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Biomedical subjects

L B Pearce

Publications and source records attributed to L B Pearce.

33 records · Page 2Linked to original sources
PubMed1989

A superfusion system designed to measure release of radiolabeled neurotransmitters on a subsecond time scale.

A new method for subsecond measurement of release of neurotransmitters from nerve terminal preparations (e.g., synaptosomes) in vitro is described. Synaptosomes were prelabeled with [3H]GABA via a Na-dependent GABA uptake system. The prelabeled nerve terminals are retained on small glass fiber filters in a superfusion chamber accessed by three high speed, solenoid-driven valves. Microcomputer-programmed circuitry controls the timing of valve operation. Each valve controls the delivery of a separate solution to the chamber, permitting rapid and independent control of membrane potential, [Ca2+]e, and drug delivery. The minimal dead volume of the chamber and the relatively high solution flow rate afford time resolution for release of at least 60 ms. This time resolution was necessary to observe the most rapid of at least three components of GABA release.

Animals↗
PubMed1988

Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation.

Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic 3',5'-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releasable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol.(mg of disk protein)-1.min-1] may be sufficient to produce a 1 microM change in local cytoplasmic [Ca] within 20 ms. The Ca:Na selectivity ratio is approximately 0.5:1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4 microM vs 2.8 microM) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25 microM l-cis-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.

Amiloride↗
PubMed1986

Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain.

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P1-PST) was subsequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P1-PST proceeds via a sequential ordered, bi-substrate reaction mechanism, where 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the leading substrate. The true Km and Kia values for PAPS were both 0.35 microM, while the true Km value for phenol was 2.8 microM.

Arylsulfotransferase↗
PubMed1986

Substance B: an endogenous brain factor that reverses presynaptic inhibition of acetylcholine release.

Evidence is presented of the existence of a substance in brain (substance B) that reverses presynaptic inhibition of evoked acetylcholine (AcCho) release. Addition of guinea pig brain synaptosomal incubation medium or whole brain extracts to assays in which 1,1-dimethyl-4-phenylpiperazinium-evoked release of [3H]AcCho from guinea pig myenteric plexus synaptosomes reversed purinergic, muscarinic, and alpha 2-adrenergic agonist inhibition. However, the extracts did not increase basal or evoked release of AcCho. Results of chromatography on Bio-Gel P-2 of a concentrated whole brain extract suggest this factor has an Mr of 700. Substance B was resistant to boiling under acid or alkaline conditions as well as incubation with phospholipase C and various proteases; ashing, however, completely destroyed activity. This endogenous factor was also found to antagonize agonist-mediated inhibition of electrically evoked contractions of myenteric plexus-longitudinal muscle strips from the guinea pig ileum. The demonstration of substance B with its ability to reverse presynaptic inhibition of AcCho release reveals one mode of presynaptic modulation.

2-Chloroadenosine↗
PubMed1986

Isolation of a factor that reverses presynaptic inhibition of acetylcholine release.

A factor (Substance B) has been isolated from brain which reverses the presynaptically-modulated inhibition of evoked ACh release from both guinea-pig myenteric plexus-longitudinal muscle synaptosomes and the intact strip. Inhibitory modulating agents whose activity is reversed by Substance B include oxotremorine, 2-chloroadenosine, clonidine, and morphine. In addition to brain, Substance B is also present in heart and ileum but not in liver or kidney. As determined by Biogel P2 chromatography, this factor appears to have a molecular weight of around 700. It is not destroyed by preincubation with periodate, amylase, adenosine deaminase, pronase, trypsin, phospholipase C or carboxypeptidase Y.

2-Chloroadenosine↗
PubMed1986

Isolation of a factor (substance B) that antagonizes presynaptic modulation: pharmacological properties.

An aqueous extract of brain has recently been shown to contain a factor (substance B) that antagonizes presynaptic inhibition of evoked [3H]acetylcholine release from guinea pig ileal synaptosomes. This factor antagonized the inhibition of electrically evoked contractions of the intact guinea pig longitudinal muscle-myenteric plexus preparation by a variety of pharmacological agents including clonidine, 2-chloroadenosine, morphine and phencyclidine. pA2App values for substance B antagonism of these four agonists were very similar. Schild plot analysis suggests a functional competition between substance B and each of these receptor agonists. In the absence of any agonist, substance B had a minimal effect on the force of contraction. This reversal of inhibition by substance B was not altered by the ganglionic blocking agent hexamethonium. Substance B was not able to reverse the inhibition of contractions elicited by atropine. In addition to its localization in brain, the factor was also found in appreciable quantities in the heart and the ileum, but not in the liver or kidney. These results indicate that an endogenous neuromodulator exists in innervated tissues that antagonizes a subcellular mechanism(s) involved in mediating inhibition of neurotransmitter release by alpha adrenergic, opiate and purinergic agonists, as well as phencyclidine.

