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Biomedical subjects

R A Harris

Publications and source records attributed to R A Harris.

At least 289 records · Page 16Linked to original sources
PubMed1991

A transient osmotic permeabilization method for the introduction of impermeant molecules into functional brain membrane vesicles.

Mouse brain membrane vesicles (microsacs) were transiently permeabilized by hypo-osmotic shock. This permeabilization method resulted in the encapsulation of both [14C]sucrose and exogenous alkaline phosphatase. The efficiency of this method was estimated by [14C]sucrose encapsulation experiments to be approximately 81%. External membrane binding experiments with the lectin [3H]concanavalin A demonstrate that the microsacs were not inverted by permeabilization. Following permeabilization, the functional integrity of a ligand-gated ion channel, the GABAA receptor complex, was investigated. Muscimol-stimulated 36Cl-uptake experiments show that this receptor retains its functional properties including blockade by the receptor antagonist bicuculline and potentiation by the allosteric modulators flunitrazepam and pentobarbital. The osmotic permeabilization technique described here provides several advantages over other permeabilization methods. These advantages include a high trapping efficiency, the encapsulation of not only small solutes but large membrane impermeant compounds such as enzymes and the functional preservation of at least one transmembrane protein. Furthermore, this method does not require specialized equipment and does not result in large, permanent holes in the plasma membrane.

Alkaline Phosphatase↗
PubMed1991

Ultimate limits in ultrasonic imaging resolution.

According to elementary theory, the resolution of an ultrasonic imaging system increases with the ultrasonic frequency. However, frequency is limited by frequency-dependent attenuation. For imaging at any required depth, resolution improvement beyond the limit imposed by ultrasonic frequency can be obtained by increasing the ultrasonic intensity. This is itself, however, dependent on safety considerations and the effects of nonlinearity. In homogeneous media, image resolution increases with decreasing f-number. Particularly at low f-numbers, however, tissue inhomogeneity leads to a deterioration in image quality. Inhomogeneity may also be considered in terms of phase aberration. It has been found that for a given aperture, image degradation due to phase aberration is worse at higher frequencies. Schemes have been proposed for correction of this problem, but so far model systems do not lend themselves to clinical application. Deconvolution is unsatisfactory, speed correction is impracticable and synthetic aperture scanning and holography are virtually useless in biological tissues. Ultrasound-computed tomography has had only limited success. Speckle reduction can improve target detectability, but at the expense of resolution. Time-frequency control provides a useful partial solution to the problem of resolution reduction resulting from attenuation. It is clear that improved resolution would result in significant clinical benefits. An optimisation system for aperture size and ultrasonic frequency is proposed with signal averaging for resolution enhancement of a defined object area. This would have a compact ultrasonic beam and would allow frame rate to be traded for resolution, by means of signal averaging.

Humans↗
PubMed1991

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Amino Acid Sequence↗
PubMed1991

Purification and comparative study of the kinases specific for branched chain alpha-ketoacid dehydrogenase and pyruvate dehydrogenase.

Rat heart branched chain alpha-ketoacid dehydrogenase kinase (BCKDH kinase) and pyruvate dehydrogenase kinase (PDH kinase) were purified from their respective complexes to apparent homogeneity. BCKDH kinase consisted of one subunit with molecular weight 44,000-45,000 Da, whereas PDH kinase consisted of two subunits with molecular weight 48,000 Da (alpha) and 45,000 Da (beta) as previously shown for the bovine kidney enzyme (Stepp et al., 1983, J. Biol. Chem. 258, 9454-9458). Proteolysis maps of BCKDH kinase and the two subunits of PDH kinase were different, suggesting that all subunits are different entities. The alpha subunit of the rat heart PDH kinase could be cleaved selectively by chymotrypsin with concomitant loss of kinase activity, as previously shown for the bovine kidney enzyme, suggesting that the catalytic activity of PDH kinase resides in the alpha subunit. The beta subunit appeared to be a different entity unique to the PDH kinase. Both kinases exhibited marked substrate specificity toward their respective complexes and would not inactivate heterologous complexes. The kinases possessed slightly different substrate specificity toward histones. BCKDH kinase preferentially phosphorylated histones in the order f1 greater than f2B much greater than f2A much greater than f3. The relative order for PDH kinase was the same, but f2A and f3 were considerably better substrates than they were for BCKDH kinase. These observations suggest that the kinases have different requirements for the structure of the protein at their phosphorylation sites.

