Evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network.

The increasing use of influenza virus neuraminidase (NA) inhibitors (NIs) necessitates the development of reliable methods for assessing the NI susceptibility of clinical isolates. We evaluated three NA inhibition assays against a panel of five clinical isolates each of influenza virus A/H1N1, A/H3N2, and B strains and four viruses with a defined resistance genotype (R292K, H274Y, R152K, and E119V). For fluorometric enzyme assay (FA) 1 (FA-1), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNANA) at 100 microM was used as the substrate, with pretitration of the virus input. For FA-2, MUNANA at 200 microM was used as the substrate, with a fixed 1:10 dilution of input virus. For the chemiluminescence (CL) assay, the 1,2-dioxetane derivative of sialic acid at 100 microM was used as the substrate, with pretitration of the virus. Four different operators repeated the assays several times in a blinded fashion with both zanamivir and oseltamivir carboxylate (GS4071) to determine intra- and interassay variations. Mean 50% inhibitory concentration (IC(50)) values were lower and generally less variable with the CL assay. FA-1 displayed greater variation than the CL assay or FA-2 and the highest IC(50) values with zanamivir; FA-2 showed the highest values with oseltamivir, particularly for influenza virus B, and was more variable with zanamivir than was the CL assay. All three assays detected 40-fold or greater changes in IC(50) values for the resistant viruses with at least one drug. Mixing experiments, whereby increasing fractions (0, 20, 40, 60, 80, and 100%) of NA from a known NI-resistant virus were mixed with the corresponding NI-sensitive parental NA, indicated that the resolution of IC(50) values was clearer with the CL assay than with FA-2 for two of the resistant variants (R152K and E119V). The FA and CL methods were reliable for the detection of NI resistance, but all assays have certain limitations. Based on reproducibility, ease of automation, time required for the assay, and greater sensitivity, the CL assay was selected for future susceptibility testing of influenza virus isolates circulating globally.

Beta-adrenergic receptors regulate astrogliosis and cell proliferation in the central nervous system in vivo.

Astrocytes express several cell surface receptors including the beta 2 -adrenergic receptor. To explore whether beta-adrenergic receptors (beta-ARs) directly regulate astrogliosis and glial scar formation, we evaluated the effects of beta-AR activation and blockade on astrocyte hypertrophy and cell proliferation in rabbit optic nerves in vivo. Artificial cerebrospinal fluid (CSF), isoproterenol (ISO; a beta-agonist), or propranolol (PROP; a beta-antagonist) were infused via osmotic minipumps into non-injured and crushed optic nerves for 14 days. Changes in nerve cell numbers and astroglial hypertrophy were monitored by ethidium bromide nuclear staining and glial fibrillary acidic protein (GFAP) immunohistochemistry, respectively. In non-injured nerves infused with CSF or PROP, there were no alterations in GFAP-immunoreactivity or cell numbers compared to normal optic nerves; however, in non-injured nerves infused with ISO, there was a significant increase in both GFAP-immunoreactivity and cell number. In crushed optic nerves, there was a significant increase in both GFAP-immunoreactivity and cell number compared to normal nerves, and this increase was not altered by infusion of either CSF or ISO. In contrast, PROP infusion significantly reduced the crush-induced increase in GFAP-immunofluorescence and cell number. These findings suggest that a) beta-AR activation, in the absence of injury, can promote astroglial hypertrophy and cell proliferation; b) after injury, beta-AR activation drives injury-induced astrogliosis and cell proliferation; c) astrocyte beta-ARs are maximally stimulated after neuronal injury; and d) neuronal regeneration may be influenced, both positively and negatively, through the pharmacological manipulation of glial receptors.

Physiological regulation of G4 AChe in fast-twitch muscle: effects of exercise and CGRP.

This work addresses the physiological regulation of the tetrameric (G4) form of acetylcholinesterase (AChe) in end-plate regions of anterior gracilis muscles from adult male Sprague-Dawley rats subjected to short-term low-intensity treadmill exercise. Experiments involved analyses of muscle AChe molecular form activities, endogenous calcitonin gene-related peptide (CGRP) levels, and the effect of exogenous CGRP on AChe forms after exercise. Animals were exercised twice per day for 1 or 2 days. Daily training sessions of 135 min (10 min of walking alternating with 5 min of resting) were separated by a 105-min resting period. Results show that exercise causes a slight decline in endogenous CGRP and a selective increase in G4 AChe that is partially reversed by treatment with exogenous CGRP. These findings indicate that CGRP influences the mechanism(s) by which G4 AChe in intact fast-twitch anterior gracilis muscles adapts to enhanced motor activity. They are also consistent with the hypothesis that, in addition to acetylcholine, neurogenic CGRP participates in the regulation of G4 AChe at the neuromuscular junction.

