rate of efflux increased with ethanol concentration until, in the presence of 1.5 M ethanol, the response to nicotine was indistinguishable from that of the full agonist carbamylcholine. The concentration-response curves for nicotine were bell-shaped, showing activation at low concentrations and inhibition at higher concentrations. Increasing concentrations of ethanol increased the apparent affinity of nicotine for channel activation and decreased its apparent affinity for channel inhibition. These actions broadened the bell-shaped curve, increasing the maximum response until it was equivalent to that of a full agonist. The apparent affinity of nicotine for its inhibitory site, derived from the aforementioned data, agreed with that determined independently by measuring the inhibition by nicotine of initial rates of ion efflux in response to acetylcholine. A value for the apparent affinity of nicotine for channel opening was estimated from the dependence of this parameter on ethanol concentration. When combined, these two parameters predicted the bell-shaped concentration-response curve for the action of nicotine. The results presented in this study are consistent with the notion that the efficacy of nicotine is determined by its relative affinities for channel activation and channel inhibition, but they do not rule out other contributions.

Animals↗
PubMed1992

Is cytomegalovirus associated with renal disease in AIDS patients?

Although it has been suggested that cytomegalovirus (CMV) infection of the kidney might facilitate the development of human immunodeficiency virus-associated nephropathy (HIVAN) or other morphologic renal changes in patients with AIDS, no systematic study has been performed on kidneys from AIDS patients. We examined 75 autopsy kidneys, two renal biopsy specimens, and a nephrectomy specimen from 78 HIV-infected patients (five with HIVAN) for the presence of CMV. Immunocytochemistry (ICC) utilizing a monoclonal antibody against the late antigen of CMV and in situ hybridization (ISH) with a biotinylated DNA probe for CMV sequences were used. The detection system for both ICC and ISH was streptavidin-conjugated alkaline phosphatase with Fast Red TR chromogen. CMV was detected in only 10 of the 78 kidneys examined (12.8%): eight by both methods, one by ISH only, and another by ICC only. All 10 positive kidneys were obtained from autopsies of patients with AIDS. The average number of positive cells (in approximately 15 x 10 mm sections) was 22 with ICC and 10 with ISH. Glomerular intracapillary cells (possibly endothelial cells) were the most commonly stained, followed by positive cells in the interstitium and peritubular capillaries. Relatively few tubular epithelial cells were stained. The majority of positive cells by either ICC or ISH did not show nuclear or cytoplasmic inclusions; however, only two of the 10 positive kidneys did not contain cells with typical Cowdry type-A intranuclear CMV inclusions. The most frequent pathologic finding in the kidneys positive for CMV by either ICC or ISH was acute tubular necrosis (in six of 10, 60%).(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Associated Nephropathy↗
PubMed1992

Conditions affecting the immunohistochemical detection of HIV in fixed and embedded renal and nonrenal tissues.

A number of studies have suggested that HIV infection can be detected in a variety of routinely fixed archival tissues using antibodies to various viral proteins. In order to study this immunocytochemical approach, paraffin sections were examined with a large panel of commercially available monoclonal antibodies against the various HIV proteins (5 antibodies to p24, 1 to p17, 1 to gp41, and 1 to gp120) using a streptavidin-biotin method. A polyclonal antibody against p24 was also tested. Formalin-fixed, paraffin-embedded HIV infected CEM E5 T cells were used as positive controls. Tissues from AIDS patients included 31 kidneys, 8 lymph nodes, 2 spleens and 3 brains. Non-AIDS tissues examined were 6 renal biopsies with focal segmental glomerulosclerosis, 5 with interstitial nephritis, 6 reactive lymph nodes, and a brain with encephalitis, all from patients not known to be at high risk for HIV infection. Additional negative controls included: 1) replacement of primary antibody with a hybridoma derived mouse monoclonal IgG1 standard, 2) omission of the primary antibody, and 3) sections of formalin-fixed paraffin-embedded CEM E5 T cells cultures not infected with HIV. Competition experiments with excess recombinant p24 protein were also performed. False positive staining with the IgG1 standard or with the antibodies to HIV proteins was frequently seen in tissues with pathologic findings (inflammation, hyalin degeneration), particularly following protein digestion. Protein digestion also had a major impact on specific staining. Digestion with proteinase K abolished specific staining for the core proteins of the virus (p17, p24) on the positive control sections.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Associated Nephropathy↗
PubMed1991

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II).

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.

Amino Acid Sequence↗
PubMed1991

A 20-month olestra feeding study in dogs.