Animals↗
PubMed1991

Metoclopramide and renal vascular resistance.

We have studied the effect of i.v. metoclopramide on renal vascular resistance in nine healthy volunteers. Peak systolic and end-diastolic frequencies were measured using duplex Doppler ultrasound of a renal interlobar artery, before and after the administration of i.v. metoclopramide 10 mg, and the resistance index derived. There was no significant change in mean arterial pressure or resistance index following metoclopramide.

Adult↗
PubMed1991

Cerebellar GABAB receptors modulate function of GABAA receptors.

Interactions between GABAA and GABAB receptors were studied using muscimol-stimulated uptake of 36Cl- by membrane vesicles from mouse cerebellum. Baclofen inhibited muscimol-stimulated 36Cl- uptake and this action was more pronounced with longer flux times (30 vs. 3 s) and after predesensitization of GABAA receptors. Baclofen also inhibited 36Cl- flux by cortical membranes but was more effective with cerebellar preparations. The action of baclofen was stereoselective, calcium-dependent, and blocked by the GABAB receptor antagonist 2-OH-saclofen. It was mimicked by GTP-gamma-S but not by GDP-beta-S, which suggests that baclofen may be acting via a G protein. The action of baclofen was inhibited by U73122, an inhibitor of phospholipase C. However, the potassium channel blockers tetraethylammonium or Ba2+ did not affect the action of baclofen. The results show that activation of GABAB receptors can inhibit the function of GABAA receptors and suggest that this action involves either a nondesensitizing subtype of GABAA receptor or the rate or recycling of desensitized to nondesensitized receptors. We speculate that this action of baclofen results from activation of phospholipase C and phosphorylation of a subtype of GABAA receptor by protein kinase C.

Animals↗
PubMed1991

Establishment of latency in vitro by the herpes simplex virus type 1 mutant in1814.

The herpes simplex virus type 1 (HSV-1) mutant in1814 possesses an insertion mutation that abolishes trans-activation of immediate early (IE) transcription by the virion protein Vmw65. Interactions between in1814 and the host cell were examined by use of an in vitro latency system which relies on infection of human foetal lung (HFL) cells at 42 degrees C to prevent lytic growth of virus. Mutant in1814 was retained in HFL cells after infection at low m.o.i. and incubation at 42 degrees C, and was reactivated by superinfection of monolayers with viruses that express the HSV-1 IE protein Vmw110. Moreover, latency was established by in1814 in an analogous manner at 37 degrees C. The low cytotoxicity of in1814 enabled an investigation of latency after infection at high m.o.i. (five particles per cell) to be undertaken. At 42 degrees C, or at 37 degrees C in the presence of an inhibitor of DNA synthesis, in1814 DNA was maintained at low abundance (one to eight copies per infected cell) in a non-linear configuration. The absence of trans-activation by Vmw65 therefore predisposes HSV to latency, as opposed to lytic growth, in HFL cells, resulting in the retention of the genome in a form resembling that found in vivo.

Cell Line↗
PubMed1991

Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 36Cl- uptake by brain microsacs.

The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.

Animals↗
PubMed1991

Modulation of gamma-aminobutyric acidA receptor-operated chloride channels by benzodiazepine inverse agonists is related to genetic differences in ethanol withdrawal seizure severity.

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.

Animals↗
PubMed1991

Chronic ethanol treatment alters brain levels of gamma-aminobutyric acidA receptor subunit mRNAs: relationship to genetic differences in ethanol withdrawal seizure severity.