A role for calcitonin gene-related peptide in the regulation of rat skeletal muscle G4 acetylcholinesterase.

Calcitonin gene-related peptide (CGRP) acts as an anterograde trophic agent which regulates skeletal muscle acetylcholine receptor function. We examined whether CGRP also influences other synaptic transmission-related molecules, i.e. acetylcholinesterase (AChE) forms. Results show that: (a) CGRP associated with rat anterior gracilis muscle endplates declines following obturator nerve transection; (b) exogenous CGRP treatment has a selective, innervation-like effect on the globular tetramer (G4) of AChE in gracilis motor endplates; and (c) this effect is reversed by the CGRP receptor antagonist hCGRP8-37. We conclude that exogenous CGRP, and/or a biologically active CGRP fragment(s), influences G4 AChE levels through specific CGRP-CGRP receptor interactions. This conclusion is consistent with the notion that motor nerve-derived CGRP participates in the trophic control of G4 AChE at the neuromuscular junction.

Axoplasmic transport of calcitonin gene-related peptide in rat peripheral nerve as a function of age.

Calcitonin gene-related peptide (CGRP) has been implicated in the trophic regulation of acetyl-choline receptors and G4 acetylcholinesterase at the rat neuromuscular junction. Since these latter molecules exhibit significant changes with advancing age, we examined the possibility that certain aspects of CGRP transport are also influenced by aging. Double nerve ligations and CGRP radio-immunoassay of 3-mm nerve segments permitted the assessment of the peptide's apparent transport rates in sciatic nerves from 3-, 12-, and 24-month-old Fischer 344 rats. Results confirm that CGRP is conveyed by anterograde axoplasmic transport; more importantly, they suggest that CGRP is also transported retrogradely, but in smaller amounts and at slower rates. In addition, our findings indicate that the apparent rates of CGRP transport in both directions significantly decline with advancing age. These data are consistent with the notion that changes in CGRP delivery may contribute to age-related changes in junctional acetylcholine receptors and acetylcholinesterase.

Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles.

This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: low-intensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by alpha-bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G1 + G2 AChE; the amount of A12 AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G4 AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.

A role for acetylcholine-nicotinic receptor interactions in the selective increase of rat skeletal muscle G4 acetylcholinesterase following short-term denervation.

The present work addresses the effects of short-term denervation on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in anterior gracilis muscles from adult male Sprague-Dawley rats. It examines possible relationships between AChE isoform changes and other denervation phenomena, and evaluates the importance of acetylcholine (ACh)-nicotinic receptor interactions in selectively modulating the activity of G4 AChE. Results confirm that denervation causes a specific, transient increase in G4 AChE and show that: most of the increment can be explained by the hydrophobic species of this isoenzyme; changes in AChE isoforms markedly precede the onset of spontaneous electromechanical activity (fibrillation), as well as acetylcholine receptor (AChR) proliferation; and the G4 AChE response is eliminated when AChRs are blocked by alpha-bungarotoxin treatment performed before but not after (24 h) denervation. These data point to the absence of direct causal relationships between the G4 AChE increment and fibrillation, AChR proliferation, or changes in the release of this isoform from denervated muscle. In turn, they suggest the participation of AChR activation in triggering the G4 AChE response and emphasize the possible role of ACh-AChR interactions in modulating the production of this isoenzyme in not only denervated but also innervated fast-twitch muscles.

Selective increase of tetrameric (G4) acetylcholinesterase activity in rat hindlimb skeletal muscle following short-term denervation.

Acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes in gracilis muscles from adult Sprague-Dawley rats were studied 24-96 h after obturator nerve transection. Results show a selective denervation-induced increase in the globular G4 isoform, which is predominantly associated with the plasmalemma. This enzymatic increase was (a) transient (occurring between 24 and 60 h) and accompanied by declines in all other identifiable AChE isoforms; (b) observed after concurrent denervation and inactivation of the enzyme with diisopropylfluorophosphate, but not following treatment with cycloheximide; and (c) more prominent in the extracellular compartment of muscle endplate regions. Aside from this transient change, G4 activity did not fall below control levels, indicating that at least the short-term maintenance of G4 AChE (i.e., at both normal and temporarily elevated levels) does not critically depend on the presence of the motor nerve. In addition, this isoform's activity increases in response to perturbations of the neuromuscular system that are known to produce elevated levels of acetylcholine (ACh), such as short-term denervation and exercise-induced enhancement of motor activity. The present study is consistent with the hypothesis that individual AChE isoforms in gracilis muscle are subject to distinct modes of neural regulation and suggests a role for ACh in modulating the activity of G4 AChE at the motor endplate.

Coated vesicles from developing and adult rat skeletal muscles contain multiple molecular forms of acetylcholinesterase.

The main purpose of this work was to determine which of the multiple isoforms of acetylcholinesterase (AChE) are associated with clathrin-coated vesicles (CVs) from developing and adult rat skeletal muscles. CV-enriched preparations were obtained by subcellular fractionation/equilibrium sedimentation and further purified by immunoadsorption to anti-clathrin IgG-coated Staphylococcus aureus cells. Analysis of individual AChE isoforms by velocity sedimentation ultracentrifugation showed that a) while both globular (G-forms) and asymmetric (A-forms) AChE were detected in all subcellular fractions evaluated, the CV-enriched fraction contained a higher proportion of A-forms (mainly the A12 species); b) most of the AChE activity contained in such a CV fraction was recovered following immunoadsorption; c) alkaline extraction conditions (pH 8.5) which depolymerize clathrin were necessary to detect a large proportion of A-forms in both the CV-enriched and immunoprecipitated preparations, while most of the G-forms (especially G1 + G2 AChE) were detected following extraction at pH 6.8; and d) comparison of AChE isoform profiles from neonate and adult muscle CV-enriched fractions showed a greater concentration of A-forms in the former. These data suggest that considerable amounts of A12 and, to a lesser extent, G4 AChE are sequestered within muscle CVs which may be destined for the plasmalemma. Our findings also indicate that the relative proportions of AChE isoenzymes in rat muscle CVs vary according to the extent of muscle development and lend support to the contention that CVs participate in the externalization of functionally important AChE isoenzymes.

Acetylcholinesterase molecular forms in rat heart.

A comparison of the molecular forms of acetylcholinesterase (AChE) in adult rat atria and ventricles was undertaken. The major forms present in both the atria and ventricles were globular 4S (G1) and 10S (G4) and asymmetric 16S (A12) with minor contributions from 6S (G2) and 12S (A8). Although the total specific AChE activity was higher in atrial samples, no differences in the proportions of the major AChE forms between the atrial and ventricular samples were seen. For example, 16S AChE accounted for 8% to 10% of the total AChE activity in all examined regions of the heart. Cardiac 16S AChE was shown to be soluble in high ionic strength buffer. The addition of EDTA to the extraction buffer resulted in no further solubilization of 16S AChE, indicating that only Type I asymmetric AChE is present in the heart. Additionally, 16S AChE did not require Triton X-100 for extraction. In contrast, 35% of the globular AChE required non-ionic detergent for extraction, which indicates that a percentage of globular AChE in rat heart is membrane-associated.

Permanent hippocampal mossy fiber hyperdevelopment following prenatal ethanol exposure.

Prenatal exposure to a liquid diet containing 35 percent ethanol derived calories during days 1-21 of gestation resulted in significant alterations in the topographical organization of the zinc-rich mossy fiber system of the rat hippocampus. Using the Timm's sulfide silver histochemical method, permanent alterations were found in the position of the mossy fiber terminal field. Aberrant distal infrapyramidal mossy fiber terminal staining occurred at midtemporal hippocampal levels in ethanol-exposed rats but not in the normal or pair-fed controls; the distribution of distal infrapyramidal mossy fibers in pair-fed rats was even more restricted than in normal rats suggesting that the aberrant mossy fiber topography was the result of the ethanol exposure rather than undernutrition. The abnormal terminal field was found in ethanol-exposed rats up to nine months of age (the oldest animals tested). The results indicate that ethanol exposure in utero, during a period of brain development roughly equivalent to the first and second human trimesters, can produce permanent developmental alterations in brain circuitry.