Three groups of beagle dogs (five/sex/group) were fed olestra, a mixture of octa-, hepta- and hexa-esters of sucrose formed with long-chain fatty acids, at 0, 5 or 10% of the diet for 20 months. The objective of the study was to assess the potential chronic toxicity of olestra in a non-rodent species. The feed was supplemented with vitamins A and E to ensure that the diets were nutritionally adequate and comparable for all groups. The levels of supplementation were established in a 91-day feeding study. Survival was 100% and growth was not affected by olestra. Olestra-fed animals consumed more feed than controls, apparently to compensate for the caloric dilution of the diet by olestra, but the increases were generally not statistically significant. No biologically significant changes were seen in haematological or serum biochemical parameters or in vitamin D and vitamin K status of the animals. Histopathology revealed no olestra-related effects. Isolated incidences of soft stools, apparently resulting from the large amounts of undigested olestra, were noted in olestra-fed animals. The results of this study indicate that olestra was not toxic when fed to dogs at up to 10% of the diet for 20 months.

Animal Feed↗
PubMed1991

Serum 25-hydroxyvitamin D concentrations of free-living subjects consuming olestra.

The effect of olestra on vitamin D status was assessed in a 6-wk, double-blind, placebo-controlled study involving 202 free-living adults. Subjects consumed a total of 20 g/d of olestra or triglycerides in cookies eaten at each meal. A 20-micrograms ergocalciferol capsule was taken with each morning meal. Serum 25-hydroxyergocalciferol (25-OHD2) concentrations rose from approximately 5.7 to 39.0 and 31.7 nmol/L in the placebo and olestra groups, respectively, at week 6. At week 6, 25-OHD2 contributed 46-54% to total serum 25-OHD concentration compared with 11% at baseline. The 19% decrease in serum 25-OHD2 concentrations produced by olestra in this study is equivalent to a decrease of approximately 1.2 nmol/L under nonsupplemented dietary conditions. Ingesting 20 g olestra/d in the diet is thus not expected to affect vitamin D nutritional status.

25-Hydroxyvitamin D 2↗
PubMed1991

Short chain and long chain alkanols have different sites of action on nicotinic acetylcholine receptor channels from Torpedo.

At nicotinic acetylcholine receptors, short chain n-alcohols (alkanols) have excitatory actions, whereas long chain alkanols inhibit channel activity. This study tests a previously proposed unitary hypothesis that suggests that these contrasting actions can be accounted for by interaction at just one hydrophobic site within the ion channel lumen. All alkanols bind to this site, but only long chain alkanols are large enough to completely block the channel. Short chain alkanols are too small to cause any channel occlusion, and in binding to the site they stabilize the open state of the receptor and enhance ion flux. In this study, we assay integrated agonist-stimulated ion efflux over 15 msec, as a measure of receptor activity. In nicotinic acetylcholine receptor-rich membrane vesicles from Torpedo, we show that, in contradiction to this elegant model, long chain and short chain alkanols appear to act at different sites. Firstly, ethanol and octanol do not compete for a single site on the receptor. Secondly, alkanol chain length dependencies for inhibition and for flux enhancement are significantly different. Thirdly, intermediate length alkanols do not partially inhibit channels, as required by the model; high concentrations of these alkanols completely inhibit the response. Fourthly, careful measurements, including determination of the free alkanol concentration, of inhibitory potencies of alkanols from propanol to decanol show no evidence for a steric contribution to the ability of an alkanol to inhibit the ion channel. Furthermore, our results suggest that the inhibitory effect of long chain alkanols may be mediated by a discrete site on nicotinic acetylcholine receptors, whereas there is no evidence that a protein site is involved in the excitatory mechanism of short chain alkanols. Indeed, it seems more likely that short chain alkanols may have a nonspecific site of action.

Acetylcholine↗
PubMed1990

A study of the effect of general anesthetics on lipid-protein interactions in acetylcholine receptor enriched membranes from Torpedo nobiliana using nitroxide spin-labels.

Stearic acid, phosphatidylcholine, and phosphatidylglycerol nitroxide spin-labels were used to probe the effect of 1-hexanol, urethane, diethyl ether, and ethanol on lipid-protein interactions in nicotinic acetylcholine receptor (nAcChoR) rich membranes from Torpedo nobiliana. For stearic acid spin-labeled at the C-14 position of the sn-1 acyl chain, 1-hexanol induced little change (over a wide concentration range, 0-16.7 mM) in either the ESR line shape or the proportion of motionally restricted spectral component from labels probing the protein interface. The main effect of 1-hexanol was limited to an increase in the mobility of stearic acid spin-labels probing the non-protein-associated environment. In contrast, for C-14 phosphatidylcholine spin-label, 1-hexanol decreased the fraction of spin-labels motionally restricted at the protein interface from 0.33 without 1-hexanol to 0.20 with 16.7 mM 1-hexanol, with no change in the line shape of the spectral component of these labels. The ESR spectral line shape of the fluid component due to phosphatidylcholine labels in sites away from the protein interface displayed a gradual decrease in spectral anisotropy on addition of increasing amounts of 1-hexanol. At a concentration of 1-hexanol that desensitizes half the receptors, the fraction of motionally restricted phosphatidylcholine spin-label is reduced by approximately 15%. The effect of 1-hexanol on phosphatidylglycerol spin-labels was intermediate between these two cases. Similar effects were measured with other general anesthetics, including urethane, diethyl ether, and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthetics↗