Chronic ethanol treatment is known to alter the function of the gamma-aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the alpha 1, alpha 3, alpha 6, gamma 2s, gamma 2L, and gamma 3 receptor subunits in withdrawal seizure-prone and -resistant (WSP and WSR, respectively) mice fed an ethanol-containing liquid diet or a control diet. Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit-specific probes. Chronic ethanol treatment decreased the content of alpha 1 mRNA in WSP but not WSR mice and decreased the content of alpha 6 mRNA in WSR but not WSP mice. The content of gamma 3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of alpha 3 and alpha 6 mRNA than the WSR line. Thus, a decrease in the content of alpha 1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of alpha 6 and alpha 3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for gamma 2S and gamma 2L subunits do not appear to be altered in ethanol dependence.

Animals↗
PubMed1991

Neuroadaptive responses to chronic ethanol.

Cellular responses of neuronal tissue to chronic ethanol exposure are reviewed. Evidence for adaptive responses to the acute actions of ethanol is available for five systems: GABA-activated chloride channels, voltage-sensitive calcium channels, NMDA-activated cation channels, receptors coupled through stimulatory guanine nucleotide binding proteins, and membrane lipid order. We suggest that at least some of these adaptive responses occur because of ethanol actions at the level of gene expression.

Alcohol Drinking↗
PubMed1991

Effect of chronic ethanol treatment and selective breeding for sensitivity to ethanol on calcium release induced by inositol trisphosphate or ethanol from brain and liver microsomes.

Our previous work showed that ethanol increases the resting intracellular free calcium concentration (CAi) in synaptosomes and releases calcium from an inositol trisphosphate (IP3)-insensitive calcium store of brain microsomes. In this report, we investigated the effects of chronic ethanol treatment and selective breeding for hypnotic sensitivity to ethanol on IP3 and ethanol-stimulated calcium release from brain and liver microsomes. Chronic ethanol treatment did not alter IP3-stimulated calcium release from brain microsomes or ethanol-stimulated calcium release from brain or liver microsomes. Chronic ethanol treatment increased the spontaneous release of calcium from brain but not liver microsomes. In microsomes isolated from cerebellum or cerebral cortex of long-sleep (LS) and short-sleep (SS) mice, ethanol and IP3 released calcium in a concentration dependent manner. The amount of calcium released by ethanol and IP3 was larger in microsomes isolated from cerebellum than microsomes from cerebral cortex. However, the amount of calcium released by ethanol and IP3 did not differ between the two lines in either brain area. These results do not support the idea that the hypnotic effects of ethanol are due to ethanol-induced calcium release from a nonmitochondrial intracellular calcium store in brain tissue. The development of ethanol tolerance and dependence also does not appear to be associated with altered ability of ethanol to release calcium from non-mitochondrial intracellular stores; however, effects of chronic ethanol exposure on spontaneous release of intracellular calcium could alter neuronal function in ethanol dependence.

Alcohol Drinking↗
PubMed1991

Ethanol-induced changes in chloride flux are mediated by both GABA(A) and GABA(B) receptors.

Low concentrations of ethanol (10-30 mM) in the presence of a GABAB receptor agonist, baclofen, promoted 36Cl- uptake into membrane vesicles (microsacs) prepared from mouse cortex. Neither ethanol nor baclofen alone altered chloride influx. The GABAB antagonists, phaclofen and 2-hydroxy-saclofen, completely blocked the increase in chloride flux produced by ethanol in the presence of either baclofen or GABA. Ethanol increased the chloride conductance produced by the GABAA agonists muscimol, isoguvacine, imidazolacetic acid and amino-propane sulfonic acid and this action of ethanol was blocked by phaclofen. The specific GABAA antagonist, bicuculline, blocked ethanol-induced increase in chloride flux in the presence of either baclofen or GABA. GABA-activated chloride channels were also studied in Xenopus oocytes expressing mouse brain mRNA. In this preparation, GABA action was enhanced by ethanol, pentobarbital, and diazepam, and 2-hydroxy-saclofen partially antagonized the action of ethanol without altering the effects of pentobarbital or diazepam. These results suggest that ethanol enhancement of GABAA receptor-chloride channel function also requires activation of GABAB receptors.

