Ronald J. Bradley

Cardiovascular effects of morphine and opioid peptides following intracisternal administration in chloralose-anesthetized rats.

Beta-Endorphin (0.9--2.0 nmol), morphine (11--250 nmol) and D-ala2-met-enkephalinamide (17--33 nmol) administered intracisternally produced preferential vasodepressor responses and bradycardia. Leu- (1.8--180 nmol), met-enkephalin (17--520 nmol) and alpha-endorphin (5.7--57 nmol) administered in the same way produced preferential vasopressor effects and the latter two peptides also produced bradycardia. Results obtained with naloxone (300 nmol) given intracisternally indicate that the pressor and depressor actions as well as the bradycardia are mediated through opiate receptors. The results indicate that opioid peptides may be involved in central cardiovascular control.

Antipsychotic drugs affect glucose uptake and the expression of glucose transporters in PC12 cells.

1. Adherence of the PC12 cell line to poly-l-lysine (PLL) on tissue culture dishes stimulated glucose transport into the cells. Fluphenazine, chlorpromazine, clozapine and haloperidol inhibited glucose uptake in this system after a short (30 min) preincubation with drug. The IC50s for this effect were typically in the range of 5-40 microM. 2. Following longer exposures of the drugs (24 hr), there was a significant increase (approximately 3-fold) in the cellular levels of the glucose transporter (GLUT) isoforms, GLUT1 and GLUT3. 3. Long-term incubation (48 hr), especially with the phenothiazine drugs, was accompanied by a marked reduction in cell growth and proliferation. The rank ordering of the potencies of the drugs was essentially the same for these various effects: fluphenazine > chlorpromazine > clozapine approximately haloperidol. 4. It is suggested that the effects on glucose transport reported here may complicate the interpretation of positron emission tomography (PET) studies that rely on the uptake of radiolabeled glucose analogs to measure the physiological response to these drugs.

Juvenile myasthenia gravis

We studied 32 children with myasthenia gravis over a period of 12 years. The mean age at onset was 7.7 years. Presentation was ocular in 63% of patients. Another major disease in addition to myasthenia occurred in 44% of patients; a seizure disorder was the most commonly associated disease. Serum IgG antibody to nicotinic acetylcholine receptor was present in 53% of patients and did not correlate with severity of disease or treatment. Medical management was effective in 63%; thymectomy was effective in only 28%. We conclude that myasthenia gravis appears commonly before age 10 and is associated with the risk of some disease other than hyperthyroidism. Serum IgG nicotinic acetylcholine receptor antibody is present less frequently than in normal adults, and vigorous medical management should be attempted before thymectomy.

The effects of alcohols and diols at the nicotinic acetylcholine receptor of the neuromuscular junction

A series of straight chain aliphatic alcohols from ethanol to octanol were tested at voltage-clamped frog endplates. In the presence of high concentrations of ethanol (greater than 1 M) the individual current responses to ionophoretic pulses of ethanol were reduced in amplitude and the dose-response curve for acetylcholine was shifted to the right. All the alcohols tested had this effect and their potency increased with the length of the carbon chain. The results were interpreted to indicate that as the molecular weight of the alcohol increased, its potency as a channel blocker also increased. The diol derivative of ethanol, which is ethylene glycol (ethanediol), was totally inactive up to 400 mM. However, 1,3-propanediol was a more potent blocker than propanol. After dose-response curves were carried out in high doses of ethanol and propanediol, the number of receptors was found to be permanently reduced. This effect could be due to irreversible denaturation of the receptor and therefore reversible denaturation could account for some of the reversible blocking effects caused by such drugs. An additional effect on the receptor was observed in that low concentrations of ethanol and propanol reduced the apparent dissociation constant for acetylcholine, thus increasing the amplitude of individual responses and shifting the dose-response curve to the left.