2-Chloroadenosine↗
PubMed1985

Cellular localization of MAO A and B in brain: evidence from kainic acid lesions in striatum.

The cellular localization of the two forms of monoamine oxidase (MAO A and MAO B) was studied by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid to produce selective degeneration of striatal neurons and subsequent proliferation of astrocytes. The results demonstrated a persistent loss of 15-20% in MAO A activity, whereas MAO B activity decreased initially by 25% and then increased to more than twice the control value by 54 days after lesions. The changes in activity were compared to parallel estimates of the postsynaptic neuronal enzyme markers glutamic acid decarboxylase (GAD) and neuron-specific enolase (NSE), astroglial enzyme markers glutamine synthetase (GS) and non-neuronal enolase (NNE), and the presynaptic enzyme marker DOPA decarboxylase (DDC). The results suggest that a small amount of striatal MAO A is present in kainic acid-sensitive postsynaptic striatal neurons and that MAO B is probably localized in both neurons and astrocytes.

Animals↗
PubMed1985

Purification and kinetic characterization of a dopamine-sulfating form of phenol sulfotransferase from human brain.

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.

Adenosine Triphosphate↗
PubMed1985

Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods.

Recently, evidence has been published which suggests that [Husain, M., Edmondson, D. E., & Singer, T.P. (1982) Biochemistry 21, 595-600] monoamine oxidase [amine:oxygen oxidoreductase (MAO), EC 1.4.3.4] deaminates phenylethylamine and benzylamine via two distinct kinetic pathways which involve either binary or ternary complex formation, respectively. These conclusions were drawn largely from stopped-flow kinetic analysis performed on purified enzyme removed from its native membrane and in the presence of the inhibitory detergent Triton X-100. In this study, d-amphetamine and alternative substrates were used as steady-state probes of the kinetics of deamination by the B form of human brain MAO using native membrane-bound enzyme. Initial velocity studies showed mixed-type patterns for amphetamine inhibition of phenylethylamine, tryptamine, and tyramine when either amine or oxygen was the varied substrate. Slope and intercept vs. amphetamine concentration replots were linear in all cases except for phenylethylamine (hyperbolic); Ki values obtained from linear replots of slope or intercept values were comparable. In contrast, amphetamine was a competitive inhibitor of benzylamine deamination when amine concentration was varied and uncompetitive when oxygen concentration was varied; slope and intercept replots were linear for both. When benzylamine was the alternative substrate inhibitor and tyramine and tryptamine deamination was measured, mixed-type inhibition patterns were obtained when either amine or oxygen concentration was varied; replots of slope and intercept were linear in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗
PubMed1983

Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside.

Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.

Brain↗
PubMed1981

Renal tubular transport and metabolism of organic cations by the rabbit.

The renal tubular transport of the organic cations tetraethylammonium (TEA), N1-methylnicotinamide (NMN), and choline was studied in anesthetized rabbits by the urinary clearance technique. The clearance ratio of [14C]TEA/inulin was 5.72 +/- 0.44 and the clearance ratio of [14C]TEA/p-aminohippuric acid (PAH) was 0.98 +/- 0.02. The clearance ratio of the 14C label/inulin when [14C]NMN was being infused was only 1.31. The clearance ratio of choline/inulin was less than 1 at choline infusion loads from 0 to about 6 mumol . kg-1 . min-1, which produced a plasma choline level of 100 microM. At higher infusion rates the urinary clearance ratio of choline/inulin rose to a maximum of 2 at plasma choline levels of 300-500 microM. Renal metabolism of choline and NMN were revealed by the use of the isolated perfused rabbit kidney. [14C]Choline was extensively metabolized by the kidney into betaine. The renal metabolite of [14C]NMN comigrated with nicotinamide in electrophoresis. The relatively low urinary clearance of the label associated with NMN in the rabbit presents a striking difference from its transport in dog and rat. Studies using isolated perfused segments are done using tissue from rabbits primarily. These data from the intact rabbit kidney may be used to guide future studies of organic cation transport with isolated perfused segments.

Animals↗
PubMedDate not supplied

Botulinum a toxin for spasmodic torticollis: multiple vs single injection points per muscle.

Eighty-six injections in 49 patients with adult onset spasmodic torticollis were evaluated for efficacy with respect to single point per muscle versus multiple point per muscle injection techniques. Parameters of the syndrome assessed were pain, posture deformity, range of cervical motion, disfigurement, cervical muscle hypertrophy, activity limitation, and degree of involuntary movement. The multiple point per muscle injection strategy appeared superior to the single injection per muscle technique with respect to pain (p less than 0.002, chi-square), posture deformity (p less than 0.001), range of motion (p less than 0.001), and improvement in activity endurance (p less than 0.001). No significant differences were noted with respect to cervical muscle hypertrophy or degree of involuntary movements, although the injections were considered beneficial in both groups to these syndrome components.

Adult↗