Alcohol Drinking↗
PubMed1991

Mammalian genetics in the study of alcohol and anesthetic actions.

Rodent lines differing in genetic sensitivity to ethanol have been selected using a variety of behavioral end points to assess ethanol action. The use, albeit limited, of these lines to study anesthetic action is presented. In addition, one pair of lines has been selected for differences in sensitivity to nitrous oxide anesthesia, and studies with these lines are reviewed. Several of these selected lines of rodents have been used to study neurochemical mechanisms of ethanol action, and the importance of combining classical and molecular genetic approaches is discussed. Genetic studies support the concept of multiple mechanisms of action of ethanol and indicate that these mechanisms are not necessarily the same for ethanol, barbiturates, or gaseous anesthetics.

Alcohols↗
PubMed1991

Effects of various secretagogues on [Ca2+]i in cultured human nasal epithelial cells.

Recent studies conducted on cultured canine tracheal cells have suggested that a complex relationship exists between cAMP and Ca2+ in the control of electrolyte secretion. The goal of this study was to determine if the Ca2+ second messenger system functions in a similar fashion in cultured human nasal epithelial cells, a tissue in which control of electrolyte secretion is known to be disrupted in the genetic disease, cystic fibrosis. Human nasal epithelial tissue was obtained as a by-product of surgery and put into monolayer cell culture. After 4-5 days in culture, cells were loaded with fura-2 and intracellular free Ca2+ measured as previously reported. We found that bradykinin increased intracellular free Ca2+ in all cells tested, whereas isoproterenol increased intracellular free Ca2+ in only half the cells tested, suggesting that more than one transporting cell type may be present in this tissue. Epinephrine and prostaglandin E2 had no effect on intracellular free Ca2+. We found that the voltage-sensitive Ca2+ channel blocker verapamil had no effect on the bradykinin-induced change in intracellular free Ca2+. Removal of Ca2+ from the bathing saline only slightly attenuated the increase in intracellular free Ca2+ that resulted from stimulation with bradykinin. We conclude that the source of the increased intracellular free Ca2+, observed during stimulation with secretagogues, was primarily intracellular stores.

Bradykinin↗
PubMed1991

Reversal of alcohol dependence and tolerance by a single administration of flumazenil.

Chronic exposure to ethanol is associated with the development of tolerance to the acute effects of ethanol and a withdrawal syndrome characterized by anxiety and seizure susceptibility. In the present study we examined the ability of flumazenil (Ro15-1788), a benzodiazepine receptor antagonist, to reverse neuronal and behavioral manifestations of ethanol tolerance and dependence. A single injection of flumazenil (10 mg/kg, 14 hr before withdrawal) to mice administered a liquid diet containing ethanol for 10 days, reduced seizure severity during withdrawal from ethanol. Acute tolerance to ethanol-induced hypothermia was not sensitive to flumazenil treatment, but tolerance and diazepam-induced cross-tolerance to the ataxic effects of ethanol were reversed by a single injection of flumazenil given 2 to 26 hr before evaluation of tolerance. At a biochemical level, the ability of benzodiazepine inverse agonists (e.g., Ro15-4513) to reduce the activity of gamma-aminobutyric acid (GABA) receptor-operated chloride channels may represent a neuronal manifestation of ethanol dependence (Buck and Harris, 1990). Flumazenil treatment of ethanol-dependent mice 14 hr before isolation of brain membrane vesicles partially reversed the augmentation of Ro15-4513 inhibition of muscimol-stimulated 36Cl- uptake in vitro. These results demonstrate that brief occupation of benzodiazepine receptors by an antagonist may reset the cellular mechanisms responsible for the development of ethanol tolerance and dependence, and support the hypothesis that increased sensitivity to benzodiazepine inverse agonists is involved in the development of ethanol dependence.

Alcoholism↗
PubMed1991

A general method for quantitative PCR analysis of mRNA levels for members of gene families: application to GABAA receptor subunits.

We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.

Animals↗