Purification and characterization of nicotinic acetylcholine receptors from muscle

The nicotinic acetylcholine receptor was purified from normal and denervated rat skeletal muscle. The purification protocol included alpha-cobratoxin biospecific adsorption, ion exchange chromatography, and gel filtration steps. The highest specific activity achieved was 7.5 pmol of 125I-alpha-bungarotoxin binding sites per microgram protein. Sodium dodecyl sulfate gel electrophoresis of purified AChR revealed subunits with molecular weights of 42,000 and 66,000 daltons and a minor component with a molecular weight of 52,000 daltons. Normal muscle AChR is comprised of one toxin binding component. Upon denervation a second component appears, but both components are increased as a consequence of denervation. A dissociation constant of 1.5 x 10(-8)M was determined for d-tubocurarine from receptor from both normal and denervated muscle. A dissociation constant of 1 x 10(-7)M for acetylcholine, perhaps analogous to the high affinity acetylcholine binding observed in electric fish receptor, was determined.

INTERACTION OF HUMAN ANTIBODY AND MURINE MONOCLONAL ANTIBODY WITH MUSCLE ACETYLCHOLINE RECEPTOR

Immunological analyses of the nicotinic acetylcholine receptor (AChR) have led to important discoveries concerning its structure and function. The AChR is the target of an autoimmune response in myasthenia gravis (MG) , a debilitating neuromuscular disease. Antibodies which bind to the receptor can be detected in the serum of SO-90% of myasthenia However, the titer of anti-AChR immunoglobulins correlates poorly with disease severity 6 * suggesting a complex pathogenesis in MG. Myasthenic antibodies have been used to detect immunological differences between junctional receptor (JR) and extrajunctional receptor (EJR) isolated from mammalian EJR appears to contain antigenic determinants which are not found on JR. In addition, antireceptor antibodies have been employed to characterize the subunit composition of electric fish receptor. Antibodies against each of the four elasmobranch AChR polypeptide chains react with native AChR from Electrophorus electricus (electric eel) and mammalian skeletal muscle, suggesting that AChR from all three sources shares a common subunit structure.9 Animals immunized with purified AChR often display signs of weakness which are characteristic of human MG.10l1 These findings have led to the development of an animal model of the disease known as experimental myasthenia gravis (EMG) . Recently, monoclonal antibodies (MAbs) against the AChR have been produced by somatic cell hybridization techniques.12+ l3 MAbs have been shown to produce EMG in rats 14, l5 and guinea pigs.14 The studies described in this report were aimed at immunological characterization of the receptor and delineation of its role in MG. Specifically, we wanted to investigate the nature of the differences between JR and EJR and to examine the role of the different AChR subunits as antigenic sites in MG.

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel

SummaryA study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.

Characterization of a nicotinic acetylcholine receptor from rabbit skeletal muscle and reconstitution in planar phospholipid bilayers

Abstract The nicotinic acetylcholine receptor from rabbit skeletal muscle was isolated by affinity chromatography and characterized by 125 I- α -Bungarotoxin binding and gel filtration chromatography. Quantal conductance events were observed when this material was added to planar phospholipid bilayers. These changes were stimulated by carbamylcholine and antagonized by curare, Butx, dithiothreitol and concanavalin A. The receptor preparation was found to bind 0.2 nMoles 125 I- α-Bungarotoxin per mg protein and the molecular weight was estimated to be 390,000.

Overlap myasthenic syndrome: combined myasthenia gravis and Eaton-Lambert syndrome

A patient with a known history of pernicious anemia had the combined features of autoimmune myasthenia gravis (MG) and the Eaton-Lambert syndrome (ELS). Initially, this patient had all the features typical of MG, and after thymectomy developed all the typical features of ELS. In view of the coexistence of two autoimmune neuromuscular transmission disorders in one patient, we termed this disorder “overlap myasthenic syndrome.”

Quantum conductance changes in lipid bilayer membranes associated with incorporation of acetylcholine receptors.

CONSIDERABLE attention has focused on the isolation and characterisation of the nicotinic acetylcholine (ACh) receptor from electroplax1–4. This receptor is identified by its ability to bind cholinergic ligands, and its molecular weight and amino acid content have been estimated5,6. Similar extraction procedures have also been applied to nicotinic receptors from mammalian brain7 but it is not known whether the coupling of ACh to the receptor results in the opening of transmembrane channels, the activation of ionic carrier mechanisms or some other mechanism that modifies membrane permeability. Little is known about the ionic permeability changes associated with the function of the single receptor. It has been reported8 that crude acetylcholinesterase (AChE) preparations from electroplax contain material capable of inducing transient membrane permeability changes in lipid bilayers in response to ACh or carbachol, but the relationship to ACh receptor function is unclear.